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WO2025137511A9 - Procédés intégrés et itératifs de conception, d'identification et d'optimisation de peptides lasso - Google Patents

Procédés intégrés et itératifs de conception, d'identification et d'optimisation de peptides lasso

Info

Publication number
WO2025137511A9
WO2025137511A9 PCT/US2024/061377 US2024061377W WO2025137511A9 WO 2025137511 A9 WO2025137511 A9 WO 2025137511A9 US 2024061377 W US2024061377 W US 2024061377W WO 2025137511 A9 WO2025137511 A9 WO 2025137511A9
Authority
WO
WIPO (PCT)
Prior art keywords
lasso
peptide
binding
lasso peptide
target molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/061377
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English (en)
Other versions
WO2025137511A1 (fr
Inventor
Mark J. Burk
Rajan CHAUDHARI
Matthew Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lassogen Inc
Original Assignee
Lassogen Inc
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Filing date
Publication date
Application filed by Lassogen Inc filed Critical Lassogen Inc
Publication of WO2025137511A1 publication Critical patent/WO2025137511A1/fr
Publication of WO2025137511A9 publication Critical patent/WO2025137511A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • lasso peptides that have been discovered and optimized for binding selectivity, binding affinity, biological activity, and other properties using the methods described herein. 3. BACKGROUND [0003] Peptides serve as useful tools and leads for drug development since they often combine high affinity and specificity for their target receptor with low toxicity (Henninot, A., et al., J. Med Chem., 2018, 61, 1382-1414). However, the clinical use of peptides as efficacious drugs has been limited due to undesirable physicochemical and pharmacokinetic properties, including poor solubility and cell permeability, low bioavailability, and instability due to rapid proteolytic degradation under physiological conditions.
  • a computer-based method for identifying a lasso peptide having optimal binding with a target molecule can include: (a) providing one or more three-dimensional (3D) model structures of lasso peptides; (b) performing conformational analysis on the one or more 3D model structures of lasso peptides using molecular dynamic simulation algorithms to obtain at least two conformational states of the one or more 3D model structures of lasso peptides; (c) docking the at least two conformational states of the one or more 3D model structures of lasso peptides onto a 3D model structure of the target molecule at a lasso-binding site of the target molecule to form at least two 3D model structures of the lasso peptide-target molecule complex; (d) performing conformational analysis on the at least two 3D model structures of the lasso peptide-target molecule complex using molecular dynamic simulation algorithms to obtain
  • the method provided herein further includes step (a-1): mapping a target-binding epitope onto different locations of the one or more 3D model structures of lasso peptides.
  • the method provided herein further 2 ACTIVE 705331286v1 includes computationally grafting the target-binding epitope onto a mapped location of the selected lasso peptide, thereby generating a grafted lasso peptide binder candidate.
  • the at least two conformational states of the one or more 3D model structures of lasso peptides are selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 or 50 or more different conformational states.
  • the favored free energy comprises a lower free energy compared to a different 3D model structure of the lasso peptide-target molecule complex.
  • the favored free energy is the lowest free energy of the two or more 3D model structure of the lasso peptide-target molecule complex.
  • step (a) further comprises retrieving one or more known 3D structures of lasso peptides from a protein structure database.
  • step (a) further comprises computationally modeling the 3D structure of a lasso peptide based on atomic coordinates of the lasso peptide.
  • the atomic coordinates of the lasso peptides are obtained from a protein structure database or scientific literature.
  • the protein structure database is selected from worldwide Protein Data Bank (wwPDB), Cambridge Structure Database, Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database.
  • wwPDB worldwide Protein Data Bank
  • NCBI National Center for Biotechnology Information
  • BMRB Biological Magnetic Resonance Data Bank
  • the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction, or 3-dimensional electron microscopy (3D-EM).
  • the X-ray crystallography is serial femtosecond crystallography.
  • the 3D-EM is cryogenic electron microscopy (cryo-EM).
  • step (a) further comprises computationally modeling the 3D structure of the lasso peptide based on X-ray diffraction data and/or nuclear magnetic resonance (NMR) data of the lasso peptide and atomic coordinates of a reference lasso peptide, and wherein the lasso peptide has at least 50% amino acid sequence identity to the reference lasso peptide.
  • the X-ray diffraction data of the lasso peptide are obtained by subjecting a crystal of the lasso peptide to 3 ACTIVE 705331286v1 X-ray crystallography analysis.
  • step (a) comprises: (i) obtaining atomic coordinates of the lasso peptide based on the X-ray diffraction data; and (ii) refining the atomic coordinates of the lasso peptide based on the atomic coordinates of the reference lasso peptide.
  • the nuclear magnetic resonance (NMR) data of the lasso peptide comprises NMR chemical shift, J-coupling constant, and resonance intensity obtained by subjecting a solution of the lasso peptide to NMR analysis.
  • step (a) comprises: (i) creating an ensemble of structural models of the lasso peptide based on the NMR data; (ii) obtaining mean atomic coordinates of the lasso peptide based on the ensemble of structural models; and (iii) refining the atomic coordinates of the lasso peptide based on the atomic coordinates of the reference lasso peptide.
  • step (a) comprises: computationally modeling the 3D structure of a lasso peptide based on the amino acid sequence of the lasso peptide and atomic coordinates of a reference lasso peptide; and wherein the lasso peptide has at least 50 percent (%) amino acid sequence identity to the reference lasso peptide.
  • computationally modeling the 3D structure of the lasso peptide is performed by homology modeling.
  • homology modeling is performed in combination with a protein structure prediction algorithm, wherein optionally the protein structure prediction algorithm is trRosetta or AlphaFold or AlphaFold 2.
  • creating the 3D model structure of the target molecule comprises computationally modeling the 3D structure of the target molecule based on X-ray diffraction data and/or nuclear magnetic resonance data of the 4 ACTIVE 705331286v1 target molecule and atomic coordinates of a reference polypeptide; wherein the reference polypeptide has at least 50% amino acid sequence identity to the reference polypeptide.
  • the X-ray diffraction data of the target molecule are obtained by subjecting a crystal of the target molecule to X-ray crystallography analysis.
  • creating the 3D model structure of the target molecule comprises: (i) obtaining atomic coordinates of the target molecule based on the X-ray diffraction data; (ii) refining the atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iii) computationally modeling the 3D structure of the target molecule based on the refined atomic coordinates.
  • the nuclear magnetic resonance (NMR) data of the target molecule comprises NMR chemical shift, J-coupling constant, and resonance intensity obtained by subjecting a solution of the target molecule to NMR analysis.
  • creating the 3D model structure of the target molecule comprises: (i) creating an ensemble of structural models of the target molecule based on the NMR data; (ii) obtaining mean atomic coordinates of the target molecule based on the ensemble of structural models; (iii) refining the mean atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iv) computationally modeling the 3D structure of the target molecule based on the refined mean atomic coordinates.
  • creating the 3D model structure of the target molecule comprises computationally modeling the 3D structure of the target molecule based on the amino acid sequence of the target molecule and atomic coordinates of one or more reference polypeptide; and wherein the amino acid sequence of the target molecule is at least about 50% identical to the amino acid sequence of the reference polypeptide.
  • computationally modeling the 3D structure of the target molecule is performed using homology modeling.
  • homology modeling is performed in combination with a protein structure prediction algorithm, wherein optionally the protein structure prediction algorithm is trRosetta or AlphaFold or AlphaFold 2.
  • the lasso-binding site is selected from a ligand binding site, a substrate binding site, a catalytic binding site, a co- factor binding site, a precursor binding site, an orthosteric binding site, an allosteric binding site, an open conformation binding site, an active conformation binding site, an inactive conformation binding site, and a closed conformation binding site of the target molecule.
  • step (c) further comprises, for each 3D model structure of the lasso peptide: (c-1) positioning a docked portion of at least 5 ACTIVE 705331286v1 one conformational state of the 3D model structure of the lasso peptide relative to the lasso- binding site of the 3D model structure of the target molecule in a first docked pose, thereby forming a docked system; (c-2) scoring the docked system based on structural complementarity between the docked portion and the lasso-binding site; (c-3) repositioning the docked portion relative to the lasso-binding site to a second docked pose, and repeating step (c-2); (c-4) repeating step (c-3) for one or more times; and (c-5) selecting the docked system having the highest score as the optimal docked system before proceeding to step (d).
  • the docked system comprises one or more complementary binding pairs; and wherein the complementary binding pair comprises a first binding moiety on the lasso peptide and a second binding moiety on the target molecule.
  • the first binding moiety is on a first amino acid residue of the lasso peptide
  • the second binding moiety is on a second amino acid residue of the target molecule; and wherein the distance between any atom of the first amino acid residue and any atom of the second amino acid residue is less than about 5 ⁇ ngströms, and optionally less that about 4 ⁇ ngströms, and optionally less than about 3 ⁇ ngströms.
  • step (c-2) comprises calculating a total binding free energy of the docked system using one or more molecular mechanics force field functions; and assigning a score to the docked system based on the total binding free energy, and wherein the score relates to the total binding free energy.
  • step (c-3) comprises identifying the second docked pose using an energy minimizing function before repositioning the docked portion into the second docked pose; wherein the docked system is predicted to have a lower binding free energy in the second docked pose than the first docked pose based on the energy minimizing function.
  • the energy minimizing function is selected from the steepest descent algorithm, conjugate gradients algorithm, L- BFGS (limited-memory Broyden-Fletcher-Goldfarb-Shanno) algorithm, and genetic algorithms.
  • the docked portion comprises at least one amino acid residue from the ring portion, the loop portion, and/or the tail portion of the lasso peptide.
  • step (a-1) comprises: (a-1-1) in the optimal docked system, identifying one or more lasso-binding moieties in the lasso- 6 ACTIVE 705331286v1 binding site of the target molecule; (a-1-2) selecting one or more amino acid residues comprising one or more target-binding moieties complementary to the one or more lasso- binding moieties; and (a-1-3) mapping an optimal set of positions in the amino acid sequence of the selected lasso peptide for grafting the one or more selected amino acid residues; wherein the grafting places the one or more target-binding moieties at suitable spatial locations and orientations for binding with the complementary lasso-binding moieties on the target molecule.
  • step (a-1-3) comprises: (a-1-3-1) computationally grafting the one or more selected amino acid residues into the amino acid sequence of the selected lasso peptide at a first set of positions; (a-1-3-2) calculating a total binding free energy of the optimal docked system using one or more molecular force field functions; (a-1- 3-3) modifying at least one position in the first set of positions thereby obtaining an adjusted set of positions, and computationally grafting the one or more selected amino acid residues into the amino acid sequence of the selected lasso peptide at the adjusted set of positions; (a- 1-3-4) repeating step (a-1-3-2); (a-1-3-5) repeating steps (a-1-3-3) and (a-1-3-4) sequentially for one or more times; and (a-1-3-6) selecting the adjusted set of positions associated with the lowest total binding free energy as the optimal map of positions.
  • step (a-1-3-3) comprises identifying the adjusted set of positions using an energy minimizing function before modifying the first set of positions, wherein the docked system having the one or more selected amino acid residues grafted into the amino acid sequence of the selected lasso peptide at the adjusted set of positions is predicted to have a lower binding free energy than at the first set of positions based on the energy minimizing function.
  • the energy minimizing function is selected from the steepest descent algorithm, conjugate gradients algorithm, L-BFGS (limited-memory Broyden- Fletcher-Goldfarb-Shanno) algorithm, and genetic algorithms.
  • the target-binding epitope corresponds to a fragment or fragments of a naturally-existing ligand of the target molecule, wherein the fragment or fragments are capable of binding with the lasso-binding site of the target molecule and forming a ligand-target interface.
  • step (a-1) comprises aligning the docked portion of the 3D model structure of the lasso peptide with a 3D model structure of the fragment or fragments in the ligand-target interface. In some embodiments, step (a-1) comprises adjusting the spatial position, conformation and/or 7 ACTIVE 705331286v1 orientation of at least one target-binding moiety in the docked portion to mimic a corresponding binding moiety of the fragment or fragments in the ligand-target interface.
  • the method further comprises: (f) mutating one or more amino acid residues of the lasso peptide binder candidate to produce a first set of lasso peptide binder variants; and (g) ranking the first set of lasso peptide binder variants based on a predicted binding affinity for binding with the target molecule.
  • step (f) further comprises adjusting conformation of the first set of lasso peptide binder variants to produce a second set of lasso peptide binder variants; and wherein step (g) comprises ranking the second set of lasso peptide binder variants based on the predicted binding affinity for binding with the target molecule.
  • mutating the amino acid residue of the lasso peptide binder candidate comprises replacing the side chain of the amino acid residue of the lasso peptide binder candidate with the side chain of a second amino acid that is different from the mutated amino acid residue.
  • the second amino acid is a naturally-occurring or a non-natural amino acid.
  • mutating the amino acid residue of the lasso peptide binder candidate comprises modifying one or more chemical moieties on the side chain of the mutated amino acid residue. In some embodiments, at least one modified chemical moiety is a target-binding moiety.
  • the mutated amino acid residue is in the: (i) ring portion of the lasso peptide; (ii) loop portion of the lasso peptide; (iii) tail portion of the lasso peptide; or (iv) any combination of (i) to (iii).
  • the method further comprises: (h) synthesizing the lasso peptide binder candidate, grafted lasso peptide binder candidate or one or more lasso peptide binder variants having the highest rankings.
  • step (h) synthesizing the lasso peptide binder candidate or the one or more lasso peptide binder variants is performed using a cell-free or cell-based synthesis method.
  • the method further comprises creating a database of optimized lasso peptide structures or intermediates thereof generated in any one of steps (a) to (h). 5.
  • FIG.1 is a schematic illustration of the of a lasso peptide with the characteristic lasso (lariat) topology containing a loop, ring and tail. Amino acids are shown as balls.
  • FIG.2A is a schematic illustration of an iterative workflow including the steps generally referred to as “design,” “build,” “test,” and “evolve” according to the present disclosure, which workflow enables the creation of lasso peptide as therapeutic outputs from digital inputs through the integration of rational in silico peptide design and evolution methods.
  • FIG.2B is a schematic illustration of the workflow for computationally identifying lasso peptides having optimal binding with a target polypeptide. 9 ACTIVE 705331286v1
  • FIG.3A illustrates an example of grafting of a 5-amino acid linear epitope from the chemokine CCL5 into the ring portion of a lasso peptide structure.
  • FIG.3B illustrates examples of epitope grafting into the loop, ring and tail portions of a lasso peptide, respectively.
  • FIG.4 illustrates an example showing grafting a conformational or 3-dimensional (3D) epitope containing discontinuous segments of amino acid residues from a natural ligand (e.g., hormones, growth factors, chemokines, cytokines) into a lasso peptide structure.
  • a natural ligand e.g., hormones, growth factors, chemokines, cytokines
  • FIG.5 is a schematic illustration of cell-free and cell-based methods for producing lasso peptides as part of an iterative workflow involving in silico modeling and experimental testing to validate and optimize biological activities and other properties of lasso peptides according to the present disclosure.
  • FIG.6 illustrates computationally modeled structures of a lasso peptide binding in a GPCR pocket from in silico modeling showing “tail down, loop up” (left) and “loop down, tail up” (right) binding configurations. This figure exemplifies an initial modeling result from a study aimed at determining the size and shape fit between 37 known lasso peptide structures and the receptor pocket.
  • FIG.7A shows a homology model of C-C chemokine receptor type 8 (CCR8) docked with its natural ligand C-C Motif Chemokine Ligand 1 (CCL1).
  • FIG.7B shows a homology model of C-C chemokine receptor type 4 (CCR4) docked with its natural ligand C-C Motif Chemokine Ligand 17 (CCL17).
  • FIG.8 shows computational models of all-Gly backbone structures for lasso peptides having SEQ ID NOS: 1 to 37, based on atomic coordinates obtained from the PDB database. SID stands for SEQ ID.
  • FIGS.9A and 9B illustrate two lasso peptides that are mapped onto C-C Motif Chemokine Ligand 17 (CCL17) in the binding pocket of C-C chemokine receptor 4 (CCR4) using in silico modeling.
  • FIG.9A shows a lasso peptide having SEQ ID NO:21 mapped onto the 30s loop of CCL17; and FIG.9B shows a lasso peptide having SEQ ID NO:2 mapped onto the N-terminus of CCL17.
  • FIG.10 illustrates plate-based calcium flux assay for testing functional inhibition of target proteins CCR8 and CCR4, showing selectivity for CCR8.
  • FIG.11A Illustrates the lasso peptide structure of SEQ ID NO: 76 showing the amino acids as balls and the critical W10 residue in the loop.
  • FIG.11B shows a computational model of the complex between SEQ ID NO: 75 and ETBR, depicting the position of W10 in the energetically favored loop-down configuration.
  • FIG.12 illustrates the general two-step lasso peptide evolution process based on evolving the lasso peptide precursor peptide and subsequent cyclization using the lasso peptidase and lasso cyclase enzymes. Black balls represent mutated amino acid residues.
  • FIG.13 illustrates a five-step method involving computational modeling, conformational analysis, and model optimization for predicting lasso analogs with high integrin binding affinity.
  • FIG.14A shows ribbon diagrams of the apo form of the ⁇ v ⁇ 6 extracellular headpiece in a bent closed conformation where the computational model was constructed using atomic coordinates from PBD structure file 5FFG.
  • FIG.14B shows ribbon diagrams of the apo form of the ⁇ v ⁇ 6 extracellular headpiece in an open extended conformation where the computational model was constructed using atomic coordinates from PBD structure file 5NET
  • FIG.15A illustrates the lasso structure of SEQ ID NO: 109.
  • FIG.15B shows a model of the lasso peptide with SEQ ID NO: 109 docked into the ligand binding site of integrin ⁇ v ⁇ 6 and depicting the key binding interactions with the loop-grafted epitope RGDL.
  • FIG.16 illustrates iterative workflow for creating novel lasso peptides with high target affinity and optimized properties. 6. DETAILED DESCRIPTION [0052] The features of this invention are set forth specifically in the appended claims. A better understanding of the features and benefits of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized. To facilitate a full understanding of the disclosure set forth herein, a number of terms are defined below.
  • the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.
  • the term “substantially” means that something takes place, as a function or activity, to provide the expected outcome or result to a large degree and to a great extent, but still not to the fullest extent. For example, if a lasso peptide is substantially purified, the lasso peptide is isolated and purification steps afford the lasso peptide at purity level above 90% and as high as 99.99%. 12 ACTIVE 705331286v1 [0058] The term “substantially all” refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.
  • oligonucleotide and “nucleic acid” refer to oligomers of deoxyribonucleotides (e.g., DNA) or ribonucleotides (e.g., RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • oligonucleotide analogs including PNA (peptidonucleic acid), and analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like).
  • PNA peptidonucleic acid
  • analogs of DNA used in antisense technology phosphorothioates, phosphoroamidates, and the like.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer, M.A., et al., Nucleic Acid Res., 1991, 19, 5081-1585; Ohtsuka, E. et al., J. Biol. Chem., 1985, 260, 2605-2608; and Rossolini, G.M., et al., Mol. Cell. Probes, 1994, 8, 91-98).
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • a cell that produces a lasso peptide of the present disclosure may include a bacterial and archaea host cell into which nucleic acids encoding the lasso peptide component have been introduced. Suitable host cells are disclosed below.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double- stranded polynucleotide sequences is referred to as the 5’ direction.
  • RNA transcripts The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.” 13 ACTIVE 705331286v1 [0061]
  • amino acid refers to naturally occurring and non-naturally occurring alpha-amino acids, as well as alpha-amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring alpha-amino acids.
  • Naturally encoded amino acids are the 22 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid. glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine).
  • Amino acid analogs or derivatives refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and a side chain R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides may be referred to by their commonly accepted single-letter codes.
  • the terms “non- natural amino acid” or “non-proteinogenic amino acid” or “unnatural amino acid” or “non- canonical” refer to alpha-amino acids that contain different side chains (different R groups) relative to those that appear in the twenty-two common or naturally occurring amino acids listed above.
  • these terms also can refer to amino acids that are described as having D-stereochemistry or R-stereochemistry, rather than L-stereochemistry or S- stereochemistry of natural amino acids, despite the fact that some amino acids do occur in the D-stereochemical form in nature (e.g., D-alanine and D-serine).
  • the term “sterics” refers to the spatial volume occupied by an atom or a group of atoms in a molecule and as measured in cubic Angstroms. Experimentally, the sterics of an atom or groups of atoms are measured in various ways, such as how an atom or group of atoms influences the equilibrium between axial and equatorial conformations of monosubstituted cyclohexanes.
  • the 20 common amino acids can be divided into five volume classes based on the sterics. Particularly, the “very large” class includes F, W, and Y having steric measurements in the range of about 189 to 228 cubic Angstroms.
  • the “large” class includes I, L, M, K, and R having steric measurements in the range of about 162 to 174 cubic Angstroms.
  • the “medium” class includes V, H, E, and Q having steric measurements in the range of about 138 to 154 cubic Angstroms
  • the “small” class includes C, P, T, D, and N having steric measurements in the range of about 108 to 117
  • the “very small” class includes A, G, and S having steric measurements in the range of about 60 to 90 cubic 14 ACTIVE 705331286v1 Angstroms.
  • polypeptide and protein are used interchangeably herein to refer to a polymer of greater than about fifty (50) amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a protein, and vice versa.
  • the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog.
  • the terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • lasso peptide and “lasso” are used interchangeably herein, and are used to refer to a class of peptide or polypeptide having the general lariat-like topology as exemplified in FIG.1.
  • the lariat-like topology can be generally divided into a ring portion, a loop portion, and a tail portion.
  • a region on one end of the peptide forms the ring around the tail on the other end of the peptide
  • the tail is threaded through the ring
  • a middle loop portion connects the ring and the tail, together forming the lariat-like topology.
  • a ring-forming amino acid can located at the N- or C-terminus of the lasso peptide (“terminal ring-forming amino acid”), or in the middle (but not necessarily the center) of a lasso peptide (“internal ring-forming amino acid”).
  • G1-D9 cyclized as used herein when referring to a lasso peptide, means that the lasso peptide has a N-terminal ring-forming amino acid of a glycine residue (G1) and an internal ring-forming amino acid of an aspartate residue at position 9 (D9), where the amino group of G1 and the side chain carboxyl group of D9 form an isopeptide bond, thus forming the ring portion of the lasso peptide.
  • G1-D9 cyclized as used herein when referring to a lasso peptide, means that the lasso peptide has a N-terminal ring-forming amino acid of a glycine residue (G1) and an internal ring-forming amino acid of an aspartate residue at position 9 (D9), where the amino group of G1 and the side chain carboxyl group of D9 form an isopeptide bond, thus forming the ring portion of the lasso peptide.
  • the fragment of a lasso peptide between and including the two ring-forming amino acid residues is the ring portion; the fragment of a lasso peptide between the internal ring-forming amino acid and where the peptide threaded through the plane of the ring is the loop portion; and the 15 ACTIVE 705331286v1 remaining fragment of a lasso peptide starting from where the peptide is threaded through the plane of the ring is the tail portion.
  • lasso peptide may further include intra-peptide disulfide bonding, such as disulfide bond(s) between the tail and the ring, between the ring and the loop, and/or between different locations within the tail.
  • intra-peptide disulfide bonding such as disulfide bond(s) between the tail and the ring, between the ring and the loop, and/or between different locations within the tail.
  • lasso peptide or “lasso” refers to both naturally-existing peptides and artificially designed or produced peptides that have the lariat-like topology as described herein.
  • lasso peptide or “lasso” also refers to non-naturally occurring analogs, derivatives, or variants of a naturally occurring lasso peptide, which analogs, derivatives or variants are also lasso peptides themselves.
  • lasso precursor peptide or “precursor peptide” as used herein refers to a precursor that is processed into or otherwise forms a lasso peptide.
  • a lasso precursor peptide comprises at least one a lasso core peptide portion.
  • a lasso precursor peptide comprises one or more amino acid residues or amino acid fragments that do not belong to a lasso core peptide, such as a leader sequence that facilitates recognition of the lasso precursor peptide by one or more lasso processing enzymes.
  • the lasso precursor peptide is enzymatically processed into a lasso peptide by removing the amino acid residues or fragments that do not belong to a lasso core peptide.
  • a lasso precursor peptide is the substrate of an enzyme that cleaves off the additional amino acid residues or fragments from a lasso precursor peptide to produce the lasso peptide.
  • lasso peptidase As used herein, the enzyme capable of catalyzing this reaction is referred to as the “lasso peptidase.”
  • the term “lasso core peptide” or “core peptide” refers to the peptide or the peptide segment of the precursor peptide that is processed into or otherwise forms a lasso peptide having the lariat-like topology.
  • a core peptide may have the same amino acid sequence as a lasso peptide, but has not matured to have the lariat-like topology of a lasso peptide.
  • core peptides can have different lengths of amino acid sequences.
  • the core peptide is at least about 9 amino acid long.
  • the core peptide is at least about 10 amino acid long.
  • the core peptide is at least about 11 amino acid long.
  • the core peptide is at least about 25 amino acid long. In some embodiments, the core peptide is at least about 30 amino acid long. In some embodiments, the core peptide is at least about 35 amino acid long. In some embodiments, the core peptide is at least about 40 amino acid long. In some embodiments, the core peptide is at least about 45 amino acid long. In some embodiments, the core peptide is at least about 50 amino acid long. In some embodiments, the core peptide is at least about 55 amino acid long. In some embodiments, the core peptide is at least about 60 amino acid long. In some embodiments, the core peptide is at least about 65 amino acid long.
  • a computer modeling system defines a lasso peptide backbone by three parameters X, Y, Z, wherein X designates the number of glycine residues in the ring portion of the lasso peptide, Y designates the number of glycine residues in the loop portion of the lasso peptide, and Z designates the number of glycine residues in the tail portion of the lasso peptide.
  • a computer modeling system is able to provide a plurality of lasso peptide backbone structures each having a different combination of X, Y, and Z values, resulting in different three-dimensional shapes.
  • a lasso peptide backbone structure can be created by first computationally modeling the 3D structure of a lasso peptide according to the present disclosure, and then computationally removing all side changes of amino acid residues in the lasso peptide except for the internal ring forming residue.
  • a computer modeling system uses a lasso peptide backbone as the starting structure for building a lasso peptide, or as a tool for simulating lasso-target interaction as described in various embodiments herein.
  • lasso peptide analog or “lasso peptide variant” are used herein interchangeably and refer to a derivative of a natural lasso peptide that has been modified or changed relative to its original structure or atomic composition.
  • the lasso peptide analog can (i) have at least one amino acid substitution(s), insertion(s) or deletion(s) as compared to the sequence of a lasso peptide; (ii) have at least one different 17 ACTIVE 705331286v1 modification(s) to the amino acids as compared to a lasso peptide, such modifications include but are not limited to acylation, biotinylation, O-methylation, N-methylation, amidation, glycosylation, pegylation, esterification, halogenation, amination, hydroxylation, dehydrogenation, prenylation, lipidoylation, heterocyclization, phosphorylation; (iii) have at least one unnatural amino acid(s) as
  • production of a lasso peptide analog may occur by introducing a modification into the gene of a lasso precursor or core peptide, followed by transcription and translation and cyclization using cell- free or cell-based methods, as described herein, leading to a lasso peptide containing that modification.
  • production of a lasso peptide analog may occur by introducing a modification into a lasso precursor or core peptide, followed by cyclization of each using cell-free or cell-based methods, as described herein, leading to a lasso peptide containing that modification.
  • production of a lasso peptide analog may occur by introducing a modification into a pre-formed lasso peptide, leading to a lasso peptide containing that modification.
  • lasso peptide analogs are designed using structural information and in silico modeling algorithms, including docking and performing conformational analysis on 3D model structures using molecular dynamics simulation algorithms to obtain conformational states of 3D model structure of lasso peptides, and such “designed” lasso peptides may be produced by cell-free or cell-based methods.
  • lasso peptide analogs are designed de novo using computer algorithms, including artificial intelligence, machine learning, deep learning, neural nets, etc., and such “designed” lasso peptides may be produced by cell-free or cell-based methods.
  • the term “lasso peptide library” as used herein refers to a collection of at least two lasso peptides or lasso peptide analogs, or combinations thereof, which may be pooled together as a mixture or kept separated from one another. In some embodiments, the lasso peptide library is kept in vitro, such as in tubes or wells.
  • the lasso 18 ACTIVE 705331286v1 peptide library may be created by biosynthesis of at least two lasso peptides or lasso peptide variants using a cell-free system.
  • the lasso peptide library may be created by biosynthesis of at least two lasso peptides or lasso peptide variants using a cell- based system.
  • the lasso peptides or lasso peptide variants of the library may be mixed with one or more component of the cell-free or cell-based systems.
  • the lasso peptides or lasso peptide variants may be purified from the cell- free or cell-based systems.
  • the lasso peptides or lasso peptide variants may be partially purified. In some embodiments, the lasso peptides or lasso peptide variants may be substantially purified. In some embodiments, the lasso peptides may be isolated. In some embodiments, the lasso peptide library may be created by isolating at least two lasso peptides from their natural environment. In some embodiments, the lasso peptides may be partially isolated. In some embodiments, the lasso peptides may be substantially isolated.
  • isotopic variant of a lasso peptide refers to a lasso peptide analog that contains an unnatural proportion of an isotope at one or more of the atoms that constitute such a peptide.
  • an “isotopic variant” of a lasso peptide analog contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen ( 1 H), deuterium ( 2 H), tritium ( 3 H), carbon-11 ( 11 C), carbon-12 ( 12 C) carbon-13 ( 13 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O) fluorine-17 ( 17 F), fluorine-18 ( 18 F), phosphorus-31 ( 31 P), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S),
  • an “isotopic variant” of a lasso peptide is in a stable form, that is, non- radioactive.
  • an “isotopic variant” of a lasso peptide contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen ( 1 H), deuterium ( 2 H), carbon-12 ( 12 C), carbon-13 ( 13 C), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-16 ( 16 O) oxygen-17 ( 17 O), oxygen-18 ( 18 O) fluorine-17 ( 17 F), phosphorus-31 ( 31 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-37 ( 37 Cl), bromine-79 ( 79 Br), bromine-81 ( 81 Br), and iodine-127 ( 127 I).
  • an “isotopic variant” of a lasso peptide is in an unstable form, that is, radioactive.
  • an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, tritium ( 3 H), carbon-11 ( 11 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), fluorine-18 ( 18 F), phosphorus-32 ( 32 P), 19 ACTIVE 705331286v1 phosphorus-33 ( 33 P), sulfur-35 ( 35 S), chlorine-36 ( 36 Cl), iodine-123 ( 123 I) iodine-125 ( 125 I), iodine-129 ( 129 I) and iodine-131 ( 131 I).
  • any hydrogen can be 2 H, as example, or any carbon can be 13 C, as example, or any nitrogen can be 15 N, as example, and any oxygen can be 18 O, as example, where feasible according to the judgment of one of skill in the art.
  • an “isotopic variant” of a lasso peptide contains an unnatural proportion of deuterium.
  • structures of compounds (including peptides) depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • ⁇ For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
  • the term “evolution” or “molecular evolution” or “evolve” as used herein refers to varying the sequence of a parent protein or peptide by introducing one or more mutations in the oligonucleotide sequence encoding the amino acid sequence of the protein or peptide.
  • a parent lasso peptide is evolved by introducing one or more mutations within the oligonucleotide sequence encoding a parent lasso peptide. In another embodiment, a parent lasso peptide is evolved by introducing one or more mutations within the amino acid sequence of the parent lasso peptide. In another embodiment, a parent lasso peptide is evolved by introducing one or more mutations within the amino acid sequence of the parent lasso peptide, including the introduction of natural or non-natural amino acids.
  • biosynthetic gene cluster refers to one or more nucleic acid molecule(s) independently or jointly comprising one or more coding sequences for a precursor and processing machinery capable of maturing the precursor into a biosynthetic end product.
  • the coding sequences can comprise multiple open reading frames (ORFs) each independently coding for one component of the precursor and processing machinery.
  • the coding sequences can comprise an ORF coding for two or more components of the precursor and processing machinery fused together, as further described herein.
  • a biosynthetic gene cluster can be identified and isolated from the genome of an organism. Computer-based analytical tools can be used to mine genomic information and identify biosynthetic gene clusters encoding lasso peptides.
  • a biosynthetic gene cluster can be assembled by artificially producing and combining the nucleic acid components of the gene cluster, using genetic manipulating methods and technology known in the art.
  • the processing enzymes encoded by the lasso peptide gene cluster convert the lasso precursor peptide into a matured lasso peptide having the lariat-like topology. Particularly, the lasso peptidase removes from the precursor peptide the additional portion that is not the lasso core peptide, and the lasso cyclase cyclizes a terminal portion of the core peptide around a terminal tail portion to form the lariat-like topology.
  • Some lasso gene clusters further encode for additional protein elements that facilitates the post-translational modification, including a facilitator protein known as the post-translationally modified peptide (RiPP) recognition element (RRE, also referred to as Gene E or B1).
  • a lasso peptide biosynthetic gene clusters may encode two or more of lasso peptidase, lasso cyclase and RRE as different domains in the same protein. Some lasso gene clusters further encode for lasso peptide transporters, kinases, or proteins that play a role in immunity, such as isopeptidase. (Burkhart, B.J., et al., Nat. Chem. Biol., 2015, 11, 564–570; Knappe, T.A. et al., J. Am. Chem. Soc., 2008, 130, 11446-11454; Solbiati, J.O. et al. J.
  • lasso peptide biosynthesis component refers to a protein comprising one or more of (i) a lasso peptidase, (ii) a lasso cyclase, and (iii) RRE.
  • cell-free biosynthesis and “CFB” are used interchangeably herein and refer to an in vitro (outside the cell) biosynthetic process for the production of one or more peptides or proteins.
  • cell-free biosynthesis occurs in a “cell-free biosynthesis reaction mixture” or “CFB reaction mixture” which provides various components, such as RNA, proteins, enzymes, co-factors, natural products, small molecules, organic molecules, to carry out protein synthesis outside a living cell.
  • 21 ACTIVE 705331286v1 the CFB reaction mixture can comprise one or more cell extracts or supplemented cell extracts, or commercially available cell-free reaction media (e.g. PURExpress®).
  • in vitro transcription and translation and “in vitro TX- TL” are used interchangeably and refer to a biosynthetic process outside an intact cell, where genes or oligonucleotides are transcribed into messenger ribonucleic acids (mRNAs), and mRNAs are translated into proteins or peptides.
  • in vitro TX-TL machinery refers to the components that act in concert to carry out the in vitro TX-TL.
  • the in vitro TX-TL machinery is provided in the form of one or more cell extract, or one or more supplemented cell extract that comprises the in vitro TX-TL machinery.
  • condition suitable for lasso formation may refer to, for example, a condition suitable for the expression of one or more protein products in a bacterial host (e.g., a lasso precursor peptide, or a lasso processing enzyme). Exemplary suitable conditions included are not limited to a suitable culturing condition of the bacterial host that enable the protein synthesis and transportation in the host cell.
  • the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi.
  • the term also includes cell cultures of any species that can be cultured for the production of a biochemical.
  • naturally occurring biological materials such as nucleic acid molecules, oligonucleotides, amino acids, polypeptides, peptides, metabolites, small molecule natural products, host cells, and the like, refers to materials that are found in or isolated directly from Nature and are not changed or manipulated by humans.
  • naturally occurring refers to organisms, cells, genes, biosynthetic gene clusters, enzymes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature.
  • wild-type refers to organisms, cells, genes, biosynthetic gene clusters, enzymes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature (in the wild).
  • non-naturally occurring or “non-natural” or “unnatural” or “non- native” or “non-canonical” as used herein refer to a material, substance, molecule, cell, enzyme, protein, peptide, or amino acid that is not known to exist or is not found in Nature or that has been structurally modified and/or synthesized by humans.
  • non-natural or “unnatural” or “non-naturally occurring” when used in reference to a microbial organism or microorganism or cell extract or gene or biosynthetic gene cluster of the invention is intended to mean that the microbial organism or derived cell extract or gene or biosynthetic gene cluster has at least one genetic alteration not normally found in a naturally occurring strain or a naturally occurring gene or biosynthetic gene cluster of the referenced species, including wild-type strains of the referenced species.
  • Genetic alterations include, for example, introduction of expressible oligonucleotides or nucleic acids encoding polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the 23 ACTIVE 705331286v1 microbial organism’s genetic material.
  • Such modifications include, for example, nucleotide changes, additions, or deletions in the genomic coding regions and functional fragments thereof, used for heterologous, homologous or both heterologous and homologous expression of polypeptides.
  • Additional modifications include, for example, nucleotide changes, additions, or deletions in the genomic non-coding and/or regulatory regions in which the modifications alter expression of a gene or operon.
  • Exemplary polypeptides include enzymes, proteins, or peptides within a lasso peptide biosynthetic pathway.
  • the terms “non- naturally occurring” or “non-natural” or “unnatural” or “non-native” or “non-canonical” are used to refer to amino acids that are introduced into a polypeptide sequence to modify the properties of the polypeptide.
  • the term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a lasso precursor peptide, or lasso processing enzymes as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of DNA
  • immunoblotting for expression of gene products or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • PCR polymerase chain reaction
  • the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., a lasso precursor peptide as described herein), and it is further understood that 24 ACTIVE 705331286v1 expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into a microbial organism or into a cell extract for cell-free expression. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism or into a cell extract for cell-free activity.
  • the source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism or into a cell extract for cell-free expression of activity. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in a microbial host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism or into a cell extract.
  • heterologous refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism or organism used to produce a cell-free extract. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.
  • isolated when used in reference to a microbial organism or a biosynthetic gene, or a biosynthetic gene cluster, or a protein, or an enzyme, or a peptide, is intended to mean an organism, gene or biosynthetic gene cluster, protein, enzyme, or peptide that is substantially free of at least one component relative to the referenced microbial organism, gene, biosynthetic gene cluster, protein, enzyme, or peptide as it is found in nature or in its natural habitat.
  • the term includes a microbial organism, gene, biosynthetic gene 25 ACTIVE 705331286v1 cluster, protein, enzyme, or peptide that is removed from some or all components as it is found in its natural environment.
  • an isolated microbial organism, gene, biosynthetic gene cluster, protein, enzyme, or peptide is partly or completely separated from other substances as it is found in nature or as it is grown, stored, or subsisted in non-naturally occurring environments (e.g., laboratories).
  • isolated microbial organisms genes, biosynthetic gene clusters, proteins, enzymes, or peptides include partially pure microbes, genes, biosynthetic gene clusters, proteins, enzymes, or peptides, substantially pure microbes, genes biosynthetic gene clusters, proteins, enzymes, or peptides, and microbes cultured in a medium that is non-naturally occurring, or genes or biosynthetic gene clusters cloned in non-naturally occurring plasmids, or proteins, enzymes, or peptides purified from other components and substances present their natural environment, including other proteins, enzymes, or peptides.
  • an “isolated nucleic acid” is a nucleic acid, for example, an RNA, a DNA, or a mixed nucleic acid, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding a lasso peptide precursor peptide as described herein are isolated or purified.
  • the term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • substantially anaerobic when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media.
  • binding or “binding” or “binding interaction” or “bonding interaction” refer to an interaction or set of interactions between molecules including, for example, to form a complex. The sum total of the binding interactions between molecules determines the binding free energy associated forming a complex between two or more molecules.
  • ACTIVE 705331286v1 Interactions can be, for example, covalent binding interactions, or non-covalent interactions including hydrogen bonding interactions, ionic or electrostatic bonding interactions, polar interactions, dipolar interactions, induced dipolar interactions, pi stacking interactions, hydrophobic interactions, and/or van der Waals interactions.
  • a complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces.
  • the strength of the total non-covalent interactions between a single target-binding site of a binding molecule (e.g., a ligand), and a single target site of a target molecule (e.g., a protein) is the affinity of the binding molecule or functional fragment for that target site.
  • the ratio of dissociation rate (k off ) to association rate (k on ) of a binding molecule to a monovalent target site (k off /k on ) is the dissociation constant K D , which is inversely related to affinity. The lower the KD value, the higher the affinity of the binding molecule.
  • KD The value of KD varies for different complexes of lasso peptides or target proteins depends on both k on and k off .
  • the dissociation constant K D for a binding molecule e.g., a lasso peptide
  • the affinity at one binding site does not always reflect the true strength of the interaction between a binding molecule and the target molecule.
  • binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding molecule such as a lasso peptide) and its binding partner (e.g., a target protein). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., lasso peptide and target protein).
  • the affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.
  • the “K D ” or “K D value” may be measured by assays known in the art, for example by a binding assay.
  • the KD may be measured in a radioimmunoassay (RIA), for 27 ACTIVE 705331286v1 example, performed with the lasso peptide of interest and its target protein.
  • RIA radioimmunoassay
  • the KD or KD value may also be measured by using surface plasmon resonance assays by Biacore ® , using, for example, a Biacore ® TM-2000 or a Biacore ® TM-3000, or by biolayer interferometry using, for example, the Octet ® QK384 system.
  • An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a Biacore ® T-200 or a Biacore ® 8K+, or the Octet ® QK384 system.
  • target refers to a peptide or polypeptide, for which it is desirable to produce a lasso peptide that specifically binds thereto.
  • target protein refers to a peptide or polypeptide, for which it is desirable to produce a lasso peptide that specifically binds thereto.
  • lasso-binding site refers to the region on a target protein or peptide that contains at least one amino acid residue with which a lasso peptide interacts to form the binding with the target protein.
  • a lasso-binding site of a target protein can contain surface groupings of binding moieties (e.g., chemical active groups on amino acids or sugar side chains) responsible for binding with the lasso peptide.
  • the binding moieties can locate on the same amino acid residue or different amino acid residues in the lasso-binding site.
  • the lasso-binding site of a target protein can have specific three-dimensional structural characteristics (e.g., a groove or pocket) where the binding moieties disperse.
  • different lasso peptides may bind to different target sites or compete for binding with the same target site of a target protein.
  • a lasso peptide specifically binds to a target molecule or a target site thereof.
  • a lasso- binding site can be a ligand binding site, a steric hindrance binding site, an orthosteric binding site, an allosteric control binding site, a conformational change binding site, a channel, or a catalytic site.
  • orthosteric site or “orthosteric binding site” is a term of art that refers to the primary binding site on a receptor that is recognized by the endogenous ligand or agonist for that receptor.
  • the orthosteric site in the M1 receptor is the site that acetylcholine binds.
  • allosteric site or “allosteric binding site” refers to the specific binding site other than the orthosteric site on a receptor molecule that, upon binding of an allosteric effector molecule, influences the function or activity of that receptor.
  • epitope and “binding epitope” are used interchangeably herein to indicate generally the set of one or more amino acid residues on a binding molecule (e.g., a 28 ACTIVE 705331286v1 lasso peptide) that is responsible for the binding interaction between the binding molecule and a target molecule (e.g., a target protein).
  • an epitope can be linear or conformational, based on their structure and interaction with the binding target.
  • a conformational epitope is formed by the three-dimensional conformation adopted by the interaction of discontinuous segments of amino acid residue(s).
  • a linear epitope is formed by the interaction of contiguous amino acid residues.
  • a linear epitope is not determined solely by the primary structure of the epitope amino acids directly involved in binding interactions. Residues that flank such amino acid residues, as well as more distant amino acid residues of the antigen can affect the ability of the primary structure epitope residues to adopt the epitope’s three-dimensional conformation required for optimal binding.
  • an epitope of a peptide or polypeptide can be “grafted” into another peptide or polypeptide, for example by inserting the sequence (e.g., in case of a linear epitope) or sequences (e.g., in case of a conformational epitope) of an epitope into the sequence of the peptide or polypeptide, such that the grafted peptide or polypeptide retains the binding capability and specificity of the epitope.
  • an epitope of a peptide or polypeptide can be “grafted” into another peptide or polypeptide, for example by replacing a segment (e.g., in case of a linear epitope) or segments (e.g., in case of a conformation epitope) of the original sequence of the peptide or peptide with the sequence or sequences of the epitope, such that the grafted peptide or polypeptide retain the binding capability and specificity of the epitope.
  • arginine-glycine-aspartate is a linear epitope composed of three amino acids that together bind to certain integrins on the surface of cells.
  • the RGD epitope was grafted into the loop region of the lasso peptide microcin J25, and while the original lasso peptide microcin J25 did not bind to integrins, the RGD-grafted microcin J25 bound with high affinity to certain integrins (Hegemann, J.D., et al., J. Med. Chem.2014, 57, 5829 ⁇ 5834).
  • the term “target-binding epitope” as used herein refers to the amino acid residue or the group of amino acid residues of a lasso peptide that binds to a target molecule.
  • a target-binding epitope as disclosed herein can be a linear epitope that contains a fragment of continuous amino acids (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 continuous amino acids) of the lasso peptide (FIG.3A and FIG.3B), or a conformational epitope that consists of amino acid residues in two or more non-continuous regions of the lasso peptide (FIG.4).
  • a target-binding epitope can contain surface groupings of binding moieties (e.g., chemical active groups on amino acids or sugar side chains) responsible for binding with the target protein.
  • the binding moieties can locate on the same 29 ACTIVE 705331286v1 amino acid residue or different amino acid residues forming the target-binding epitope.
  • the target-binding epitope of a lasso peptide can have specific three- dimensional structural characteristics (e.g., an edge or a hump) where the binding moieties are arranged and displayed.
  • a lasso peptide can have more than one different target-binding epitopes that bind with different target sites of a target protein, or bind with different target proteins.
  • the term “structurally opposite to” refers to at least two amino acids residing on the target molecule and the lasso peptide, respectively, where the at least two amino acids are located and oriented in a 3-dimensional space in such a way that enables a binding interaction between the at least two amino acids.
  • a lysine residue on a target molecule may be located in space at the correct distance and orientation to engage in a binding interaction with an aspartate residue on the lasso peptide.
  • the two amino acid residues in this example, lysine and aspartate can referred to as being structurally opposite to each other.
  • a lasso peptide which binds a target molecule of interest is one that binds the target molecule with sufficient affinity such that the lasso peptide is useful, for example, as a diagnostic or therapeutic agent in targeting a cell or tissue expressing the target molecule, and does not significantly cross-react with other molecules.
  • the extent of binding of the lasso peptide to a “non-target” molecule will be less than about 10% of the binding of the lasso peptide to its particular target molecule, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.
  • FACS fluorescence activated cell sorting
  • the term “specific binding,” “specifically binds to,” or “is specific for” a particular polypeptide or a fragment on a particular polypeptide target means binding that is measurably different from a non-specific interaction.
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target.
  • binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • the term “specific binding,” “specifically binds to,” or “is specific for” a particular polypeptide or a fragment on a particular polypeptide target as used herein refers to binding where a molecule binds to a particular polypeptide or fragment on a particular polypeptide without substantially binding to any other polypeptide or polypeptide fragment.
  • a lasso peptide that binds to a target molecule has a dissociation constant (KD) 30 ACTIVE 705331286v1 of less than or equal to 100 ⁇ M, 80 ⁇ M, 50 ⁇ M, 25 ⁇ M, 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 n
  • a target protein is said to specifically bind to a lasso peptide, for example, when the dissociation constant (K D ) is ⁇ 10 -7 M.
  • the lasso peptides specifically bind to a target protein with a KD of from about 10 -7 M to about 10 -12 M.
  • the lasso peptides specifically bind to a target protein with high affinity when the K D is ⁇ 10 -8 M or K D is ⁇ 10 -9 M.
  • the lasso peptides may specifically bind to a purified human target protein with a KD of from 1 x 10 -9 M to 10 x 10 -9 M as measured by Biacore ® .
  • the lasso peptides may specifically bind to a purified human target protein with a K D of from 0.1 x 10 -9 M to 1 x 10 -9 M as measured by KinExATM (Sapidyne, Boise, ID).
  • the lasso peptides specifically bind to a target protein expressed on cells with a K D of from 0.1 x 10 -9 M to 10 x 10 -9 M.
  • the lasso peptides specifically bind to a human target protein expressed on cells with a K D of from 0.1 x 10 -9 M to 1 x 10 -9 M. In some embodiments, the lasso peptides specifically bind to a human target protein expressed on cells with a K D of 1 x 10 -9 M to 10 x 10 -9 M. In certain embodiments, the lasso peptides specifically bind to a human target protein expressed on cells with a K D of about 0.1 x 10 -9 M , about 0.5 x 10 -9 M, about 1 x 10 -9 M, about 5 x 10 -9 M, about 10 x 10 -9 M, or any range or interval thereof.
  • the lasso peptides specifically bind to a non-human target protein expressed on cells with a K D of 0.1 x 10 -9 M to 10 x 10 -9 M. In certain embodiments, the lasso peptides specifically bind to a non-human target protein expressed on cells with a KD of from 0.1 x 10 -9 M to 1 x 10 -9 M. In some embodiments, the lasso peptides specifically bind to a non-human target protein expressed on cells with a KD of 1 x 10 -9 M to 10 x 10 -9 M.
  • the lasso peptides specifically bind to a non-human target protein expressed on cells with a KD of about 0.1 x 10 -9 M, about 0.5 x 10 -9 M, about 1 x 10 -9 M, about 5 x 10 -9 M, about 10 x 10 -9 M, or any range or interval thereof.
  • the term “preferential binding” or “preferentially binds to” a particular polypeptide or a fragment on a particular target molecule with respect to a reference molecule means binding of the target molecule is measurably higher than binding of the reference molecule, while the reference 31 ACTIVE 705331286v1 molecule may or may not also bind to the lasso peptide.
  • Preferential binding can be determined, for example, by determining the binding affinity.
  • a lasso peptide that preferentially binds to a target molecule over a reference molecule can bind to the target molecule with a KD less than the KD exhibited relative to the reference molecule.
  • the lasso peptide preferentially binds a target molecule with a KD less than half of the K D exhibited relative to the reference molecule.
  • the lasso peptide preferentially binds a target molecule with a K D at least 10 times less than the K D exhibited relative to the reference molecule.
  • the lasso peptide preferentially binds a target molecule with a K D with K D that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the K D exhibited relative to the reference molecule.
  • the ratio between the KD exhibited by the lasso peptide when binding to the reference molecule and the KD exhibited when binding to the target molecule is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10 3 fold, at least 10 4 fold, or at least 10 5 fold.
  • a lasso peptide that specifically or preferentially binds to a target protein can be identified, for example, by immunoassays (e.g., ELISA, fluorescent immunosorbent assay, chemiluminescence immune assay, radioimmunoassay (RIA), enzyme multiplied immunoassay, solid phase radioimmunoassay (SPRIA), a surface plasmon resonance (SPR) assay (e.g., Biacore ® ), a biolayer interferometry assay, a fluorescence polarization assay, a fluorescence resonance energy transfer (FRET) assay, Dot-blot assay, fluorescence activated cell sorting (FACS) assay, or other techniques known to those of skill in the art.
  • immunoassays e.g., ELISA, fluorescent immunosorbent assay, chemiluminescence immune assay, radioimmunoassay (RIA), enzyme multiplied immunoassay, solid phase radioimmuno
  • a lasso peptide binds specifically to a target protein when it binds to the target protein with higher affinity than to any cross-reactive target molecule as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs).
  • RIA radioimmunoassays
  • ELISAs enzyme linked immunosorbent assays
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background.
  • the term “compete” when used in the context of lasso peptides means competition as determined by an assay in which the lasso peptide (or binding fragment) thereof under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand of the target molecule) to a common target molecule.
  • a reference molecule e.g., a reference ligand of the target molecule
  • Numerous types of competitive binding assays can be used to determine if a test lasso peptide competes with a reference ligand for binding to a target molecule.
  • assays that can be employed include solid phase direct or indirect RIA, solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-53), solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J.
  • Solid phase direct labeled assay solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988)), solid phase direct label RIA using I-125 label (see, e.g., Morel et al., 1988, Mol. Immunol.25:7-15), and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol.32:77-82).
  • such an assay involves the use of a purified target molecule bound to a solid surface, or cells bearing either of an unlabeled test target-binding lasso peptide or a labeled reference target-binding protein (e.g., reference target-binding ligand).
  • Competitive inhibition may be measured by determining the amount of label bound to the solid surface in the presence of the test target-binding lasso peptide.
  • the test target-binding molecule is present in excess.
  • Target-binding lasso peptides identified by competition assay include lasso peptides binding to the same target site as the reference and lasso peptides binding to an adjacent target site sufficiently proximal to the target site bound by the reference for steric hindrance to occur. Additional details regarding methods for determining competitive binding are described herein. Usually, when a competing lasso peptide is present in excess, it will inhibit specific binding of a reference to a common target molecule by at least 30%, for example 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.
  • binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.
  • blocking lasso peptide or an “antagonist” lasso peptide is one which inhibits or reduces biological activity of the target molecule it binds.
  • blocking lasso peptide or antagonist lasso peptide may substantially or completely inhibit the biological activity of the target molecule.
  • inhibitor refers to partial (such as, 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (i.e., 100%) inhibition.
  • IC50 refers an amount, concentration, or dosage of a compound that results in 50% inhibition of a maximal response in an assay that measures such response.
  • EC 50 refers an amount, concentration, or dosage of a compound that results in for 50% of a maximal response in an assay that measures such response.
  • attenuate refers to partial (such as, 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (i.e., 100%) reduction in a property, activity, function, effect, or value.
  • the term “selective inhibition of,” “selectively inhibits,” “selective antagonism of,” “selectively antagonizes,” “selective attenuation of,” or “selectively attenuates” a target molecule or a signaling pathway mediated by a target molecule means inhibition of the target molecule activity is measurably stronger than inhibition of a reference molecule activity. Selective inhibition can be determined, for example, by determining the IC 50 value.
  • a lasso peptide that selectively inhibits or attenuates a target molecule can exhibit an IC50 value less than the IC50 exhibited relative to a reference molecule.
  • the lasso peptide selectively inhibits or attenuates a target molecule with an IC 50 less than half of the IC 50 exhibited relative to the reference molecule.
  • the lasso peptide selectively inhibits or attenuates a target molecule with an IC50 that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the IC 50 exhibited relative to the reference molecule.
  • the ratio between the IC50 exhibited by the lasso peptide with respect to the reference molecule and the IC50 exhibited with respect to the target molecule is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 10 3 fold, at least 10 4 fold, or at least 10 5 fold.
  • the phrase “substantially similar” or “substantially the same” denotes a sufficiently high degree of similarity between two numeric values (e.g., one associated with a lasso peptide of the present disclosure and the other associated with a reference ligand) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by the values (e.g., K D values).
  • the difference between the two values may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5%, as a function of the value for the reference ligand.
  • the phrase “substantially increased,” “substantially reduced,” or “substantially different,” as used herein, denotes a sufficiently high degree of difference between two numeric values (e.g., one associated with a lasso peptide of the present disclosure and the other associated with a reference ligand) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by the values. For example, the difference between said two values can be greater than about 10%, greater than about 20%, greater than about 30%, 34 ACTIVE 705331286v1 greater than about 40%, or greater than about 50%, as a function of the value for the reference ligand.
  • the term “assaying” is meant the creation of experimental conditions and the gathering of data regarding a particular result of the exposure to specific experimental conditions.
  • enzymes can be assayed based on their ability to act upon a detectable substrate.
  • a lasso peptide can be assayed based on its ability to bind to a particular target molecule or molecules.
  • modulating or “modulate” as used herein refers to an effect of altering a biological activity (i.e. increasing or decreasing the activity), especially a biological activity associated with a particular biomolecule such as a, enzyme or cell surface receptor.
  • an inhibitor of a particular biomolecule modulates the activity of that biomolecule, e.g., an enzyme, by decreasing the activity of the biomolecule, such as an enzyme.
  • activity is typically indicated in terms of an inhibitory concentration (IC 50 ) of the compound for an inhibitor with respect to, for example, an enzyme or a cell surface receptor.
  • IC 50 inhibitory concentration
  • the referenced components may be contacted in any particular order or combination and the particular order of recitation of components is not limiting.
  • “contacting A with B and C” encompasses embodiments where A is first contacted with B then C, as well as embodiments where C is contacted with A then B, as well as embodiments where a mixture of A and C is contacted with B, and the like.
  • such contacting does not necessarily require that the end result of the contacting process be a mixture including all of the referenced components, as long as at some point during the contacting process all of the referenced components are simultaneously present or simultaneously included in the same mixture or solution.
  • the term “partially” means that something takes place, as a function or activity, to provide the expected outcome or result in part and to a limited extent, not to the fullest extent. For example, if a lasso peptide is partially purified, the lasso peptide is isolated and purification steps afford the lasso peptide at purity level that is greater than about 20% and less than about 90%.
  • computational modeling may be used interchangeably and refer to methods of 35 ACTIVE 705331286v1 applying computational algorithms or programs to predict, simulate, analyze, and assess the structure and properties of a molecule, especially with respect to the interaction between one molecule and another, such as a lasso peptide interacting with a target protein.
  • the term “docking” refers to the use of computer algorithms to fit a model ligand compound structure, such as a model three-dimensional structure of a lasso peptide, into the space of a binding site of a model target protein, and adjusting the models according to chemical and physical principles to simulate and predict the energy of their interactions.
  • amino acid residue interactions between the lasso peptide and the target protein are analyzed and energy is minimized to predict the lowest energy or highest-affinity binding.
  • the three-dimensional structure of the target protein is known or can be computationally created so that a plurality of model three-dimensional structures of the lasso peptide can be docked onto a known or predicted lasso-binding site of the target protein.
  • interactions between amino acid residues of a plurality of lasso peptides and the target protein are analyzed and energy is minimized to predict and rank the lowest-energy or highest-affinity binding lasso peptide using scoring functions.
  • rigid docking methods are used to predict and rank the binding affinities of docked lasso peptides.
  • flexible docking methods are used to predict and rank the binding affinities of docked lasso peptides.
  • Structural complementarity forms the means to molecular recognition that allows the structure of one molecule to interact with another molecule similarly to matching pieces in a puzzle, to splines or tenons dovetailed into their corresponding grooves in a machine, or to a lock and its corresponding key.
  • the phrase “structural complementarity” as used herein is meant to encompass chemical compatibility as well as spatial compatibility.
  • peptides or proteins in general by virtue of their architectural and functional diversity, can interact with one another based on structural complementarity which combines (i) a suitable and matching placement or positioning of one or more chemically-corresponding associating groups which are selected as members of a binding pair that can form one or more bonds with their corresponding member of the binding pair, and (ii) the overall structural features of the compound which depend mostly on the core structure of the molecule to which the associating groups are attached.
  • atomic coordinate is a term of art and as used herein refers to a mathematical description of the location of an atom in 3-dimensional (3D) space using a 36 ACTIVE 705331286v1 coordinate system.
  • Exemplary types of atomic coordinates that can be used in connection with the present disclosure includes but are not limited to Cartesian coordinates (X, Y, Z), internal or “Z” matrix coordinates, redundant internal coordinates, symmetry-adapted coordinates, polar coordinates, Quaternion coordinates.
  • atomic coordinates are used to define a model of atomic structure of a molecule (e.g., a peptide or a protein) that fits experimental data.
  • atomic structures archived in databases such as the wwPDB are models that have been constructed to be as consistent as possible with available experimental data.
  • atomic coordinates of a protein or peptide molecule can be obtained based on diffraction of X-rays by the atoms of a protein or peptide crystal.
  • the diffraction data are typically used to calculate an electron density map, which is used to establish the positions of the individual atoms within the unit cell of the crystal.
  • atomic coordinates of a protein or peptide molecule can be derived from chemical shift, J coupling, and magnetic relaxation data based on the nuclear overhauser effect (NOE).
  • nuclear magnetic resonance (NMR) data regarding a given molecule provide information about distances between atoms and local conformations, which information allows an ensemble of atomic structure models to be constructed, each model with slightly different conformations and atomic coordinates, for the molecule in solution.
  • NMR data affords information about the conformational flexibility of a molecule or portions of a molecule. Averaged atomic coordinates can be obtained from the ensemble of atomic structure models.
  • molecular modeling is a term of art and refers to the use of a computer algorithm to generate a predicted structural model of a molecule, including a protein or peptide, or a set of interacting molecules, such as a lasso peptide and a target protein.
  • a structural model generated as such is herein referred to as a “model structure,” such as a three-dimensional (3D) model structure.
  • Molecular modeling can be performed with a collection of computer-based techniques for deriving, representing, simulating, predicting, and manipulating the structures and behaviors of molecules, as well as reactions and interactions between molecules, and those properties that are dependent on these three-dimensional atomic structures.
  • Molecular modeling implements molecular mechanics calculations based on equilibrium bond lengths, bond angles, partial charge values, force constants, and van der Waals parameters, which collectively are referred to as force fields. Deviations from these equilibrium force field functions, along with non-bonded van der Waals and electrostatic interactions, will increase or decrease the energy of a system. 37 ACTIVE 705331286v1 Energy minimization and optimization algorithms are used to find the lowest energy arrangements of interacting atoms or molecules that are defined by force fields.
  • homology modeling is a term of art and refers to a procedure that generates a previously unknown protein structure by “fitting” its sequence (target) into a known structure (template), given a certain level of sequence homology (at least 30%) between target and template. Homology modeling thus predicts the 3D structure of a query protein through the sequence and structure alignment with one or more template proteins of known 3D structure.
  • the process of homology modeling involves four steps: template identification, sequence alignment, model building, and model refinement (Meier and Soding, PLoS Comput Biol.2015; 11(10): e1004343. doi: 10.1371/journal.pcbi.1004343).
  • protein structure prediction refers to the use of algorithms for the prediction of tertiary protein structure based on primary sequence data, conformational modeling, and energy minimization. Numerous advanced algorithms have been developed for accurate protein structure prediction, including trRosetta (Yang, J., et al., Improved protein structure prediction using predicted interresidue orientations. Proc. Nat. Acad. Sci., USA.2020, 117(3), 1496-1503), AlphaFold (Senior, A.W., et al., Improved protein structure prediction using potentials from deep learning.
  • molecular replacement is a term of art and refers to a process implemented to overcome the phase problem associated with X-ray diffraction data regarding a molecule. In molecular replacement, a known structure model very similar in structure to the one crystallized, is computationally placed in the crystallographic unit cell.
  • diffraction data phases can be calculated and used to start the process of interpreting the electron density map in order to solve the unknown structure and define atomic coordinates (Evans, P., McCoy, A. An introduction to molecular replacement. Acta Cryst. Bio. Cryst., 2008, D64, 1-10.).
  • configuration when used in the context of a three-dimensional (3D) structure of a molecule, refers to the fixed 3D relationship of atoms or groups of atoms of a molecule that is defined by the bonds between the atoms or groups of atoms.
  • the term “orientation” when used in the context of spatial relationship between two or more molecules refers to the position of a molecule or a portion of the molecule in a three-dimensional (3D) space relative to another molecule or a portion of such other molecule.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. 6.3 Lasso Peptides [00125] Bacterially-derived lasso peptides are emerging as a class of natural molecular scaffolds for drug design (Hegemann, J.D. et al., Acc. Chem.
  • Lasso peptides are members of the larger class of natural ribosomally synthesized and post-translationally modified peptides (RiPPs).
  • Lasso peptides are derived from a precursor peptide, comprising a leader sequence and core peptide sequence, which is cyclized through formation of an isopeptide bond between the N-terminal amino group of the linear core peptide and the side chain carboxyl groups of glutamate or aspartate residues located at positions 7, 8, 9, or 10 of the linear core peptide.
  • the resulting macrolactam ring is formed around the C-terminal linear tail, which is threaded through the ring leading to the 39 ACTIVE 705331286v1 characteristic lasso (also referred to as lariat) topology of general structure 1 as shown in FIG.1, which is held in place through sterically bulky side chains below and sometimes above the plane of the ring, and sometimes containing disulfide bonds between the tail and the ring or alternatively only in the tail.
  • ACTIVE 705331286v1 characteristic lasso (also referred to as lariat) topology of general structure 1 as shown in FIG.1 which is held in place through sterically bulky side chains below and sometimes above the plane of the ring, and sometimes containing disulfide bonds between the tail and the ring or alternatively only in the tail.
  • Lasso peptide gene clusters typically consist of three main genes, one coding for the precursor peptide (referred to as Gene A), and two for the processing enzymes, a lasso peptidase (referred to as Gene B or B2) and a lasso cyclase (referred to as Gene C) that close the macrolactam ring around the tail to form the unique lariat structure.
  • the precursor peptide consists of a leader sequence that binds to and directs the enzymes that carry out the cyclization reaction, and a core peptide sequence which contains the amino acids that together form the nascent lasso peptide upon cyclization.
  • lasso peptide gene clusters contain additional genes, such as those that encode for a small facilitator protein called a RIPP recognition element (RRE, also referred to as Gene E or B1), those that encode for lasso peptide transporters, those that encode for kinases, or those that encode proteins that are believed to play a role in immunity, such as an isopeptidase (Burkhart, B.J., et al., Nat. Chem. Biol., 2015, 11, 564–570; Knappe, T.A. et al., J. Am. Chem. Soc., 2008, 130, 11446- 11454; Solbiati, J.O. et al. J.
  • RRE RIPP recognition element
  • the ultimate lasso peptide directly derives from a core peptide that typically comprises a linear sequence ranging from about 11 to 50 amino acids in length.
  • the macrolactam ring of a lasso peptide may contain 7, 8, 9, or 10 amino acids, while the loop and tail vary in length.
  • RODEO genomic sequence mining algorithm
  • Lasso peptides represent a new type of molecular diversity which could serve as a vast source of novel products for use in the pharmaceutical, diagnostic, agricultural, and consumer industries. While the discovery of new activities and functions can occur by exploring the naturally occurring lasso peptides, a large percentage (>95%) of natural lasso peptides remain as prophetic entities predicted on the basis of genome sequence analyses. Lasso peptide development is constrained by the lack of effective methods to rapidly convert virtual lasso peptide biosynthetic gene cluster sequences into actual molecules that can be characterized and screened for biological activity and new methods for advancing lasso peptides are required.
  • lasso peptides are new methods for the identification, design, production, and optimization of lasso peptides.
  • the constrained nature of the three-dimensional lariat topology of a lasso peptide scaffold is suited for rational in silico design of non-natural sequences and structures that possess desirable properties.
  • an integrated set of technologies, methods, and tools that are implemented iteratively to develop new lasso peptides.
  • DBTE design-build-test-evolve
  • CADD computer-aided drug design
  • CADD is usually used for three major purposes: (1) filter large compound libraries into smaller sets of predicted active compounds that can be tested experimentally; (2) guide the optimization of lead compounds, whether to increase its affinity 41 ACTIVE 705331286v1 or optimize drug metabolism and pharmacokinetics (DMPK) properties including absorption, distribution, metabolism, excretion, and the potential for toxicity (ADMET); (3) design new compounds, either by “constructing” starting molecules one functional group at a time or by piecing together fragments into new chemotypes.
  • DMPK drug metabolism and pharmacokinetics
  • ADMET potential for toxicity
  • CADD can be classified into two general categories: structure-based and ligand- based.
  • Structure-based CADD relies on the knowledge of the target protein structure to calculate interaction energies for all compounds tested, whereas ligand-based CADD exploits the knowledge of known active and inactive molecules through chemical similarity searches or construction of predictive, quantitative structure-activity relation (QSAR) models (Kalyaanamoorthy and Chen, Drug Discovery Today, 2011, 16(17-18), 831–839).
  • Structure- based CADD is generally preferred where high-resolution structural data of the target proteins are available, e.g., for soluble proteins that can be structurally analyzed by X-ray crystallography or NMR spectrometry.
  • Ligand-based CADD is generally preferred when no or little structural information is available, often for membrane protein targets.
  • the central goal of structure-based CADD is to design compounds that bind tightly to the target, i.e., with large reduction in free energy, improved DMPK/ADMET properties, and are target specific, i.e., have reduced off-target effects (Pinzi, S., Rastelli, G. Int. J. Mol. Sci.2019, 20, 4331; doi:10.3390/ijms20184331).
  • a successful application of these methods will result in a compound that has been validated in vitro and in vivo and its binding location has been confirmed, ideally through a cocrystal structure.
  • One of the common uses in CADD is the screening of virtual compound libraries, also known as virtual high-throughput screening (vHTS).
  • vHTS comes in many forms, including chemical similarity searches by fingerprints or topology, selecting compounds by predicted biologic activity through QSAR models or pharmacophore mapping, and virtual docking of compounds into target of interest, known as structure-based docking. These methods allow the ranking of “hits” from the virtual compound library for acquisition. The ranking can reflect a property of interest such as 42 ACTIVE 705331286v1 percent similarity to a query compound or predicted biologic activity, or in the case of docking, the lowest energy scoring poses for each ligand bound to the target of interest. Often initial hits are rescored and ranked using higher level computational techniques that are too time consuming to be applied to full-scale vHTS.
  • vHTS does not aim to identify a drug compound that is ready for clinical testing, but rather to find leads with chemotypes that have not previously been associated with a target. This is not unlike a traditional HTS where a compound is generally considered a hit if its activity (e.g., Kd, IC50, or EC50) is close to 10 micromolar.
  • a potential drug is first developed into a “lead” with higher affinity, some understanding of its structure-activity-relation, and initial tests for DMPK/ ADMET properties.
  • the computational optimization of a hit compound can involve a structure-based analysis of docking poses and energy profiles for hit analogs, ligand-based screening for compounds with similar chemical structure or improved predicted biologic activity, or prediction of favorable DMPK/ADMET properties.
  • the comparably low cost of CADD compared with chemical synthesis and biologic characterization of large libraries of compounds make these methods attractive to focus, reduce, and diversify the chemical space that is explored (Enyedy and Egan, J Comput Aided Mol Des., 2008; 22:161–168).
  • De novo drug design is another tool in CADD methods, but rather than screening libraries of previously synthesized compounds, it involves the design of novel compounds.
  • a structure generator is needed to sample the space of chemicals.
  • the construction algorithms are generally defined as either linking or growing techniques.
  • Linking algorithms involve docking of small fragments or functional groups such as rings, acetyl groups, esters, etc., to particular binding sites followed by linking fragments from adjacent sites.
  • Growing algorithms begin from a single fragment placed in the binding site to which fragments are added, removed, and changed to improve activity. Similar to vHTS, the role of de novo drug design is not to design the single compound with nanomolar activity and acceptable DMPK/ADMET properties but rather to design a lead compound that can be subsequently improved. [00135] Advances in the algorithms for in silico modeling have led to the creation of increasingly powerful tools for analyzing, designing, and optimizing potential new drugs.
  • GPCRs G-protein-coupled receptors
  • a ligand-based strategy in contrast, can either consider the three-dimensional or the topological structure of one or more known ligands.
  • target protein structures or homology models are preferred for defining binding sites and predicting ligand binding interactions.
  • Lasso peptides are scaffolds for amino acids and provide unique structural and geometric constraints in terms of arrangements of its amino acid residues in space for optimal interaction with a target protein.
  • lasso-binding sites are identified 44 ACTIVE 705331286v1 through a search for target protein amino acids that are arranged in a manner that would allow engagement with complementary amino acids displayed on a 3D lasso peptide scaffold.
  • Amino acids that are structurally complementary to a target protein set of amino acids in a predicted binding site are computationally arranged on a plurality of different lasso peptide scaffolds to predict scaffold-amino acid combinations that are scored to identify the lowest energy binding interactions (e.g., highest predicted binding high affinity).
  • Lasso peptides offer a uniquely constrained 3-dimensional structure that is amenable to designing new biologically active molecules through predictive in silico modeling.
  • lasso peptides having optimal binding are identified using computational algorithms that enable in silico docking of lasso peptide structures into a binding site of a target protein.
  • lasso peptides are designed using computational algorithms that enable in silico docking of lasso peptide structures into a binding site of a target protein.
  • lasso peptides are identified using computational algorithms that enable in silico docking of lasso peptide structures into a lasso-binding site of protein structures (FIG.2B).
  • lasso peptides are identified and binding interactions are refined using computational algorithms that enable in silico docking and multiple rounds of conformational dynamic modeling of lasso peptide-protein target structures that are scored and ranked on the basis of predicted binding affinity of a lasso peptide bound to a protein structure.
  • lasso peptides are identified using computational algorithms that enable in silico docking of lasso peptide structures into a lasso-binding site of protein structures and the lasso peptide-protein interactions are further refined and ranked using artificial intelligence algorithms.
  • lasso peptides are identified and binding interactions are refined using computational and artificial intelligence algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso-binding site of protein structures.
  • lasso peptides are identified, and binding interactions are refined, using computational algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso binding site of protein structures.
  • lasso peptides are identified using computational algorithms that enable in silico docking of lasso peptide structures into a lasso binding site of protein structures and the lasso peptide- 45 ACTIVE 705331286v1 protein interactions are further refined and ranked using artificial intelligence algorithms.
  • lasso peptides are identified and binding interactions are refined using computational and artificial intelligence algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso-binding site of protein structures.
  • lasso peptides having optimal binding with a target molecule are identified using a combination of in silico docking algorithms and conformational analysis on 3D model structures of lasso peptides using molecular dynamics simulation algorithms.
  • Such methods can include providing one or more three-dimensional (3D) model structures of lasso peptides, performing a first conformational analysis on these structures using molecular dynamics simulation algorithms, docking the conformational states of the 3D model structures on to a 3D model structure of a target molecule, performing a second conformational analysis on these structures using molecular dynamics simulation algorithms, and then selecting the 3D model structure of the lasso peptide-target molecule complex having a favored free energy.
  • Such a method can identify lasso peptide binder candidates having optimal binding with a target molecule. These methods can be performed on multiple 3D model structures to provide at least two conformational states of the 3D model structures.
  • the at least two conformational states of the one ore more 3D model structures of lasso peptides can be selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, 30, 35, 40, 45, or 50 or more different conformational states.
  • the method for identifying a lasso peptide having optimal binding with a target molecule includes: (a) providing one or more three-dimensional (3D) model structures of lasso peptides; (b) performing conformational analysis on the one or more 3D model structures of lasso peptides using molecular dynamic simulation algorithms to obtain at least two conformational states of the one or more 3D model structures of lasso peptides; (c) docking the at least two conformational states of the one or more 3D model structures of lasso peptides onto a 3D model structure of the target molecule at a lasso- binding site of the target molecule to form at least two 3D model structures of the lasso peptide-target molecule complex; (d) performing conformational analysis on the at least two 3D model structures of the lasso peptide-target molecule complex using molecular dynamic simulation algorithms to obtain at least two conformational states of the 3D model structure of the lasso peptide
  • lasso peptides having optimal binding with a target molecule are identified, or lasso peptides are identified and binding interactions with target proteins are refined, using in silico docking algorithms (Pagadala, N.S., et al., Biophys Rev, 2017, 9, 91–102) including, but not limited to, CABS-dock (Kurcinski, M., et al., Protein Science, 2020, 29, 211–222), Macromodel (Mohamadi, F., et al., J. Comput. Chem, 1990, 11, 440-467), FlexPepDock (London, N.
  • lasso peptides having optimal binding with a target molecule are identified, or lasso peptides are identified and binding interactions with target proteins are refined, using in silico docking algorithms together with conformational analysis on 3D model structures of lasso peptides using molecular dynamics simulation algorithms, including but not limited to the publicly available or commercial programs, GROMACS, AMBER, CHARMM, and Schroedinger’s MAESTRO.
  • lasso peptides are identified and binding interactions with target proteins are modeled and analyzed by using artificial intelligence, deep learning, or machine learning algorithms for in silico virtual screening and/or de novo design to define the overall lasso peptide structural topologies encompassing the loop, ring, and tail size, as well as amino acid residues that suitably fit inside and engage the lasso-binding site of a target protein in order to optimize lasso peptide binding affinity predictions that may be refined further through iterative in silico docking and molecular dynamics simulations approaches.
  • one or more different artificial intelligence, deep learning, or machine learning algorithms are used, including but not limited to, support vector machines (Warmuth, M.K., et al., J. Chem. Inf. Comput. Sci., 2003, 43, 667-673), random forest (Deshmukh A.L., et al., Mol. Biosyst., 2017, 13, 1630–1639), k-nearest neighbors (Luo, M., et al., Mol. Inf., 2016, 35, 36 – 41), as well as neural networks with or without autoencoders, such as convolutional neural network (Jimenez, J., et al., J. Chem. Inf.
  • lasso peptides are identified and binding interactions with target proteins are optimized using in silico docking algorithms together with conformational molecular dynamics algorithms and/or artificial intelligence, deep learning, or machine learning algorithms, and lasso peptides are simultaneously optimized for multiple properties, such as solubility, cell permeability, cellular activity, or stability using artificial intelligence algorithms that include, but are not limited to, fuzzy-logic design simulations (Warszawski, S., et al., J. Mol.
  • the structures of all known natural lasso peptide sequences are predicted using computational algorithms, including but not limited to PEP-FOLD3 (Lamiable, A., et al., Nucl.
  • lasso peptides containing epitope grafted segments are identified using computational algorithms that enable in silico docking of lasso peptide structures into a binding site of a target protein structures.
  • lasso peptides containing epitope grafted segments are identified and binding interactions are optimized using computational algorithms that enable in silico docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in pocket of protein structures.
  • lasso peptides containing epitope grafted segments are identified using computational algorithms that enable in silico docking of lasso peptide structures into a binding site of a target protein structures and the lasso peptide-protein interactions are further refined and ranked using artificial intelligence algorithms.
  • lasso peptides containing epitope grafted segments are identified and binding interactions are optimized using computational and artificial intelligence algorithms that enable iterative in silico 48 ACTIVE 705331286v1 docking and conformational dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso binding site of protein structures.
  • a binding epitope is computationally grafted into a lasso peptide structure.
  • epitope-grafted lasso peptides are used as a basis for in silico docking and virtual screening against protein targets of interest.
  • a linear or continuous binding epitope is computationally grafted into a lasso peptide structure.
  • a conformational epitope is computationally grafted into a lasso peptide structure.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of binding epitopes are computationally grafted into the loop of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of binding epitopes are computationally grafted into the ring of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of binding epitopes are computationally grafted into the tail of a lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of binding epitopes are computationally grafted into the loop, ring, and/or tail of a lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of conformational epitopes are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop and ring of a lasso peptide structure to create a library of lasso epitope graft variants
  • a plurality of conformational epitopes are computationally grafted into the ring and tail of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop and tail of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop, ring, and tail of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from different polypeptide ligands are experimentally grafted into a lasso peptide sequence to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are experimentally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are computationally grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop and ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of conformational epitopes are computationally grafted into the ring and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of conformational epitopes are computationally grafted into the loop and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop, ring, and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • the method comprises the steps of (a) providing one or more three-dimensional (3D) model structures of lasso peptides; (b) docking the one or more 3D model structures of lasso peptides onto a 3D model structure of the target molecule at a lasso-binding site of the target molecule, thereby obtaining an optimal docked system; (c) selecting the 3D model structure of the lasso peptide forming the optimal docked system; (d) mapping a target-binding epitope onto the selected 3D model structure of the lasso peptide; and (e) computationally grafting the target-binding epitope onto the mapped location of the selected lasso peptide, thereby generating a lasso peptide binder candidate.
  • the method comprises the steps of (a) providing one or more three-dimensional (3D) model structures of lasso peptides; (a-1) mapping a target-binding epitope onto different locations of one or more 3D model structures of lasso peptides; (b) performing conformational analysis on the one or more 3D model structures of lasso peptides using molecular dynamic simulation algorithms to obtain at least two conformational states of the one or more 3D model structures of lasso peptides; (c) docking the at least two conformational states of the one or more 3D model 50 ACTIVE 705331286v1 structures of lasso peptides onto a 3D model structure of the target molecule at a lasso- binding site of the target molecule to form at least two 3D model structures of the lasso peptide-target molecule complex;
  • the method comprises the steps of (a) providing one or more three-dimensional (3D) model structures of lasso peptides; (a-1) mapping a target-binding epitope onto different locations of one or more 3D model structures of lasso peptides; (b) performing conformational analysis on the one or more 3D model structures of lasso peptides using molecular dynamic simulation algorithms to obtain at least two conformational states of the one or more 3D model structures of lasso peptides; (c) docking the at least two conformational states of the one or more 3D model structures of lasso peptides onto a 3D model structure of the target molecule at a lasso- binding site of the target molecule to form at least two 3D model structures of the lasso peptide-target molecule complex; (d) performing conformational analysis on the at
  • providing 3D model structures of a protein or peptide can be performed by retrieving known structural information of the protein or peptide from a protein structure database, such as the worldwide Protein Data Bank (wwPDB) (website: www.wwpdb.org), the Cambridge Structure Database (CSD) (website: www.ccdc.cam.ac.uk/solutions/csd-core/components/csd), Molecular Model Database (MMDB) of National Center for Biotechnology Information (NCBI) (website: www.ncbi.nlm.nih.gov/Structure/MMDB/docs/mmdb_help.html), and the Biological Magnetic Resonance Data Bank (BMRB) (website: bmrb.io).
  • wwPDB worldwide Protein Data Bank
  • CSD Cambridge Structure Database
  • MMDB Molecular Model Database
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • BMRB Biological Magnetic Resonance Data Bank
  • the protein structure database contains readily available modeled 3D structure of a peptide or 51 ACTIVE 705331286v1 protein.
  • the protein structure database contains atomic coordinates of a peptide or protein that can be used to create a 3D model structure of the protein or peptide using a computer modeling algorithm.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises retrieving one or more known 3D structures of lasso peptide from a protein structure database (FIG.2B).
  • the protein structure database is selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises computationally modeling the 3D structure of a lasso peptide based on atomic coordinates of the lasso peptide.
  • the atomic coordinates of the lasso peptide are obtained from a protein structure database or scientific literature.
  • the protein structure database is selected from a protein structure database.
  • the protein structure database is selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises computationally modeling the 3D structure of a lasso peptide based on atomic coordinates of the lasso peptide.
  • the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction, or 3-dimensional electron microscopy (3D-EM).
  • NMR nuclear magnetic resonance
  • X-ray crystallography neutron diffraction
  • 3D-EM 3-dimensional electron microscopy
  • the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to serial femtosecond crystallography.
  • the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to cryogenic electron microscopy (cryo- EM).
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises computationally modeling the 3D structure of a lasso peptide based on atomic coordinates of the lasso peptide.
  • the computational modeling is performed by molecular replacement modeling.
  • the step of (a) providing one or more 3D model structures of lasso peptides of 52 ACTIVE 705331286v1 the present method comprises computationally modeling the 3D structure of the lasso peptide based on X-ray diffraction data and/or nuclear magnetic resonance (NMR) data of the lasso peptide and atomic coordinates of a reference lasso peptide.
  • NMR nuclear magnetic resonance
  • the X-ray diffraction data of the lasso peptide are obtained by subjecting a crystal of the lasso peptide to X-ray crystallography analysis.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises (i) obtaining atomic coordinates of the lasso peptide based on the X-ray diffraction data; and (ii) refining the atomic coordinates of the lasso peptide based on the atomic coordinates of the reference lasso peptide.
  • the step of (ii) refining the atomic coordinates of the lasso peptide is performed using a molecular replacement method.
  • the NMR data of the lasso peptide comprises NMR chemical shift, J-coupling constant, and resonance intensity obtained by subjecting a solution of the lasso peptide to NMR analysis.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises (i) creating an ensemble of structural models of the lasso peptide based on the NMR data; (ii) obtaining mean atomic coordinates of the lasso peptide based on the ensemble of structural models; and (iii) refining the atomic coordinates of the lasso peptide based on the atomic coordinates of the reference lasso peptide.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises computationally modeling the 3D structure of a lasso peptide based on the amino acid sequence of the lasso peptide and atomic coordinates of a reference lasso peptide, and wherein the lasso peptide has at least 50 percent (%) sequence identity to the reference lasso peptide.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method comprises computationally modeling the 3D structure of a lasso peptide based on the amino acid sequence of the lasso peptide and atomic coordinates of a reference lasso peptide; and wherein the lasso peptide has at least 50 percent (%) amino acid sequence identity to the reference lasso peptide.
  • computationally modeling the 3D structure of the lasso peptide is performed by a homology modeling algorithm.
  • the lasso peptide has at least about 50 percent (%) amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 53 ACTIVE 705331286v1 55% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 60% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 65% amino acid sequence identity to the reference lasso peptide.
  • the lasso peptide has at least about 70% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 75% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 80% amino acid sequence identity to the reference lasso peptide. In specific embodiments involving a reference lasso peptide, the lasso peptide has at least about 85% amino acid sequence identity to the reference lasso peptide.
  • a 3D model structure of a lasso peptide can be a lasso peptide backbone structure.
  • a lasso peptide backbone structure can be constructed by computationally modeling the 3D structure of a lasso peptide and further computationally modeling the lasso backbone structure by removing side chains of each amino acid residue that is not an internal ring-forming residue from the 3D structure of lasso peptides.
  • the step of (a) providing one or more 3D model structures of lasso peptides of the present method further comprises computationally modeling the lasso backbone structures by removing side chains of each amino acid residue that is not an internal ring-forming residue from the modeled 3D structure of lasso peptides before proceeding to step (c) of the present method.
  • step (c) of the method comprises creating the 3D model structure of the target molecule before docking the one or more 3D model structure of lasso peptide onto the 3D model structure of the target molecule.
  • the target molecule has one or more lasso binding site on its surface, and step (c) of the method comprises docking the one or more 3D model structure of lasso peptide onto the lasso- binding site of the target molecule.
  • the target molecule is capable of binding to a ligand, and the lasso-binding site of the target molecule can be the ligand-binding site where the ligand binds to the target molecule.
  • the target molecule is an enzyme capable of binding and catalyzing a reaction of the substrate.
  • the enzyme contains an active site which is the region where substrate molecules bind and undergo a chemical reaction.
  • the active site consists of amino acid residues that form temporary bonds with the substrate (substrate binding site) and residues that catalyze a reaction of that substrate (catalytic site).
  • the lasso-binding site of a target molecule can be a substrate binding site of the target molecule.
  • the lasso-binding site of a target molecule can be a catalytic site of the target molecule.
  • the target molecule can be an enzyme or non-enzyme protein that uses one or more cofactor molecules to activate, inhibit, or otherwise perform its function.
  • the target molecule is capable of binding to the cofactor molecules, and the region in the target molecule where the cofactor binds is a cofactor binding site of the target molecule.
  • the lasso-peptide binding site of a target molecule can be the cofactor binding site of the target molecule.
  • the target molecule is a receptor that has an orthosteric site where a ligand molecule binds. Accordingly, in some embodiments, the lasso-peptide binding site of a target molecule can be an orthosteric site of the target molecule.
  • the target molecule is a receptor that has at least one allosteric site where an allosteric effector molecule binds. Accordingly, in some embodiments, the lasso-peptide binding site of a target molecule can be an allosteric site of the target molecule.
  • the target molecule is capable of switching between an active conformation (open conformation) and an inactive conformation (closed conformation) (FIG.14A and FIG.14B).
  • the lasso-peptide binding site of a target molecule can be a binding site that exists in the active or open conformation of the target peptide.
  • the lasso-peptide binding site of a target molecule can be a binding site that exists in the inactive or closed conformation of the target peptide.
  • the step (c) of the present method comprises creating the 3D model structure of the target molecule before docking.
  • creating the 3D model structure of the target molecule comprises 55 ACTIVE 705331286v1 computationally modeling the 3D structure of the target molecule based on atomic coordinates of the target molecule.
  • the atomic coordinates of the target molecule are known.
  • the atomic coordinates of the target molecule can be obtained from a protein structure database or scientific literature.
  • the atomic coordinates of the target molecule can be obtained from protein structure database selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.
  • the atomic coordinates of the target molecule are obtained by subjecting the target molecule to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction analysis, or 3-dimensional electron microscopy (3D- EM).
  • NMR nuclear magnetic resonance
  • X-ray crystallography X-ray crystallography
  • neutron diffraction analysis or 3-dimensional electron microscopy (3D- EM).
  • the atomic coordinates of the target molecule are obtained by subjecting the target molecule to serial femtosecond crystallography.
  • the atomic coordinates of the target molecule are obtained by subjecting the target molecule to cryogenic electron microscopy (cryo-EM).
  • step (c) of the present method comprises creating the 3D model structure of the target molecule before docking.
  • creating the 3D model structure of the target molecule comprises computationally modeling the 3D structure of the target molecule based on X-ray diffraction data and/or nuclear magnetic resonance data of the target molecule and atomic coordinates of a reference polypeptide.
  • the X-ray diffraction data of the target molecule are obtained by subjecting a crystal of the target molecule to X-ray crystallography analysis.
  • creating the 3D model structure of the target molecule comprises: (i) obtaining atomic coordinates of the target molecule based on the X-ray diffraction data; (ii) refining the atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iii) computationally modeling the 3D structure of the target molecule based on the refined atomic coordinates.
  • the refining step is performed using a molecular replacement algorithm.
  • the atomic coordinates of the reference polypeptide are known.
  • the atomic coordinates of the reference polypeptide are obtained from a protein structure database selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.
  • the atomic coordinates of the reference polypeptide are obtained from scientific literature.
  • the nuclear magnetic resonance (NMR) data of the target molecule comprises NMR chemical shift, J-coupling constant, and resonance intensity obtained from subjecting a solution of the target molecule to NMR analysis.
  • creating the 3D model structure of the target molecule comprises (i) creating an ensemble of structural models of the target molecule based on the NMR data; (ii) obtaining mean atomic coordinates of the target molecule based on the ensemble of structural models; (iii) refining the mean atomic coordinates of the target molecule based on the atomic coordinates of the reference polypeptide; and (iv) computationally modeling the 3D structure of the target molecule based on the refined mean atomic coordinates.
  • the refining step is performed using a molecular replacement algorithm.
  • the atomic coordinates of the reference polypeptide are known.
  • the atomic coordinates of the reference polypeptide are obtained from a protein structure database selected from the Protein Data Bank (wwPDB), the Cambridge Structure Database, the Molecular Model Database of National Center for Biotechnology Information (NCBI), and the Biological Magnetic Resonance Data Bank (BMRB) database.
  • the atomic coordinates of the reference polypeptide are obtained from scientific literature.
  • step (c) of the present method comprises creating the 3D model structure of the target molecule before docking.
  • creating the 3D model structure of the target molecule comprises computationally modeling the 3D structure of the target molecule based on the amino acid sequence of the target molecule and atomic coordinates of one or more reference polypeptide.
  • computationally modeling the 3D structure of the target molecule is performed by homology modeling. In some embodiments, computationally modeling the 3D structure of the target molecule is performed by protein structure prediction. In some embodiments, computationally modeling the 3D structure of the target molecule is performed by a combination of protein structure prediction and homology modeling. In some embodiments, the atomic coordinates of the reference polypeptide are known. In some embodiments, the atomic coordinates of the reference polypeptide are obtained from a protein structure database or scientific literature.
  • the protein structural database is selected from worldwide Protein Data Bank (wwPDB), Cambridge Structure Database, 57 ACTIVE 705331286v1 Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database.
  • wwPDB worldwide Protein Data Bank
  • NCBI National Center for Biotechnology Information
  • BMRB Biological Magnetic Resonance Data Bank
  • the target molecule has at least about 65% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 70% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 75% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 80% amino acid sequence identity to the reference peptide. In specific embodiments involving a reference polypeptide, the target molecule has at least about 85% amino acid sequence identity to the reference peptide.
  • step (c) of the present method comprises docking the one or more 3D model structures of lasso peptides onto a 3D model structure of the target molecule at a lasso-binding site of the target molecule, thereby obtaining an optimal docked system.
  • the docking comprises, for each 3D model structure of the lasso peptide: (c-1) positioning a docked portion of at least one conformational state of the 3D model structure of the lasso peptide relative to the lasso-binding site of the 3D model structure of the target molecule in a first docked pose, thereby forming a docked system; (c-2) scoring the docked system based on structural complementarity between the docked portion and the lasso-binding site; (c-3) repositioning the docked portion relative to the lasso-binding site to a second docked pose, and repeating step (c-2); (c-4) repeating step (c-3) for one or more times; and (c-5) selecting the docked system having the highest score as the optimal docked system before proceeding to step (d).
  • the docked portion of the 3D model structure comprises at least one amino acid residue from the loop portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure comprises at least one amino acid residue from the ring portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure comprises at least one amino acid residue from the tail portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure comprises at least one amino acid residue from the loop portion of the lasso peptide and at least one amino acid residue from the ring portion of the lasso peptide.
  • the docked portion of the 3D model structure comprises at least one amino acid residue from the loop portion of the lasso peptide and at least one amino acid residue from the tail portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure comprises at least one amino acid residue from the ring portion of the lasso peptide and at least one amino acid residue from the tail portion of the lasso peptide. In some embodiments, the docked portion of the 3D model structure comprises at least one amino acid residue from the loop portion of the lasso peptide, at least one amino acid residue from the ring portion of the lasso peptide, and at least one amino acid residue from the tail portion of the lasso peptide.
  • the docked system comprises one or more complementary binding pairs comprising a first binding moiety located on the lasso peptide, and a second binding moiety located on the target molecule.
  • the first and second binding moieties form binding interaction with one another.
  • the binding interaction between the first and second binding moieties is non- covalent interaction.
  • the binding interaction between the first and second binding moieties is selected from hydrogen bonding interactions, ionic or electrostatic bonding interactions, pi stacking interactions, polar interactions, dipolar interactions, induced dipolar interactions, hydrophobic interactions, and van der Waals interactions.
  • the first binding moiety is located on a first amino acid residue of the lasso peptide
  • the second binding moiety is on a second amino acid residue located on the target molecule.
  • the distance between any atom of the first amino acid residue and any atom of the second amino acid residue is less than about 5 ⁇ ngströms.
  • the distance between any atom of the first amino acid residue and any atom of the second amino acid residue is less than about 4 ⁇ ngströms.
  • the distance between any atom of the first 59 ACTIVE 705331286v1 amino acid residue and any atom of the second amino acid residue is less than about 3 ⁇ ngströms.
  • step (c-2) scoring the docked system based on structural complementarity between the docked portion and the lasso-binding site comprises calculating a total binding free energy of the docked system using one or more molecular mechanics force field functions, and assigning a score to the docked system based on the total binding free energy.
  • the assigned score to a docked system negatively relates to the total binding free energy of the docked system.
  • a relatively higher score is assigned to a docked system having a relatively lower total binding free energy.
  • a relatively lower score is assigned to a docked system having a relatively higher total binding free energy.
  • Various computational modeling platforms that are known in the art can be used for calculating the free energy differences between different docked molecules and/or poses and total binding free energy of a docked system using molecular mechanics force field functions and scoring the docked system.
  • Exemplary computational modeling platforms include but are not limited to the MOE 2020.09 computational modeling platform (Molecular Operating Environment, Chemical Computing Group Inc. Canada).
  • a variety of force fields and force field parameters can be used in connection with the present disclosure, including but not limited to Amber force fields AMBER99, Amber 10EHT, ff14SB, and ff19SB, with amino acids-specific side chain and protein backbone parameters (See: Ponder, J.W., Case, D.A. “Force fields for protein simulations.” Adv. Prot.
  • scoring the docked system based on structural complementarity between the docked portion and the lasso-binding site comprises calculating a total binding free energy of the docked system using one or more molecular mechanics force field functions selected from Amber force fields AMBER99, Amber 10EHT, ff14SB, ff19SB, and the Merck force field MMFF94x, and assigning a score to the docked system based on the lower total binding free energy.
  • the score assigned to a docked system negatively relates to the total binding free energy.
  • the favored free energy includes a lower free energy compared to a different 3D model structure of the lasso peptide-target molecule complex. In some embodiments, the favored free energy is the lowest free energy of the two or more 3D model structures of the lasso peptide-target molecule complex. In some embodiments, a relatively higher score is assigned to a docked system having a relatively lower total binding free energy. In some embodiments, a relatively lower score is assigned to a docked system having a relatively higher total binding free energy.
  • step (c-3) repositioning the docked portion relative to the lasso-binding site to a second docked pose comprises identifying the second docked pose using an energy minimizing function before repositioning the docked portion, wherein the docked system is predicted to have a lower binding free energy in the second docked pose than in the first docked pose based on the energy minimizing function.
  • energy minimizing function known in the art can be used in connection with the present disclosure, including but not limited to the steepest decent algorithm (Jaidhan, B.J., et al.
  • step (a-1) mapping a target-binding epitope onto the selected 3D model structure of the lasso peptide comprises (a-1-1) in the optimal docked system, identifying one or more lasso-binding moieties in the lasso-binding site of the target molecule; (a-1-2) selecting one or more amino acid residues comprising one or more target- binding moieties complementary to the one or more lasso-binding moieties; and (a-1-3) mapping an optimal set of positions in the amino acid sequence of the selected lasso peptide 61 ACTIVE 705331286v1 for grafting the one or more selected amino acid residues; wherein the grafting places the one or more target-binding moieties at suitable
  • the binding interaction between the complementary lasso-binding moiety and the target-binding moiety is selected from covalent bonding interactions or non-covalent interactions including hydrogen bonding interactions, ionic or electrostatic bonding interactions, polar interactions, dipolar interactions, induced dipolar interactions, pi stacking interactions, H-bond-aromatic interactions, hydrophobic interactions, and van der Waals interactions.
  • step (a-1-3) mapping an optimal set of positions in the amino acid sequence of the selected lasso peptide for grafting the one or more selected amino acid residues comprises: (a-1-3-1) computationally grafting the one or more selected amino acid residues into the amino acid sequence of the selected lasso peptide at a first set of positions; (a-1-3-2) calculating a total binding free energy of the optimal docked system using one or more molecular force field functions; (a-1-3-3) modifying at least one position in the first set of positions thereby obtaining an adjusted set of positions, and computationally grafting the one or more selected amino acid residues into the amino acid sequence of the selected lasso peptide at the adjusted set of positions; (a-1-3-4) repeating step (a-1-3-2); (a- 1-3-5) repeating steps (a-1-3-3) and (a-1-3-4) sequentially for one or more times; and (a-1-3- 6) selecting the adjusted set of positions associated with the lowest total binding free energy
  • the molecular mechanics force field functions are selected from Amber force fields AMBER99, Amber 10EHT, ff14SB, ff19SB, and Merck force field MMFF94x.
  • step (a-1-3-5) wherein repeating steps (a-1-3-3) and (a- 1-3-4) sequentially for one or more times comprising repeating steps (a-1-3-3) and (a-1-3-4) for at least 1 time, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least fifteen times, at least twenty times, or at least twenty-five times.
  • step (a-1-3-3) comprises identifying the adjusted set of positions using an energy minimizing function before modifying the first set of positions to become the adjusted set of positions, wherein the docked system having the one or more selected amino acid residues grafted into the amino acid sequence of the selected lasso peptide at the adjusted set of positions is predicted to have a lower binding free energy than at the first set of positions based on the energy minimizing function.
  • the 62 ACTIVE 705331286v1 energy minimizing function is selected from the steepest descent algorithm, conjugate gradients algorithm, L-BFGS (limited-memory Broyden-Fletcher-Goldfarb-Shanno) algorithm, and genetic algorithms.
  • the target molecule has a naturally-existing ligand to which the target molecule binds.
  • the target-binding epitope to be mapped onto the 3D model structure of the lasso peptide corresponds to a continuous fragment of the naturally-existing ligand that binds to the target molecule.
  • the target-binding epitope is a linear epitope (FIG.3A).
  • the target-binding epitope has 100% amino acid sequence identity to the corresponding continuous fragment of the naturally–existing ligand of the target molecule.
  • the amino acid sequence of the target-binding epitope differ from the corresponding continuous fragment of the naturally-existing ligand of the target molecule by 1, 2, 3, 4, 5, or more than 5 amino acid residue.
  • the target-binding epitope to be mapped onto the 3D model structure of the lasso peptide comprises multiple discontinuous fragments that respectively correspond to multiple discontinuous fragments of the naturally-existing ligand to which the target molecule binds (FIG.4).
  • the target-binding epitope is a conformational epitope.
  • the multiple discontinuous fragments of the target-binding epitope has 100% amino acid sequence identity to the corresponding multiple discontinuous fragments of the naturally-existing ligand. In some embodiments, the multiple discontinuous fragments of the target-binding epitope differ from the corresponding multiple discontinuous fragments of the naturally-existing ligand by 1, 2, 3, 4, 5, or more than 5 amino acid residue. [00195] In some embodiments, the target molecule has a naturally-existing ligand, wherein the target-binding epitope corresponds to a fragment or fragments of a naturally-existing ligand of the target molecule that are capable of binding with the lasso-binding site of the target molecule and forming a ligand-target interface.
  • step (d) of the present method further comprises aligning the docked portion of the 3D model structure of the lasso peptide with a 3D model structure of the corresponding fragment or fragments of the naturally-existing ligand in the ligand-target interface.
  • the aligning comprises adjusting the spatial position, conformation and/or orientation of at least one target-binding moiety in the docked portion of the lasso peptide to mimic the spatial position, conformation and/or orientation of the corresponding binding moiety of the fragment or fragments of the naturally-existing ligand in the ligand-target interface.
  • the present method further comprises (f) mutating one or more amino acid residues of the lasso peptide binder candidate to produce a first set of lasso peptide binder variants; and (g) ranking the first set of lasso peptide binder variants based on a predicted binding affinity for binding with the target molecule.
  • step (f) further comprises adjusting conformations of the first set of lasso peptide binder variants to produce a second set of lasso peptide binder variants.
  • step (g) further comprises ranking the second set of lasso peptide binder variants based on the predicted binding affinity for binding with the target molecule.
  • mutating the amino acid residue of the lasso peptide binder candidate comprises replacing the side chain of the amino acid residue of the lasso peptide binder candidate with the side chain of a second amino acid that is different from the mutated amino acid residue.
  • the second amino acid is a naturally-occurring or a non-natural amino acid.
  • mutating the amino acid residue of the lasso peptide binder candidate comprises modifying one or more chemical moieties on the side chain of the mutated amino acid residue. In some embodiments, at least one modified chemical moiety is a target-binding moiety.
  • mutating the amino acid residue of the lasso peptide binder candidate comprises modifying one or more side chains of the lasso peptide binder candidate to complement one or more lasso-binding moieties on the target molecule; and wherein the modifying is selected from the group consisting of: (i) incorporating into the lasso peptide a neutral or basic side chain that is structurally opposite to an acidic lasso- binding moiety; (ii) incorporating into the lasso peptide a neutral or acid side chain that is structurally opposite to a basic lasso-binding moiety; (iii) incorporating into the lasso peptide a neutral or positively charged side chain that is structurally opposite to a negatively charged lasso-binding moiety; (iv) incorporating into the lasso peptide a neutral or negatively charged side chain that is structurally opposite to a positively charged lasso-binding moiety; (v) incorporating into the lasso peptide a neutral or negatively charged side chain that is
  • the mutated amino acid residue is in the (i) ring portion of the lasso peptide; (ii) loop portion of the lasso peptide; (iii) tail portion of the lasso peptide; or (iv) any combination of (i) to (iii).
  • the method further comprises step (h) synthesizing the lasso peptide binder candidate, grafted lasso peptide binder candidate or one or more lasso peptide binder variants having the highest rankings.
  • step (h) synthesizing the lasso peptide binder candidate or the one or more lasso peptide binder variants is performed using a cell-free or cell-based synthesis method.
  • the method further comprises creating a database of optimized lasso peptide structures or intermediates thereof generated in any one of steps (a) to (h).
  • de novo design of lasso peptides represents an alternative computational strategy for discovering new lasso peptides that can bind with high affinity to target proteins.
  • De novo drug design is based on stochastic structure optimization, evolutionary fitness functions, and combinatorial design principles that potentially represent a vast chemical space.
  • constraints are placed on the de novo design space.
  • receptor-based constrains or ligand-based constraints are implemented (Schneider, G., Fechner, U., Computer-based de novo design of drug-like molecules, Nat Rev Drug Discov, 2005, 4, 649- 663). All information that is related to the ligand–receptor interaction forms the primary target constraints for candidate compounds.
  • Such constraints can be gathered both from the three-dimensional receptor structure and from the structures of known ligands of the particular target.
  • Receptor-based design strategies starts with the determination of known 65 ACTIVE 705331286v1 binding sites for known ligands and potential binding sites for new ligands.
  • the binding site is then examined to derive shape constraints for a ligand, as well as specific non-covalent ligand–receptor interactions in the form of hypothetical interaction sites created through hydrogen bonding, van der Waals, electrostatic, and hydrophobic interactions.
  • Receptor groups capable of hydrogen-bonding are of special interest owing to the strongly directional nature of the two interaction partners (i.e., hydrogen-bond acceptor and donor) and often form key interaction sites, especially in protein-protein or protein-peptide complexes. These constraints allow the assignment of ligand atom positions with a complementary hydrogen-bond type within a small region of space and a defined orientation.
  • the target binding epitope is designed de novo by creating a set of amino acids that complements those in a potential lasso peptide binding site of a target protein.
  • a linear or three-dimensional (3D) arrangement of a set of amino acids is identified as a potential lasso binding site in a target protein.
  • the linear or 3D arrangement consists of 2-10 amino acids.
  • a complementary set of amino acids are identified for interacting with the amino acids in the target binding site.
  • the complementary set of amino acids have properties that facilitate attractive or non-repulsive interactions with the amino acids in the target binding site.
  • the complementary set of amino acids is grafted onto a lasso peptide scaffold.
  • methods to identify and computationally graft a target protein-binding epitope of a natural or synthetic polypeptide ligand into a lasso peptide 3-dimensional (3D) topological structure are provided herein.
  • methods to 66 ACTIVE 705331286v1 identify and experimentally graft a target protein-binding epitope of a natural or synthetic polypeptide ligand into a lasso peptide 3-dimensional (3D) topological structure.
  • lasso peptides containing epitope grafted segments are identified and modeled using computational algorithms that enable in silico docking of lasso peptide structures containing such epitope grafts into a lasso binding site of protein structures.
  • lasso peptides containing epitope grafted segments are modeled and binding interactions are optimized using computational algorithms that enable in silico docking and conformational molecular dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso binding site of protein structures.
  • lasso peptides containing epitope grafted segments are identified using computational algorithms that enable in silico docking of lasso peptide structures into a lasso binding site of protein structures and the lasso peptide-protein interactions are further refined and ranked using artificial intelligence algorithms.
  • lasso peptides containing epitope grafted segments are identified and binding interactions are optimized using computational and artificial intelligence algorithms that enable in silico docking and conformational molecular dynamic modeling of lasso peptide structures that are scored and ranked on the basis of predicted binding affinity in a lasso binding site of protein structures.
  • a binding epitope which is identified by analyzing the binding interactions between a natural or synthetic polypeptide ligand and a target protein, is computationally grafted into a lasso peptide structure.
  • a binding epitope is grafted into the loop of a lasso peptide.
  • a binding epitope is grafted into the ring of a lasso peptide.
  • a binding epitope is grafted into the tail of a lasso peptide.
  • a binding epitope is grafted into both the loop and ring of a lasso peptide.
  • a binding epitope is grafted into both the loop and tail of a lasso peptide. In some embodiments, a binding epitope is grafted into both the ring and tail of a lasso peptide. In some embodiments, a binding epitope is grafted into the loop, ring, and tail of a lasso peptide. [00206] In some embodiments, a linear or continuous binding epitope is identified whereby contiguous sections (two or more amino acid residues) of a natural or synthetic polypeptide ligand bind to a target protein, and the linear epitope is grafted into a lasso peptide structure (FIG.3A and FIG.3B).
  • a conformational binding epitope is identified whereby two or more non-contiguous sections (one or more amino acid residues) of a natural or synthetic polypeptide ligand that binds to a target protein is grafted into a lasso peptide structure (FIG.4).
  • the grafting process involves a 67 ACTIVE 705331286v1 replacement of specific amino acids in an existing natural or synthetic lasso peptide sequence with amino acids of a binding epitope.
  • the grafting process involves an insertion of amino acids into the sequence of an existing natural or synthetic lasso peptide sequence, thereby extending the length of the sequence.
  • the grafting process involves the de novo creation of lasso peptide sequences containing the binding epitopes of interest in the loop, ring, and/or tail of the lasso peptide.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of epitopes are computationally grafted into the loop of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of epitopes are computationally grafted into the ring of a lasso peptide structure to create a library of lasso epitope graft variants. In some embodiments, a plurality of epitopes are computationally grafted into the tail of a lasso peptide structure to create a library of lasso epitope graft variants. In some embodiments, a plurality of epitopes are computationally grafted into the loop and ring of a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of epitopes are computationally grafted into the loop and tail of a lasso peptide structure to create a library of lasso epitope graft variants. In some embodiments, a plurality of epitopes are computationally grafted into the ring and tail of a lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of epitopes are computationally grafted into the loop, ring, and tail of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop and ring of a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the ring and tail of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of conformational epitopes are computationally grafted into the loop and tail of a lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of conformational epitopes are computationally grafted into the loop, ring, and tail of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from different polypeptide ligands are experimentally grafted into a lasso peptide sequence to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are experimentally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are computationally grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the loop and ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the ring and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are computationally grafted into the loop and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are computationally grafted into the loop, ring, and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the loop and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of binding epitopes are synthetically grafted into the loop of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the ring of a lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the tail of a lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from different polypeptide ligands are synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are 69 ACTIVE 705331286v1 synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are synthetically grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the loop of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the loop and ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the loop and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the ring and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the loop, ring, and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. [00212] In another aspect, computationally identified lasso peptides are synthesized in a host organism that contains the enzymes of a lasso peptide biosynthetic pathway.
  • computationally identified lasso peptides are synthesized in a host organism that contains the enzymes of a lasso peptide biosynthetic pathway composed of a precursor peptide (A), and lasso peptidase (B or B2), a lasso cyclase (C), and a RiPP recognition sequence (E or B1).
  • computationally identified lasso peptides are synthesized by treating a precursor peptide (A), with at least one of a lasso peptidase (B or B2), a lasso cyclase (C), and a RiPP recognition sequence (E or B1).
  • computationally identified lasso peptides are synthesized by treating an isolated precursor peptide (A), with at least one of an isolated lasso peptidase (B or B2), an isolated lasso cyclase (C), and an isolated RiPP recognition sequence (E or B1).
  • computationally identified lasso peptides are synthesized by adding a precursor peptide (A), with host organism that contains at least one of a lasso peptidase (B or B2), a lasso cyclase 70 ACTIVE 705331286v1 (C), and a RiPP recognition sequence (E or B1).
  • computationally identified lasso peptides are synthesized by contacting the genes for a precursor peptide (A), and at least one of an isolated lasso peptidase (B or B2), an isolated lasso cyclase (C), and an isolated RiPP recognition sequence (E or B1), with a cell extract to produce the identified lasso peptide in a cell-free biosynthesis process.
  • computationally modeled and identified lasso peptides are produced as described herein and screened for biological activity in a testing step that involves contacting such lasso peptides with biological targets, proteins, or receptors and measuring a property, such as cell penetration, binding affinity, or inhibition of function or activity.
  • the testing step allows the ranking of computationally identified and experimentally produced lasso peptides for desired biological activities and/or properties.
  • computationally designed and experimentally produced lasso peptides that are ranked highest are subjected to further optimization through iterative computational refinement based on the structure of the best ranked lasso peptides.
  • computationally identified and experimentally produced lasso peptides that are ranked highest are subjected to further optimization through directed or random peptide evolution methods.
  • computationally identified and experimentally produced lasso peptides that are ranked highest are subjected to further optimization through directed or random peptide evolution methods and lasso peptides that are ranked highest after evolution are further subjected to iterative computational optimization.
  • 6.5 Methods of Producing Lasso Peptides and Lasso Peptide Analogs In silico modeling is aimed at identifying molecules that are predicted to bind selectively and with high affinity to a target protein of interest. Once identified, the lasso peptide variants are synthesized, isolated, and partially or substantially purified to enable experimental testing for validation of biological activity and other properties.
  • lasso peptides there are two main methods for producing the identified molecules, which involve: (i) cell-free biosynthesis technology, and/or (ii) cell-based production methods (FIG.5).
  • cell-free biosynthesis technology e.g., cell-free biosynthesis technology
  • FFB cell-free biosynthesis
  • CFB methods allow rapid expression of natural biosynthetic genes and pathways and facilitate targeted or phenotypic activity screening of natural products, without the need for plasmid-based cloning or in vivo cellular propagation, thus enabling rapid process/product pipelines (e.g., creation of large quantity of lasso peptide in a short time).
  • oligonucleotides linear or circular constructs of DNA or RNA
  • a minimal set of lasso peptide biosynthesis pathway genes e.g., Genes A-C in a lasso peptide biosynthetic gene cluster
  • a cell extract containing in vitro TX-TL machinery for transcribing and translating the genes into the functional enzymes and lasso precursor peptides for production of lasso peptides
  • the CFB methods can produce in a CFB reaction mixture at least one, two or more of the lasso peptide variants.
  • the method for producing a lasso peptide comprises (a) providing a CFB system comprising a minimal set of lasso peptide biosynthesis components; and (b) incubating the CFB system under a suitable condition to produce the lasso peptide.
  • the minimal set of lasso peptide biosynthesis components comprises one or more components functions to provide a lasso precursor peptide, and one or more components function to process the lasso precursor peptide into the lasso peptide.
  • the one or more components function to process the lasso precursor peptide into the lasso peptide consist of a lasso peptidase and a lasso cyclase. In some embodiments, the one or more components function to process the lasso precursor peptide into the lasso peptide consists of a lasso peptidase, a lasso cyclase and an RRE. [00218] In some embodiments, the minimal set of lasso peptide biosynthesis components comprises one or more components functions to provide a lasso core peptide, and one or more components function to process the lasso core peptide into the lasso peptide.
  • the one or more components function to process the lasso core peptide into the lasso peptide comprises one or more selected from a lasso peptidase, a lasso cyclase and an RRE. In some embodiments, the one or more components function to process the lasso core into the lasso peptide consist of a lasso cyclase.
  • the one or more components function to provide a peptide or protein (e.g., a lasso precursor peptide, a lasso core peptide, or lasso peptide biosynthetic enzymes and proteins) in a CFB system can be provided in the form of the peptide or protein are provided in the form of the peptide or protein per se.
  • 72 ACTIVE 705331286v1 At least some of the peptide or protein components in the CFB system can be natural peptides or polypeptides. In some embodiments, at least some of the peptide or protein components in the CFB system are derivatives of natural peptides or polypeptides.
  • the peptide or protein components in the CFB system are non-natural peptides.
  • the one or more peptide or protein components of the CFB system can be isolated from nature, such as isolated from microorganisms producing the lasso precursor peptides.
  • the one or more peptide or protein components of the CFB system can be synthetically or recombinantly produced, using methods known in the art.
  • the one or more peptide or protein components of the CFB system can be synthesized using the CFB system as described herein, followed by purifying the biosynthesized peptide or protein components from the CFB system.
  • the CFB system comprises one or more fusion protein, or a polynucleotide encoding the fusion protein such that the CFB system is capable of producing the fusion protein through in vitro transcription and translation (TX-TL).
  • the fusion protein comprised a lasso precursor peptide or a lasso core peptide fused to one or more lasso peptide biosynthesis components.
  • the one or more lasso peptide biosynthesis components are selected from (i) a lasso peptidase; (ii) a lasso cyclase; (iii) a RRE; or (iv) any combinations of (i) to (iii).
  • the one or more lasso peptide biosynthesis components are encoded by the same lasso peptide biosynthetic gene cluster. In other embodiments, the one or more lasso peptide biosynthesis components are encoded by different lasso peptide biosynthetic gene cluster.
  • the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. [00224] In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso cyclase.
  • the fusion protein comprises a lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a lasso cyclase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises a lasso precursor 73 ACTIVE 705331286v1 peptide fused to a lasso cyclase and a RRE.
  • the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase, a lasso cyclase and RRE.
  • the fusion protein comprises a lasso core peptide fused to a lasso peptidase.
  • the fusion protein comprises a lasso core peptide fused to a lasso cyclase.
  • the fusion protein comprises a lasso core peptide fused to a RRE.
  • the fusion protein comprises a lasso core peptide fused to a lasso peptidase and a lasso cyclase.
  • the fusion protein comprises a lasso core peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises a lasso core peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein comprises a lasso core peptide fused to a lasso peptidase, a lasso cyclase and RRE. [00225] In some embodiments, the fusion protein comprised a lasso precursor peptide or a lasso core peptide fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom through cell-free biosynthesis.
  • Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso precursor peptide or lasso core peptide in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the lasso precursor peptide or lasso core peptide in the CFB system; (iii) a peptide or polypeptide that facilitates the processing of the lasso precursor peptide or lasso core peptide into the lasso peptide; (iv) a peptide or polypeptide that improves stability of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (v) a peptide or polypeptide that improves solubility of the lasso precursor peptide or lasso core peptid
  • the fusion protein comprised a lasso precursor peptide or a lasso core peptide fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a biologically 74 ACTIVE 705331286v1 active peptide or polypeptide.
  • biologically active peptide or polypeptide that can be fused with a lasso precursor peptide or lasso core peptide include but are not limited to (i) a peptide or polypeptide capable of binding to a target molecule (e.g., an antibody or an antigen); (ii) a peptide or polypeptide that enhance cell permeability of the fusion protein; (iii) a peptide or polypeptide capable of conjugating the fusion protein to at least one additional copy of the fusion protein; (iv) a peptide or polypeptide capable of linking the fusion protein to one or more peptidic or non-peptidic molecule; (v) a peptide or polypeptide capable of modulating activity of the lasso precursor peptide or lasso core peptide; (vi) a peptide or polypeptide capable of modulating activity of the lasso peptide derived from the lasso precursor peptide or the lasso core peptide;
  • the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide is fused to the N-terminus of the lasso peptidase or the lasso cyclase.
  • the one or more additional peptide or polypeptide is fused at the C-terminus of the lasso peptidase or the lasso cyclase.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. [00228] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster.
  • peptide or polypeptide that can be fused with a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) a lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi).
  • the fusion protein comprises at least one lasso cyclase and at least one lasso peptidase. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE. [00229] In some embodiments, the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso peptidase or lasso cyclase through cell-free biosynthesis.
  • peptide or polypeptide that can be fused with the lasso peptidase or lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso peptidase or lasso cyclase in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the lasso peptidase or lasso cyclase in the CFB system; (iii) a peptide or polypeptide that improves stability of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that improves solubility of the lasso peptidase or
  • the fusion protein comprised a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide.
  • biologically active peptide or polypeptide that can be fused with a lasso peptidase or a lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).
  • the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • RRE RIPP recognition element
  • the one or more additional peptide or polypeptide is fused to the N-terminus of the RRE.
  • the one or more additional peptide or polypeptide is fused at the C-terminus of the RRE.
  • a polynucleotide encoding the fusion protein comprises a 76 ACTIVE 705331286v1 nucleic acid sequence encoding the RRE, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the RRE, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • the fusion protein comprises an amino acid linker between the RRE and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between RRE and the one or more additional peptide or polypeptide.
  • the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster.
  • peptide or polypeptide that can be fused with a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) a lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi).
  • the fusion protein comprises at least one lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso core peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE. [00233] In some embodiments, the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • RRE RIPP recognition element
  • the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the RRE through cell-free biosynthesis.
  • peptide or polypeptide that can be fused with the RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the RRE in the CFB system; (ii) a peptide or polypeptide that increases the level of translation of the RRE in the CFB system; (iii) a peptide or polypeptide that improves stability of the RRE; (vi) a peptide or polypeptide that improves solubility of the RRE; (v) a peptide or polypeptide that enables or facilitates the detection of the RRE; (vi) a peptide or polypeptide that enables or facilitates purification of the RRE; (vii) a peptide or polypeptide that enables or facilitates immobilization of the RRE; or (viii)
  • the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • RRE RIPP recognition element
  • the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide.
  • biologically active peptide or polypeptide that can be fused with a RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).
  • the lasso precursor peptide genes are fused at the 5’- terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired TX-TL products (Marblestone, J.G., et al., Protein Sci, 2006, 15, 182–189).
  • MBP maltose-binding protein
  • SUMO small ubiquitin-like modifier protein
  • the lasso precursor peptides are fused at the C- terminus of the leader sequences to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin-like modifier protein, which enhance the stability, solubility, and production of the fused MBP-lasso or SUMO-lasso precursor peptide.
  • peptides or proteins such as maltose-binding protein or small ubiquitin-like modifier protein
  • the lasso precursor peptide genes or lasso core peptide genes are fused at the 3’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, such as sequences encoding maltose-binding protein (MBP) or small ubiquitin-like modifier protein (SUMO), which enhance the stability, solubility, and production of the desired TX-TL products.
  • MBP maltose-binding protein
  • SUMO small ubiquitin-like modifier protein
  • the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the N-terminus to form conjugates with peptides or proteins, such as maltose-binding protein or small ubiquitin- like modifier protein, which enhance the stability, solubility, and production of the fused MBP-lasso or SUMO-lasso precursor peptide.
  • the lasso precursor peptide genes or lasso core peptide genes are fused at the 5’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide tags for affinity purification or immobilization, including his-tags, strep- tags, or FLAG-tags.
  • the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus of the core peptides to form conjugates with 78 ACTIVE 705331286v1 other peptides or proteins, with or without a linker, such as peptide tags for affinity purification or immobilization, including his-tags, strep-tags, or FLAG-tags.
  • lasso precursor peptides, lasso core peptides, or lasso peptides are fused to molecules that can enhance cell permeability or penetration into cells, for example through the use of arginine-rich cell-penetrating peptides such as TAT peptide, penetratin, and flock house virus (FHV) coat peptide (Brock, R., Bioconjug. Chem., 2014, 25, 863–868).
  • arginine-rich cell-penetrating peptides such as TAT peptide, penetratin, and flock house virus (FHV) coat peptide
  • a lasso precursor peptide gene or core peptide gene is fused at the 3’-terminus to oligonucleotide sequences that encode arginine-rich cell- penetrating peptides or proteins, including oligonucleotide sequences that encode penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups (Wender, P.A., et al., Adv. Drug Deliv. Rev., 2008, 60, 452–472).
  • FHV flock house virus
  • a lasso precursor peptide, lasso core peptide, or lasso peptide is fused at the C-terminus to peptides that promote cell penetration such as arginine-rich cell-penetrating peptides or proteins, including amino acid sequences that encode TAT peptide, penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups.
  • FHV flock house virus
  • the lasso precursor peptide genes or lasso core peptide genes are fused at the 5’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding natural or unnatural peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, integrin ligand binding epitopes, and the like.
  • the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus to peptides or proteins, with or without a linker, such as peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, chemokine binding epitopes, integrin ligand binding epitopes, and the like.
  • the one or more components function to provide a peptide or protein (e.g., a lasso precursor peptide, a lasso core peptide, or lasso peptide biosynthetic enzymes and proteins) in a CFB system can be provided in the form of a nucleic acid encoding the peptide or protein and in vitro TX-TL machinery capable of producing the peptide or protein via in vitro TX-TL of the coding sequences.
  • the coding nucleic acid can be DNA, RNA or cDNA.
  • one or more 79 ACTIVE 705331286v1 coding nucleic acid sequences can be contained in the same nucleic acid molecule, such as a vector. [00241] It is understood that when more than one coding nucleic acid sequences are included in a CFB system, such more than one encoding nucleic acid sequences can be introduced on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof.
  • a microbial organism or a cell extract can be engineered to express two or more exogenous nucleic acids encoding lasso precursor peptide, lasso core peptide, lasso peptidase, lasso cyclase or RRE.
  • two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism or into a cell extract
  • the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid or as linear strands of DNA, or on separate plasmids, or can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids.
  • the in vitro TX-TL machinery is purified from a host cell.
  • the in vitro TX-TL machinery is provided in the form of a cell extract of a host cell.
  • An exemplary procedure for obtaining a cell extract comprises the steps of (i) growing cells, (ii) breaking open or lysing the cells by mechanical, biological or chemical means, (iii) removing cell debris and insoluble materials e.g., by filtration or centrifugation, and (iv) optionally treating to remove residual RNA and DNA, but retaining the active enzymes and biosynthetic machinery for transcription and translation, and optionally the metabolic pathways for co-factor recycle, including but not limited to co-factors such as THF, S-adenosylmethionine, ATP, NADH, NAD and NADP and NADPH.
  • a cell extract may be further supplemented for improved performance in in vitro TX-TL.
  • a cell extract can be further supplemented with some or all of the twenty proteinogenic naturally-occurring amino acids and corresponding transfer ribonucleic acids (tRNAs), and optionally, may be supplemented with additional components, including but not limited to: (1) glucose, xylose, fructose, sucrose, maltose, or starch, (2) adenosine triphosphate (ATP), and/or adenosine diphosphate (ADP), purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and/or 80 ACTIVE 705331286v1 uridine triphosphate, or combinations thereof, (3) cyclic-adenosine monophosphate (cAMP) and/or 3-phosphoglyceric acid (3-PGA), (4) nicotinamide adenine dinucleotides NADH and/or NAD, or nicotinamide a
  • tRNAs transfer
  • the cell extracts or supplemented cell extracts can be used as a reaction mixture to carry out in vitro TX-TL. In some embodiments, supplementations or adjustments can be made to the cell extract to provide a suitable condition for lasso formation.
  • the in vitro TX-TL machinery is provided in the form of a cell extract or supplemented cell extract of a host cell.
  • the host cell is the cell of the same organism where the coding nucleic acid is derived from.
  • the coding nucleic acid sequences can be identified using one or more computer-based genomic mining tools described herein or known in the art. For example, U.S.
  • Provisional Application Nos.62/652,213 and 62/651,028 disclose thousands of sequences from lasso peptide biosynthetic gene clusters identified from various organisms, and provide GenBank accession numbers for various sequences for lasso precursor peptides, lasso peptidase, lasso cyclase and/or RRE.
  • Host organisms where the lasso peptide biosynthetic gene clusters originate can be identified based on the GenBank accession numbers, including but not limited to Caulobacteraceae species (e.g., Caulobacter sp. K31, Caulobacter henricii), Streptomyces species (e.g.
  • Burkholderiaceae species e.g., Burkholderia thailandensis E264
  • Pseudomallei species Bacillus species
  • Burkholderia species e.g., Burkholderia thailandensis MSMB43, Burkholderia o
  • the host cell is a microbial organism known to be applicable to fermentation processes.
  • Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus 81 ACTIVE 705331286v1 subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Streptomyces albus, Clostridium acetobutylicum, Vibrio natriegens, Pseudomonas fluorescens, and Pseudomonas putida.
  • Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris.
  • E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering.
  • Other particularly useful host organisms include Vibrio natriegens, and yeast such as Saccharomyces cerevisiae.
  • the CFB system is configured to produce a lasso peptide.
  • the CFB system comprises one or more components configured to provide (i) a lasso precursor peptide, (ii) a lasso peptidase, (iii) a lasso cyclase.
  • the CFB system comprises one or more components configured to provide (i) a lasso core peptide, and (ii) a lasso cyclase.
  • the CFB system further comprises one or more components configured to provide (iv) an RRE. In some embodiments, all of (i) to (iv) above are provided in the CFB system as the corresponding peptide or protein.
  • the CFB system further comprises in vitro TX-TL machinery for producing the corresponding protein from the coding nucleic acid.
  • the CFB systems can be incubated under a condition suitable for lasso formation to produce the lasso peptide.
  • the incubation condition can be designed and adjusted based on various factors known to skilled artisan in the art, including for example, condition suitable for maintain stability of components of the CFB system, conditions suitable for the lasso processing enzymes to exert enzymatic activities, and/or conditions suitable for the in vitro TX-TL of the coding sequences present in the CFB system.
  • different lasso peptidases can process the same lasso precursor peptide into different lasso core peptide by recognizing and cleaving different leader peptide off the lasso precursor.
  • different lasso cyclase can process the same lasso core peptide into distinct lasso peptides by cyclizing the core peptide at different ring-forming amino acid residues.
  • different RREs can facilitate different processing by the lasso peptidase and/or lasso cyclase, and thus lead to formation of distinct lasso peptides from the same lasso precursor peptide.
  • the CFB system comprises the lasso precursor peptide, lasso peptidase, and lasso cyclase produced from coding sequences of the same lasso peptide biosynthetic gene cluster (such as Genes A, B, and C of the same lasso peptide biosynthetic gene cluster).
  • the CFB system comprises the lasso precursor peptide, lasso peptidase, lasso cyclase, and RRE produced from coding sequences of the same lasso peptide biosynthetic gene cluster.
  • the CFB system comprises the lasso core peptide, and lasso cyclase produced from coding sequences of the same lasso peptide biosynthetic gene cluster (such as Genes A and C of the same lasso peptide biosynthetic gene cluster).
  • the CFB system comprises the lasso core peptide, lasso cyclase, and RRE produced from coding sequences of the same lasso peptide biosynthetic gene cluster.
  • the CFB system comprises the lasso core peptide, lasso cyclase, and RRE produced from coding sequences of the same lasso peptide biosynthetic gene cluster.
  • at least two of the lasso precursor peptide, lasso peptidase and lasso cyclase in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters (such as Gene A from one, and Genes B and C from another, lasso peptide biosynthetic gene cluster).
  • At least two of the lasso precursor peptide, lasso peptidase, lasso cyclase and RRE in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters.
  • the lasso core peptide and lasso cyclase in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters (such as Gene A from one, and Gene C from another, lasso peptide biosynthetic gene cluster).
  • a derivative of a natural lasso peptide at least two of the lasso core peptide, lasso cyclase and RRE in the CFB system are produced from coding sequences of different lasso peptide biosynthetic gene clusters.
  • a lasso precursor peptide is modified at the core peptide sequence, while the leader sequence is maintained the same.
  • the modified precursor peptide can then processed by corresponding lasso peptidase and/or lasso cyclase into a matured lasso peptide with modified amino acid sequence.
  • the contacting step (a) comprises adding a first nucleic acid sequence encoding the peptide into the cell-free biosynthesis reaction mixture, and where the cell-free biosynthesis reaction mixture comprises in vitro TX-TL machinery and is configured to express the peptide.
  • the contacting step (a) comprises adding a second nucleic acid sequence encoding the lasso peptide biosynthesis component to the cell-free biosynthesis reaction mixture, and where the cell-free biosynthesis reaction mixture comprises in vitro TX-TL machinery configured to express the lasso peptide biosynthesis component.
  • the lasso peptide biosynthesis component comprises a lasso peptidase.
  • the lasso peptide biosynthesis component comprises a lasso cyclase.
  • the lasso peptide biosynthesis component further comprises a post-translationally modified peptide (RiPP) recognition element (RRE).
  • RRE post-translationally modified peptide
  • the contacting step (a) comprises adding the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase.
  • the contacting step (a) comprises adding the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE.
  • the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post- translationally modified peptide (RiPP) recognition element (RRE)
  • the contacting step (a) comprises adding the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase and a fourth nucleic acid sequence encoding the RRE.
  • the cell-free biosynthesis reaction mixture comprises cell extract or supplemented cell extract.
  • CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined with genes that encode additional peptides, proteins or enzymes, including genes that encode RiPP recognition elements (RREs) or oligonucleotides that encode RREs that are fused to the 5’ or 3’ end of a lasso precursor peptide gene, a lasso core peptide gene, a lasso peptidase gene or a lasso cyclase gene.
  • RREs RiPP recognition elements
  • CFB reactions are conducted with a minimal set of lasso peptide 84 ACTIVE 705331286v1 biosynthesis components, including lasso precursor peptides, lasso peptidases, or lasso cyclase that are fused to RREs at the N-terminus or C-terminus.
  • CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with additional isolated proteins or enzymes, including RiPP recognition elements (RREs).
  • RREs RiPP recognition elements
  • CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with genes that encode additional proteins or enzymes, including genes that encode lasso peptide modifying enzymes such as N-methyltransferases, O-methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, dehydrogenases, halogenases, kinases, RiPP heterocyclases, RiPP cyclodehydratases, peptidylarginine deiminase, and prenyltransferases.
  • genes that encode lasso peptide modifying enzymes such as N-methyltransferases, O-methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, de
  • CFB reactions are conducted with a minimal set of lasso peptide biosynthesis components combined and contacted with additional isolated proteins or enzymes, including lasso peptide modifying enzymes such as N-methyltransferases, O- methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, dehydrogenases, halogenases, kinases, RiPP heterocyclases, RiPP cyclodehydratases, peptidylarginine deiminase, and prenyltransferases.
  • lasso peptide modifying enzymes such as N-methyltransferases, O- methyltransferases, biotin ligases, glycosyltransferases, esterases, acylases, acyltransferases, aminotransferases, amidases, hydroxylases, dehydrogenases,
  • CFB methods and systems provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are conducted in a CFB reaction mixture, comprising one or more cell extracts that are supplemented with all twenty proteinogenic naturally occurring amino acids and corresponding transfer ribonucleic acids (tRNAs).
  • tRNAs transfer ribonucleic acids
  • Cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components also may be supplemented with additional components, including but not limited to, glucose, xylose, fructose, sucrose, maltose, starch, adenosine triphosphate (ATP), and/or adenosine diphosphate (ADP), purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate, cyclic-adenosine monophosphate (cAMP) and/or 3-phosphoglyceric acid (3-PGA), nicotinamide adenine dinucleotides NADH and/or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and/or NADP, or combinations thereof,
  • the preparation CFB reaction mixtures and cell extracts employed for the CFB methods as provided herein comprises characterization of the CFB reaction mixtures and cell extracts using proteomic approaches to assess and quantify the proteome available for the production of lasso peptides and related molecules thereof.
  • 13 C metabolic flux analysis (MFA) and/or metabolomics studies are conducted on CFB reaction mixtures and cell extracts to create a flux map and characterize the resulting metabolome of the CFB reaction mixture and cell extract or extracts.
  • the CFB method is performed using: one or a combination of two or more cell extracts from various “chassis” organisms, such as E. coli, optionally mixed with one or a combination of two or more cell extracts derived from other species, e.g., a native lasso peptide-producing organism or relative.
  • a native lasso peptide-producing organism or relative e.g., a native lasso peptide-producing organism or relative.
  • This can give the advantage of a robust transcription/translation machinery, combined with any unknown components of the native species that might be needed for proper protein folding or activity, or to supply precursors for the lasso peptide pathway.
  • these factors if these factors are known they can be expressed in the chassis organism prior to making the cell extract or these factors can be isolated and purified and added directly to the CFB reaction mixture or cell extract.
  • CFB methods and systems provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components including the use of cell extracts for in vitro TX-TL systems express lasso peptide biosynthetic gene clusters without the regulatory constraints of the cell.
  • some or all of the lasso peptide pathway biosynthetic genes are refactored to remove native transcriptional and translational regulation.
  • some or all of the lasso peptide pathway biosynthetic genes are refactored and constructed into operons on plasmids.
  • CFB methods, systems and processes, including in vitro TX-TL systems, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are cell-free platforms that can use whole cell, cytoplasmic or nuclear extract from a single organism such 86 ACTIVE 705331286v1 as E.coli or Saccharomyces cerevisiae (S. cerevisiae) or from an organism of the Actinomyces genus, e.g., a Streptomyces.
  • CFB methods, systems and processes, including in vitro TX-TL systems, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are cell-free platforms that can use mixtures of whole cell, cytoplasmic, and/or nuclear extracts from the same or different organisms.
  • strain engineering approaches as well as modification of the growth conditions are used (on the organism from which at least one extract is derived) towards the creation of cell extracts as provided herein, to generate mixed cell extracts with varying proteomic and metabolic capabilities in the final CFB reaction mixture.
  • cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components comprise whole cell, cytoplasmic or nuclear extracts from a bacterial cell or eukaryotic cell, including insect, plant, fungal, yeast, or mammalian cells.
  • cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components comprise whole cell, cytoplasmic or nuclear extracts from a bacterial cell or eukaryotic cell, including insect, plant, fungal, yeast, or mammalian cells, and are designed, produced and processed in a way to maximize efficacy and yield in the production of desired lasso peptides or related molecules thereof.
  • cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components derive from at least two different bacterial cells, two different fungal cells; two different yeast cells, two different insect cells, two different plant cells or two different mammalian cells, or combinations of cell extracts from different species and genera thereof.
  • cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components comprises an extract derived from: an Escherichia or a Escherichia coli (E.
  • coli a Streptomyces or an Actinobacteria; an Ascomycota, Basidiomycota, or a Saccharomycetales; a Penicillium or a Trichocomaceae; a 87 ACTIVE 705331286v1 Spodoptera, a Spodoptera frugiperda, a Trichoplusia or a Trichoplusia ni; a Poaceae, a Triticum, or a wheat germ; a rabbit reticulocyte or a HeLa cell.
  • cell extracts used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components comprises a cell extract from or comprises an extract derived from: any prokaryotic and eukaryotic organism including, but not limited to, bacteria, including Archaea, eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human cells.
  • At least one of the cell extracts used in the CFB methods provided herein comprises an extract from or comprises an extract derived from: Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingi
  • At least one cell, cytoplasmic or nuclear extract used in the CFB methods, provided herein to produce lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components comprises a cell extract from or comprises an extract derived from: Acinetobacter baumannii Naval-82, Acinetobacter sp. ADP1, Acinetobacter sp.
  • Chloroflexus aggregans DSM 9485 Chloroflexus aurantiacus J-10-fl, Citrobacter freundii, Citrobacter koseri ATCC BAA-895, Citrobacter youngae , Clostridium, Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridium acidurici, Clostridium aminobutyricum, Clostridium asparagiforme DSM 15981, Clostridium beijerinckii , Clostridium beijerinckii NCIMB 8052, Clostridium bolteae ATCC BAA-613, Clostridium carboxidivorans P7, Clostridium cellulovorans 743B, Clostridium difficile, Clostridium hiranonis DSM 13275, Clostridium hylemonae DSM 15053, Clostridium kluyveri, Clostridium
  • Geobacillus themodenitrificans NG80-2 Geobacter bemidjiensis Bem, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Geobacillus stearothermophilus DSM 2334, Haemophilus influenzae, Helicobacter pylori, Homo sapiens, Hydrogenobacter thermophilus, Hydrogenobacter thermophilus TK-6, Hyphomicrobium denitrificans ATCC 51888, Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniae subsp.
  • strain JC1 DSM 3803 Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium gastri , Mycobacterium marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2155, Mycobacterium tuberculosis, Nitrosopumilus salaria BD31, Nitrososphaera gargensis Ga9.2, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Nostoc sp.
  • PCC 7120 Ogataea angusta, Ogataea parapolymorpha DL-1 (Hansenula polymorpha DL-1), Paenibacillus peoriae KCTC 3763, Paracoccus denitrificans, Penicillium chrysogenum, Photobacterium profundum 3TCK, Phytofermentans ISDg, Pichia pastoris, Picrophilus torridus DSM9790, Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonas aeruginosa PA01, Pseudomonas denitrificans, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas 90 ACTIVE 705331286v1 sp, Pseudomonas syringae pv.
  • Rhodobacter syringae B728a Pyrobaculum islandicum DSM 4184, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii OT3, Ralstonia eutropha, Ralstonia eutropha H16, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1, Rhodospirillum rubrum, Rhodospirillum rubrum ATCC 11170, Ruminococcus obeum ATCC 29174, Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Salmonella enterica, Salmonella enterica subsp.
  • enterica serovar Typhimurium str. LT2 Salmonella enterica typhimurium , Salmonella typhimurium, Schizosaccharomyces pombe, Sebaldella termitidis ATCC 33386 , Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021, Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350, Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystis str. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica, Thermoanaerobacter sp.
  • cell extracts used in the CFB methods and processes, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components e.g., including at least one of the cell, cytoplasmic or nuclear extracts, have added to them, or further comprise, supplemental ingredients, compositions or compounds, reagents, ions, trace metals, salts, or elements, buffers and/or solutions.
  • the CFB method and system of the present disclosure provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, use or fabricate environmental conditions to optimize the rate of formation or yield of a lasso peptide or related molecules thereof.
  • CFB reaction mixtures and cell extracts used in the CFB methods and systems, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are supplemented with a carbon source and other essential nutrients.
  • the CFB production system including cell extracts used in the CFB methods and processes, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components, can include, for example, any carbohydrate 91 ACTIVE 705331286v1 source.
  • sources of sugars or carbohydrate substrates include glucose, xylose, maltose, arabinose, galactose, mannose, maltodextrin, fructose, sucrose and starch.
  • CFB methods and systems provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are conducted in a CFB reaction mixture, comprising cell extracts that are supplemented with all twenty proteinogenic naturally occurring amino acids and corresponding transfer ribonucleic acids (tRNAs).
  • cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are supplemented with adenosine triphosphate (ATP), and/or adenosine diphosphate (ADP).
  • ATP adenosine triphosphate
  • ADP adenosine diphosphate
  • cell extracts used in the CFB reaction mixture are supplemented with glucose, xylose, maltose, arabinose, galactose, mannose, maltodextrin, fructose, sucrose and/or starch.
  • cell extracts used in the CFB reaction mixture are supplemented with purine and guanidine nucleotides, adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate.
  • cell extracts used in the CFB reaction mixture are supplemented with cyclic-adenosine monophosphate (cAMP) and/or 3-phosphoglyceric acid (3-PGA).
  • cAMP cyclic-adenosine monophosphate
  • 3-PGA 3-phosphoglyceric acid
  • cell extracts used in the CFB reaction mixture are supplemented with nicotinamide adenine dinucleotides NADH and/or NAD, or nicotinamide adenine dinucleotide phosphates, NADPH, and/or NADP, or combinations thereof.
  • cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are supplemented with amino acid salts such as magnesium glutamate and/or potassium glutamate.
  • cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are supplemented with buffering agents such as HEPES, TRIS, spermidine, or 92 ACTIVE 705331286v1 phosphate salts.
  • cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are supplemented with salts, including but not limited to, potassium phosphate, sodium chloride, magnesium phosphate, and magnesium sulfate.
  • cell extracts used in the CFB reaction mixture, provided herein for the synthesis of lasso peptides and related molecules thereof from a minimal set of lasso peptide biosynthetic pathway components are supplemented with folinic acid and co-enzyme A (CoA).
  • CoA co-enzyme A
  • cell extracts used in the CFB reaction mixture are supplemented with crowding agents such as PEG 8000, Ficoll 70, or Ficoll 400, or combinations thereof.
  • crowding agents such as PEG 8000, Ficoll 70, or Ficoll 400, or combinations thereof.
  • cell-free biosynthesis of lasso peptides or lasso peptide analogs is conducted with isolated peptide and enzyme components in standard buffered media, such as phosphate-buffered saline or tris-buffered saline, in each case containing salts, ATP, and co-factors required for lasso peptidase and lasso cyclase enzymatic activity.
  • standard buffered media such as phosphate-buffered saline or tris-buffered saline
  • cell-free biosynthesis of lasso peptides is conducted using genes that require transcription (TX) and translation (TL) to afford the lasso precursor peptide and/or lasso peptide biosynthetic enzymes in situ, and such in vitro biosynthesis processes are conducted in cell extracts derived from prokaryotic or eukaryotic cells (See: Gagoski, D., et al., Biotechnol. Bioeng.2016;113: 292–300; Culler, S. et al., PCT Appl. No. WO2017/031399).
  • a cell-free biosynthesis process for producing lasso peptide analogs is conducted by contacting an isolated, chemically- or biologically-synthesized precursor peptide with one or more isolated lasso peptidase, lasso cyclase, and lasso RRE.
  • a cell-free biosynthesis process for producing lasso peptide analogs is conducted by contacting an isolated, chemically- or biologically- synthesized core peptide with one or more isolated lasso peptidase, lasso cyclase, and lasso RRE.
  • lasso peptides and lasso peptide analogs include lasso peptides identified using in silico modeling 93 ACTIVE 705331286v1 methods, using a non-naturally occurring microbial organism.
  • Certain cell-based production methods involve cultivating or fermenting a microbial organism that is a natural producer of a lasso peptide of interest.
  • Alternative cell-based production methods involve cloning the genes encoding lasso peptide biosynthesis component into an appropriate vector or plasmid, introducing that vector or plasmid into a microorganism, and propagating or cultivating that organism with the necessary nutrients and under conditions for heterologous production of recombinant lasso peptides of interest (Zhang, Y., et al, Heterologous production of microbial ribosomally synthesized and post-translationally modified peptides, Front. Microbiol., 2018, doi: 10.3389/fmicb.2018.01801).
  • the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed lasso peptide pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more lasso peptide biosynthetic pathways.
  • lasso peptide biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid.
  • exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins.
  • exogenous expression of all enzymes or proteins in a pathway for production of a lasso peptide can be included, such as a lasso peptide precursor, a lasso peptide peptidase, a lasso peptide cyclase, and/or a lasso peptide RiPP recognition element (RRE).
  • RRE lasso peptide RiPP recognition element
  • a non-naturally occurring microbial organism can have one, two, three, four, five, six, seven, eight, nine or ten, up to all nucleic acids encoding the enzymes or proteins constituting a lasso peptide biosynthetic pathway disclosed herein.
  • the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize lasso peptide biosynthesis or that confer other useful functions onto the host microbial organism.
  • One such other functionality can include, for example, augmentation of the synthesis of one or more of the lasso peptide pathway precursors, such as amino acids.
  • a host microbial organism is selected such that it produces the lasso precursor peptide, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired biosynthesis precursor or increased production of a biosynthesis precursor naturally produced by the host microbial organism.
  • amino acids are produced naturally in a host organism such as E. coli.
  • a host organism can be engineered to increase production of one or more amino acids in order to increase production of lasso precursor, as disclosed herein.
  • a host organism can be engineered to produce a non-natural amino acid that is incorporated into the lasso precursor peptide (Piscotta, F.J., et al., Chem. Commun., 2015, 51, 409-412; Al-Toma, R.S., et al., ChemBioChem 2015, 16, 503 – 509).
  • a microbial organism that has been engineered to produce a desirable lasso precursor peptide can be used as a host organism and further engineered to express enzymes or proteins that processes the lasso precursor peptide into matured lasso peptides containing non-natural amino acids.
  • a non-naturally occurring microbial organism is generated from a host that contains the enzymatic capability to synthesize a lasso peptide or lasso peptide analog.
  • it can be useful to increase the synthesis or accumulation of a lasso peptide pathway intermediate or product to, for example, drive lasso peptide pathway reactions toward lasso peptide production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described lasso peptide pathway enzymes or proteins.
  • the enzyme or enzymes and/or protein or proteins of the lasso peptide pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily engineered to be non-naturally occurring microbial organisms for producing lasso peptides or lasso peptide analogs, through overexpression of one, two, three, four, five, six, seven, eight, nine, or ten, that is, up to all nucleic acids encoding a lasso peptide biosynthetic pathway enzymes or proteins.
  • a non- naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the lasso peptide biosynthetic pathway.
  • exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user.
  • endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene’s 95 ACTIVE 705331286v1 promoter when linked to an inducible promoter or other regulatory element.
  • an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent (Daniel-Ivad, M. et al., ACS Chem. Biol.2017, 12, 628 ⁇ 634), or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time.
  • an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.
  • any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention.
  • the nucleic acids can be introduced so as to confer, for example, a lasso peptide biosynthetic pathway onto the microbial organism.
  • encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer lasso peptide biosynthetic capability.
  • a non-naturally occurring microbial organism having a lasso peptide biosynthetic pathway can comprise at least one exogenous nucleic acid encoding desired enzymes or proteins, such as the linear lasso precursor peptide, or alternatively a combination of a lasso peptide peptidase and a lasso peptide cyclase.
  • desired enzymes or proteins such as the linear lasso precursor peptide
  • any combination of one or more genes encoding one or more peptides, enzymes, or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention.
  • any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, lasso peptide peptidase and a lasso peptide cyclase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.
  • the non- naturally occurring microbial organisms also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes.
  • one alternative to produce a lasso peptide other than use of the lasso peptide producers is through addition of another microbial organism capable of converting a lasso peptide pathway intermediate into a lasso peptide or lasso peptide analog.
  • One such procedure includes, for example, the fermentation of a microbial organism that produces a linear lasso precursor peptide.
  • the linear lasso precursor 96 ACTIVE 705331286v1 peptide can then be used as a substrate for a second microbial organism that converts the linear lasso precursor peptide to a lasso peptide.
  • the linear lasso precursor peptide can be added directly to another culture of the second organism or the original culture of the linear lasso precursor peptide producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.
  • a lasso peptide also can be biosynthetically produced from microbial organisms through co-culture or co-fermentation using two organisms in the same vessel, where the first microbial organism produces a linear lasso precursor peptide and the second microbial organism converts the intermediate to a lasso peptide.
  • a lasso peptide also can be biosynthetically produced by first chemically synthesizing the linear lasso precursor peptide, followed by addition of the chemically synthesized linear lasso precursor peptide to a fermentation broth using one or more organisms in the same vessel, where linear lasso precursor peptide is converted to a lasso peptide.
  • a lasso peptide also can be biosynthetically produced from microbial organisms through cell-free biosynthesis of the linear lasso precursor peptide, followed by addition of the linear lasso precursor peptide fermentation broth using one or more organisms in the same vessel, where linear lasso precursor peptide is converted into a lasso peptide.
  • a lasso peptide also can be biosynthetically produced by first chemically synthesizing the linear lasso precursor peptide, followed by addition of the chemically synthesized linear lasso precursor peptide to a broth containing the isolated biosynthetic enzymes, including but not limited to one or more of a lasso peptide peptidase, a lasso peptide cyclase, and lasso peptide RRE, wherein the linear lasso precursor peptide is converted to a lasso peptide.
  • a lasso peptide also can be biosynthetically produced by first producing the linear lasso precursor peptide by cell- free biosynthesis methods, followed by addition of the linear lasso precursor peptide to a broth containing the isolated biosynthetic enzymes, including but not limited to one or more of a lasso peptide peptidase, a lasso peptide cyclase, and lasso peptide RRE, wherein the linear lasso precursor peptide is converted to a lasso peptide.
  • the fusion protein comprised a lasso precursor peptide or a lasso core peptide fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide is fused to the N-terminus of the lasso precursor peptide or lasso core peptide. In some embodiments, the one or more additional peptide or polypeptide is fused at the C-terminus of the lasso precursor peptide or lasso core peptide.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso precursor peptide or the lasso core peptide, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso precursor peptide or the lasso core peptide, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • the fusion protein comprises an amino acid linker between the lasso precursor peptide or lasso core peptide and the one or more additional peptide or polypeptide.
  • the fusion protein does not comprise an amino acid linker between the lasso precursor peptide or lasso core peptide and the one or more additional peptide or polypeptide.
  • the fusion protein comprised a lasso precursor peptide or a lasso core peptide fused to one or more lasso peptide biosynthesis components.
  • the one or more lasso peptide biosynthesis components are selected from (i) a lasso peptidase; (ii) a lasso cyclase; (iii) a RRE; or (iv) any combinations of (i) to (iii).
  • the one or more lasso peptide biosynthesis components are encoded by the same lasso peptide biosynthetic gene cluster.
  • the one or more lasso peptide biosynthesis components are encoded by different lasso peptide biosynthetic gene cluster.
  • the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide.
  • the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase.
  • the fusion protein comprises a lasso precursor peptide fused to a lasso cyclase. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a lasso cyclase. In 98 ACTIVE 705331286v1 specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises a lasso precursor peptide fused to a lasso cyclase and a RRE.
  • the fusion protein comprises a lasso precursor peptide fused to a lasso peptidase, a lasso cyclase and RRE.
  • the fusion protein comprises a lasso core peptide fused to a lasso peptidase.
  • the fusion protein comprises a lasso core peptide fused to a lasso cyclase.
  • the fusion protein comprises a lasso core peptide fused to a RRE.
  • the fusion protein comprises a lasso core peptide fused to a lasso peptidase and a lasso cyclase.
  • the fusion protein comprises a lasso core peptide fused to a lasso peptidase and a RRE. In specific embodiments, the fusion protein comprises a lasso core peptide fused to a lasso cyclase and a RRE. In specific embodiments, the fusion protein comprises a lasso core peptide fused to a lasso peptidase, a lasso cyclase and RRE. [00284] In some embodiments, the fusion protein comprises a lasso precursor peptide or a lasso core peptide fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom through cell-free biosynthesis.
  • Examples of peptide or polypeptide that can be fused with a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso precursor peptide or lasso core peptide in the microbial organism; (ii) a peptide or polypeptide that increases the level of translation of the lasso precursor peptide or lasso core peptide in the microbial organism; (iii) a peptide or polypeptide that facilitates the processing of the lasso precursor peptide or lasso core peptide into the lasso peptide; (iv) a peptide or polypeptide that improves stability of the lasso precursor peptide or lasso core peptide or the lasso peptide derived therefrom; (v) a peptide or polypeptide that improves solubility of the lasso precursor peptide or lasso core
  • the fusion protein comprises a lasso precursor peptide or a lasso core peptide fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide.
  • biologically active peptide or polypeptide that can be fused with a lasso precursor peptide or lasso core peptide include but are not limited to (i) a peptide or polypeptide capable of binding to a target molecule (e.g., an antibody or an antigen); (ii) a peptide or polypeptide that enhance cell permeability of the fusion protein; (iii) a peptide or polypeptide capable of conjugating the fusion protein to at least one additional copy of the fusion protein; (iv) a peptide or polypeptide capable of linking the fusion protein to one or more peptidic or non-peptidic molecule; (v) a peptide or polypeptide capable of modulating activity of the lasso precursor peptide or lasso core peptide; (vi) a peptide or polypeptide capable of modulating activity of the lasso peptide derived from the lasso precursor peptide or the lasso core peptide;
  • the fusion protein comprises a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide is fused to the N-terminus of the lasso peptidase or the lasso cyclase.
  • the one or more additional peptide or polypeptide is fused at the C-terminus of the lasso peptidase or the lasso cyclase.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the lasso peptidase or the lasso cyclase, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • the fusion protein comprises an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between the lasso peptidase or the lasso cyclase and the one or more additional peptide or polypeptide. [00287] In some embodiments, the fusion protein comprises a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster.
  • peptide or polypeptide that can be fused with 100 ACTIVE 705331286v1 a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) a lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi).
  • the fusion protein comprises at least one lasso cyclase and at least one lasso peptidase. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE. [00288] In some embodiments, the fusion protein comprises a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the lasso peptidase or lasso cyclase through cell-free biosynthesis.
  • peptide or polypeptide that can be fused with the lasso peptidase or lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the lasso peptidase or lasso cyclase in the microbial organism; (ii) a peptide or polypeptide that increases the level of translation of the lasso peptidase or lasso cyclase in the microbial organism; (iii) a peptide or polypeptide that improves stability of the lasso peptidase or lasso cyclase; (vi) a peptide or polypeptide that improves solubility of the lasso peptidas
  • the fusion protein comprises a lasso peptidase or a lasso cyclase fused to one or more additional peptide or polypeptide.
  • the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide.
  • biologically active peptide or polypeptide that can be fused with a lasso peptidase or a lasso cyclase according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).
  • the fusion protein comprises a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • RRE RIPP recognition element
  • the one 101 ACTIVE 705331286v1 or more additional peptide or polypeptide is fused to the N-terminus of the RRE.
  • the one or more additional peptide or polypeptide is fused at the C-terminus of the RRE.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the RRE, wherein the 5’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • a polynucleotide encoding the fusion protein comprises a nucleic acid sequence encoding the RRE, wherein the 3’ end of the nucleic acid sequence is linked to a nucleic acid sequence encoding the one or more additional peptide or polypeptide.
  • the fusion protein comprises an amino acid linker between the RRE and the one or more additional peptide or polypeptide. In some embodiments, the fusion protein does not comprise an amino acid linker between RRE and the one or more additional peptide or polypeptide.
  • the fusion protein comprises a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • the more additional peptide or polypeptide comprises a peptide or polypeptide encoded by a lasso peptide biosynthetic gene cluster.
  • peptide or polypeptide that can be fused with a lasso precursor peptide or a lasso core peptide according to the present disclosure include but are not limited to (i) a lasso precursor peptide; (ii) a lasso core peptide; (iii) a lasso peptidase; (iv) a lasso cyclase, (v) a RRE; or (vi) any combinations of (i) to (vi).
  • the fusion protein comprises at least one lasso precursor peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso core peptide fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso cyclase fused to a RRE. In specific embodiments, the fusion protein comprises at least one lasso peptidase fused to a RRE. [00292] In some embodiments, the fusion protein comprises a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • RRE RIPP recognition element
  • the one or more additional peptide or polypeptide comprises a peptide or polypeptide that facilitates production of the RRE through cell-free biosynthesis.
  • peptide or polypeptide that can be fused with the RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide that increases the level of transcription of the RRE in the microbial organism; (ii) a peptide or polypeptide that increases the level of translation of the RRE in the microbial organism; (iii) a peptide or polypeptide that improves stability of the RRE; (vi) a peptide or polypeptide that improves solubility of the RRE; (v) a peptide or polypeptide that enables or facilitates the detection of the RRE; (vi) a peptide or polypeptide 102 ACTIVE 705331286v1 that enables or facilitates purification of the RRE; (vii) a peptide or polypeptide that enables or facilitate
  • the fusion protein comprised a RIPP recognition element (RRE) fused to one or more additional peptide or polypeptide.
  • RRE RIPP recognition element
  • the one or more additional peptide or polypeptide comprises a biologically active peptide or polypeptide.
  • biologically active peptide or polypeptide that can be fused with a RRE according to the present disclosure include but are not limited to (i) a peptide or polypeptide capable of modulating the reaction catalyzing activity of the lasso peptidase or lasso cyclase; (ii) a peptide or polypeptide capable of modulating target specificity of the lasso peptidase or lasso cyclase; (iii) an enzyme having the same or different enzymatic activity as the lasso peptidase or lasso cyclase; or any combination of (i) to (iii).
  • the lasso precursor peptide genes or lasso core peptide genes are fused at the 5’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide tags for affinity purification or immobilization, including his-tags, a strep- tags, or FLAG-tags.
  • the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus of the core peptides to form conjugates with other peptides or proteins, with or without a linker, such as peptide tags for affinity purification or immobilization, including his-tags, a strep-tags, or FLAG-tags.
  • a linker such as peptide tags for affinity purification or immobilization, including his-tags, a strep-tags, or FLAG-tags.
  • lasso precursor peptides, lasso core peptides, or lasso peptides are fused to molecules that can enhance cell permeability or penetration into cells, for example through the use of arginine-rich cell-penetrating peptides such as TAT peptide, penetratin, and flock house virus (FHV) coat peptide (Brock, R., Bioconjug. Chem., 2014, 25, 863–868).
  • FHV flock house virus
  • a lasso precursor peptide gene or core peptide gene is fused at the 3’-terminus to oligonucleotide sequences that encode arginine-rich cell- penetrating peptides or proteins, including oligonucleotide sequences that encode penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups (Wender, P.A., et al., Adv. Drug Deliv. Rev., 2008, 60, 452–472).
  • FHV flock house virus
  • a lasso precursor peptide, lasso core peptide, or lasso peptide is fused at the C-terminus to peptides that promote cell penetration such as arginine-rich cell-penetrating peptides or proteins, including amino acid sequences that encode TAT peptide, penetratin, and flock house virus (FHV) coat peptide or similar peptides that contain guanidinium groups or a combination of lysine and guanidinium groups.
  • FHV flock house virus
  • the lasso precursor peptide genes or lasso core peptide genes are fused at the 5’-terminus of the DNA template strand of the gene to oligonucleotide sequences that encode peptides or proteins, with or without a linker, such as sequences encoding peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, integrin ligand binding epitopes, and the like.
  • the lasso precursor peptides, lasso core peptides, or lasso peptides are fused at the C-terminus to peptides or proteins, with or without a linker, such as peptide epitopes that are known to bind with high affinity to antibodies, cell surface proteins, or cell surface receptors, including cytokine binding epitopes, integrin ligand binding epitopes, and the like.
  • the lasso peptide biosynthesis component comprises a lasso cyclase.
  • the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a third nucleic acid sequence encoding the lasso peptidase.
  • the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase and a fourth nucleic acid sequence encoding the RRE.
  • the lasso peptide biosynthesis component comprises a lasso peptidase, a lasso cyclase and a post-translationally modified peptide (RiPP) recognition element (RRE)
  • the method comprises introducing the second nucleic acid sequence encoding the lasso cyclase, a third nucleic acid sequence encoding the lasso peptidase, and a fourth nucleic acid sequence encoding the RRE.
  • the lasso peptide producers can be cultured for the biosynthetic production of lasso peptide.
  • host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes.
  • Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Streptomyces albus, Clostridium acetobutylicum, Vibrio natriegens, Pseudomonas 104 ACTIVE 705331286v1 fluorescens, and Pseudomonas putida.
  • Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like.
  • E. coli is a particularly useful host organisms since it is a well characterized microbial organism suitable for genetic engineering.
  • Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae.
  • Sources of encoding nucleic acids for a lasso peptide pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction.
  • Such species include both prokaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria
  • Exemplary species for such sources include, for example, Escherichia coli, Acidaminococcus fermentans, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. ADP1, Acinetobacter sp. Strain M-1, Aquifex aeolicus, Arabidopsis thaliana, Arabidopsis thaliana col, Arabidopsis thaliana col, Archaeoglobus fulgidus DSM 4304, Azoarcus sp.
  • MG1655 Eubacterium rectale ATCC 33656, Fusobacterium nucleatum, Fusobacterium nucleatum subsp. nucleatum ATCC 25586, Geobacillus thermoglucosidasius, Haematococcus pluvialis, Haemophilus influenzae, Haloarcula marismortui ATCC 43049, Helicobacter pylori, Homo sapiens, Klebsiella pneumoniae, Lactobacillus plantarum, Leuconostoc mesenteroides, marine gamma proteobacterium HTCC2080, Metallosphaera sedula, Methanocaldococcus jannaschii, Mus musculus, Mycobacterium avium subsp.
  • paratuberculosis K-10 Mycobacterium bovis BCG, Mycobacterium marinum M, Mycobacterium smegmatis MC2155, Mycobacterium tuberculosis, Mycoplasma pneumoniae M129, Nocardia farcinica IFM 10152, Nocardia 105 ACTIVE 705331286v1 iowensis (sp.
  • NRRL 5646 Oryctolagus cuniculus, Paracoccus denitrificans, Penicillium chrysogenum, , Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens, Pseudomonas fluorescens Pf-5, Pseudomonas knackmussii (B13), Pseudomonas putida, Pseudomonas putida E23, Pseudomonas putida KT2440, Pseudomonas sp, Pyrobaculum aerophilum str.
  • strain PCC6803 Syntrophus , ciditrophicus, Thermoanaerobacter brockii HTD4, Thermoanaerobacter tengcongensis MB4, Thermosynechococcus elongates, Thermotoga maritime MSB8, Thermus thermophilus, Thermus, hermophilus HB8, Trichomonas vaginalis G3, Trichosporonoides megachiliensis, Trypanosoma brucei, Tsukamurella paurometabola DSM 20162, Vibrio natriegens, Yersinia intermedia ATCC 29909, Zoogloea ramigera, Zygosaccharomyces rouxii, Zymomonas mobilis, as well as other exemplary species disclosed herein are available as source organisms for corresponding genes.
  • lasso peptide biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced 106 ACTIVE 705331286v1 reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ.
  • teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize a lasso peptide.
  • Methods for constructing and testing the expression levels of a non-naturally occurring lasso peptide-producing host can be performed, for example, by recombinant and detection methods well known in the art.
  • Exogenous nucleic acid sequences involved in a pathway for production of a lasso peptide can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E.
  • nucleic acid sequences in the genes can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired.
  • genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the active proteins.
  • An expression vector or vectors can be constructed to include one or more lasso peptide biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism.
  • Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.
  • Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • PCR polymerase chain reaction
  • immunoblotting for expression of gene products
  • expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.
  • anaerobic conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap.
  • microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration.
  • Exemplary anaerobic conditions have been described previously and are well-known in the art.
  • Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.
  • the growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism.
  • Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch.
  • Other sources of carbohydrate include, for example, renewable feedstocks and biomass.
  • Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks.
  • Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch.
  • the cell-free or cell-based biosynthesis system e.g., a CFB reaction mixture of cell culture
  • the cell-free or cell-based biosynthesis system can be maintained under aerobic or substantially aerobic conditions, where such conditions can be achieved, for example, by sparging with air or oxygen, shaking under an atmosphere of air or oxygen, stirring under an atmosphere of air or oxygen, or combinations thereof.
  • the cell-free or cell-based biosynthesis system for lasso peptides and lasso peptide analogs can be maintained under anaerobic or substantially anaerobic conditions, where such conditions can be achieved, for example, by first sparging the medium with nitrogen and then sealing the wells or reaction containers, or by shaking or stirring under a nitrogen atmosphere.
  • anaerobic conditions refer to an environment devoid of oxygen.
  • substantially anaerobic conditions include, for example, biosynthesis processes conducted such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation.
  • Substantially anaerobic conditions also include performing the biosynthesis methods and processes inside a sealed chamber maintained with an atmosphere of less than 1% oxygen.
  • the percent of oxygen can be maintained by, for example, sparging the CFB reaction or cell culture with an N 2 /CO 2 mixture or other suitable non-oxygen gas or gases.
  • the pH of the cell culture medium or CFB reaction mixture, including cell extracts, used in the biosynthesis methods and systems, provided herein for the synthesis of lasso peptides and related molecules thereof can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a buffer, a base, such as NaOH or other bases, or an acid, as needed to maintain the production system at a desirable pH for high rates and yields in the production of lasso peptides and related molecules thereof.
  • a desired pH in particular neutral pH, such as a pH of around 7 by addition of a buffer, a base, such as NaOH or other bases, or an acid, as needed to maintain the production system at a desirable pH for high rates and yields in the production of lasso peptides and related molecules thereof.
  • the cell culture medium or CFB reaction mixture including cell extracts, used in the CFB methods and systems, provided herein for the synthesis of lasso peptides and related molecules thereof can be supplemented with one or more enzymes (or the nucleic acids that encode them) of central metabolism pathways, for example, one or more (or all of the) central metabolism enzymes from the tricarboxylic acid cycle (TCA, or Krebs cycle), the glycolysis pathway or the Citric Acid Cycle, or enzymes that promote the production of amino acids.
  • TCA tricarboxylic acid cycle
  • Citric Acid Cycle a tric Acid Cycle
  • Metabolic modeling and simulation algorithms can be utilized to optimize conditions for the present biosynthesis process and to optimize lasso peptide production rates and yields in the cell-free or cell-based system. Modeling can also be used to design gene knockouts that additionally optimize utilization of the lasso peptide pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No.7,127,379).
  • OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable metabolic network which overproduces the target product.
  • the framework examines the complete metabolic and/or biochemical network in order to suggest genetic manipulations that lead to maximum production of a lasso peptide or related molecules thereof.
  • OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism.
  • the OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data.
  • OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions.
  • OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems.
  • the metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 110 ACTIVE 705331286v1 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. publication 2009/0047719, filed August 10, 2007.
  • SimPheny® Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®.
  • This computational method and system is described in, for example, U.S. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003.
  • SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system.
  • constraints-based modeling This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.
  • 111 ACTIVE 705331286v1 6.6 Methods for Screening Lasso Peptides
  • methods for screening products produced by the methods and workflow provided herein including methods for screening lasso peptide and/or lasso peptide analogs for those with desirable properties, such as therapeutic properties.
  • the target is a cell surface molecule.
  • binding of the lasso peptide or lasso peptide analog to the target activates a signaling pathway in a cell. In some embodiments, binding of the lasso peptide or lasso peptide analog to the target inhibits a cellular signaling pathway. In some embodiments, the cellular signaling pathway can be intracellular and/or intercellular. In some embodiments, the activation and/or inhibition of the cellular signaling pathway is useful for treating or preventing a diseased condition in the cell. Accordingly, lasso peptides and lasso peptide analogs screened and selected herein can be suitable for treating or preventing the diseased condition in a subject.
  • the method for screening lasso peptides or lasso peptide analogs comprises contacting a candidate lasso peptide with a target; and measuring the binding affinity between the lasso peptide or lasso peptide analog and the target.
  • the target is in purified form. In other embodiments, the target is present in a sample.
  • the method for screening lasso peptides or lasso peptide analogs comprises contacting a candidate lasso peptide with a cell expressing the target; and detecting a signal associated with a cellular signaling pathway of interest from the cell.
  • the signaling pathway is inhibited by a candidate lasso peptide or lasso peptide analog. In other embodiments, the signaling pathway is activated by a candidate lasso peptide or lasso peptide analog.
  • the target is G protein- couple receptors (GPCRs). In other embodiments, the target is an ion channel. In other embodiments, the target is a membrane protein.
  • the method for screening lasso peptides or lasso peptide analogs comprises contacting a candidate lasso peptide with a subject expressing the target; and measuring a signal associated with a phenotype of interest from the subject.
  • the phenotype is a disease phenotype.
  • binding of the lasso peptide or lasso peptide analog to the target facilitates delivery of the lasso peptide or lasso peptide analog to the target.
  • the method for screening lasso peptides or lasso peptide analogs comprises contacting a candidate lasso peptide or lasso peptide analog with a target; and detecting localization of the lasso peptide or lasso peptide analog near the target.
  • the lasso peptide or lasso peptide analog is comprised within a larger molecule and detecting localization of the lasso peptide or lasso peptide analog is performed by detecting the localization of such larger molecule or a portion thereof.
  • the larger molecule is a conjugate, a complex or a fusion molecule comprising the lasso peptide or lasso peptide analog.
  • detecting localization of the larger molecule comprising the lasso peptide or lasso peptide analog is performed by detecting a signal produced by such larger molecule.
  • detecting localization of the larger molecule comprising the lasso peptide or lasso peptide analog is performed by detecting an effect produced by such larger molecule.
  • the larger molecule comprises the lasso peptide and a therapeutic agent, and detecting localization of the larger molecule is performed by detecting a therapeutic effect of the therapeutic agent.
  • the therapeutic effect is in vivo. In other embodiments, the therapeutic effect is in vitro. Accordingly, lasso peptides and lasso peptide analogs screened and selected herein can be suitable for targeted delivery of a therapeutic agent to a target location within a subject.
  • binding of the lasso peptide or lasso peptide analog to the target facilitates purifying the target from the sample.
  • the target is comprised in a sample, and binding of the lasso peptide or lasso peptide analog to the target facilitates detecting the target from the sample.
  • detecting the target from the sample is indicative of the presence of a phenotype of interest in a subject providing the sample.
  • the phenotype is a diseased phenotype. Accordingly, lasso peptides and lasso peptide analogs screened and selected herein can be suitable for diagnosing the disease from a subject.
  • any method for screening for a desired enzyme activity e.g., production of a desired product, e.g., such as a lasso peptide or lasso peptide analog
  • a desired product e.g., such as a lasso peptide or lasso peptide analog
  • Any method for isolating enzyme products or final products e.g., lasso peptides or lasso peptide analogs, can be used.
  • methods and compositions of the invention comprise use of any method or apparatus to detect a purposefully biosynthesized organic product, e.g., lasso peptide or lasso peptide analog, or supplemented 113 ACTIVE 705331286v1 or microbially-produced organic products (e.g., amino acids, CoA, ATP, carbon dioxide), by e.g., employing invasive sampling of either cell extract or headspace followed by subjecting the sample to gas chromatography or liquid chromatography often coupled with mass spectrometry.
  • a purposefully biosynthesized organic product e.g., lasso peptide or lasso peptide analog, or supplemented 113 ACTIVE 705331286v1 or microbially-produced organic products (e.g., amino acids, CoA, ATP, carbon dioxide)
  • microbially-produced organic products e.g., amino acids, CoA, ATP, carbon dioxide
  • the methods of screening lasso peptides and lasso peptide analogs comprises screening lasso peptides and lasso peptide analogs from a lasso peptide library as provided herein.
  • the apparatus and instruments are designed or configured for High Throughput Screening (HTS) and analysis of products, e.g., lasso peptides or lasso peptide analogs, produced by CFB methods and processes as provided herein, by detecting and/or measuring the products, e.g., lasso peptides, either directly or indirectly, in soluble form by sampling a CFB cell-free extract or culture broth.
  • HTS High Throughput Screening
  • lasso peptide production methods such as cell-free or cell-based methods and processes, are automatable and suitable for use with laboratory robotic systems, eliminating or reducing operator involvement, while providing for high- throughput biosynthesis and screening.
  • the activity can be for a pharmaceutical, agricultural, nutraceutical, nutritional or animal veterinary or health and wellness function.
  • Also provided are methods screening for: a modulator of protein activity, transcription, or translation or cell function; a toxic metabolite or a protein; a cellular toxin; an inhibitor or of transcription or translation comprising: (a) providing a CFB method and a cell extract or TX-TL composition or culture broth described herein, wherein the composition comprises at least one protein-encoding nucleic acid; (b) providing a test compound; (c) combining or mixing the test compound with the cell extract or culture medium under conditions wherein the TX-TL extract or culture broth initiates or completes transcription and/or translation, or modifies a molecule (optionally a protein, a small molecule, a natural product, natural product analog, a lasso peptide, or a lasso peptide analog) and (d) determining or measuring any change in the functioning or products of the extract, or the transcription and/or translation, wherein determining or measuring a change in the protein activity, transcription or translation or cell function identifies the test compound as
  • Suitable purification and/or assays to test for the production of lasso peptides or lasso peptide analogs can be performed using well known methods. Suitable replicates such as triplicate CFB reactions or cell-based reactions, can be conducted and analyzed to verify lasso peptide production and concentrations.
  • the final lasso peptide product and any intermediates, and other organic compounds can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectrometry), LC-MS (Liquid Chromatography-Mass Spectrometry), MALDI or other suitable analytical methods using routine procedures well known in the art.
  • HPLC High Performance Liquid Chromatography
  • GC-MS Gas Chromatography-Mass Spectrometry
  • LC-MS Liquid Chromatography-Mass Spectrometry
  • MALDI Liquid Chromatography-Mass Spectrometry
  • Byproducts and residual amino acids or glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and saturated fatty acids, and a UV detector for amino acids and other organic acids (Lin et al., Biotechnol. Bioeng., 2005, 90, 775-779), or other suitable assay and detection methods well known in the art.
  • the individual enzyme or protein activities from the exogenous or endogenous DNA sequences can also be assayed using 115 ACTIVE 705331286v1 methods well known in the art.
  • the activity of phenylpyruvate decarboxylase can be measured using a coupled photometric assay with alcohol dehydrogenase as an auxiliary enzyme (See: Weiss et al., Biochem, 1988, 27, 2197-2205).
  • NADH- and NADPH- dependent enzymes such as acetophenone reductase can be followed spectrophotometrically at 340 nm (See: Sch Kunststoffen et al, J. Mol. Biol., 2005, 349, 801-813).
  • Lasso peptides and lasso peptide analogs can be isolated, separated purified from other components in the CFB reaction mixtures or culture broth using a variety of methods well known in the art.
  • Such separation methods include, for example, extraction procedures, including extraction of CFB reaction mixtures, or culture broth, or fermentation cell mass, using organic solvents such as methanol, butanol, ethyl acetate, and the like, as well as methods that include continuous liquid-liquid extraction, solid-liquid extraction, solid phase extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, dialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, ultrafiltration, medium pressure liquid chromatography (MPLC), and high pressure liquid chromatography (HPLC).
  • extraction procedures including extraction of CFB reaction mixtures, or culture broth, or fermentation cell mass, using organic solvents such as methanol, butanol, ethyl acetate, and the like
  • organic solvents such as methanol, butanol, ethyl acetate, and the like
  • methods that include continuous liquid
  • lasso peptides may be modified by engineering the sequence of a lasso peptide to incorporate a binding epitope from a natural or non-natural ligand that is known to bind to a receptor of interest (FIG.3A and FIG.3B).
  • a binding epitope is introduced into a lasso peptide by inserting into or extending the length of a natural lasso peptide sequence to include the binding epitope sequence.
  • the binding epitope is inserted into the ring of a lasso peptide, which leads to a ring that is expanded by the inserted epitope sequences.
  • the binding epitope is inserted into the loop of a lasso peptide, which leads to a loop that is expanded by 116 ACTIVE 705331286v1 the inserted epitope sequences.
  • the binding epitope Arg-Gly-Asp can be inserted into an eight-residue lasso peptide loop to create and eleven-residue loop containing the epitope.
  • the binding epitope is inserted into the tail of a lasso peptide, which leads to a tail that is expanded by the inserted epitope sequences.
  • the binding epitope is attached to the C-terminus of the tail of a lasso peptide, which leads to a tail that is extended by the inserted epitope sequences.
  • the binding epitope is introduced into the ring of a lasso peptide by replacing natural ring residues with the epitope sequence residues, which leads to a ring that is the same size but is mutated to incorporate the epitope sequences.
  • the binding epitope Arg-Gly-Asp RGD
  • RGD can be introduced into an eight-residue lasso peptide ring that lacks RGD, leading to a lasso peptide that contains an eight-residue ring containing the RGD epitope.
  • the binding epitope is introduced into the loop of a lasso peptide by replacing natural loop residues with the epitope sequence residues, which leads to a loop that is the same size but is mutated to incorporate the epitope sequences.
  • the binding epitope is introduced into the tail of a lasso peptide by replacing natural tail residues with the epitope sequence residues, which leads to a tail that is the same size but is mutated to incorporate the epitope sequences.
  • a linear or continuous binding epitope is introduced into a lasso peptide by inserting into or extending the length of a natural lasso peptide sequence to include the binding epitope sequence.
  • the linear or continuous binding epitope is inserted into the ring of a lasso peptide, which leads to a ring that is expanded by the inserted epitope sequences.
  • the linear or continuous binding epitope is inserted into the loop of a lasso peptide, which leads to a loop that is expanded by the inserted epitope sequences.
  • the binding epitope Arg-Gly-Asp RGD
  • RGD can be inserted into an eight-residue lasso peptide loop to create and eleven-residue loop containing the epitope.
  • the linear or continuous binding epitope is inserted into the tail of a lasso peptide, which leads to a tail that is expanded by the inserted epitope sequences.
  • the linear or continuous binding epitope is attached to the C-terminus of the tail of a lasso peptide, which leads to a tail that is extended by the inserted epitope sequences.
  • the linear or continuous binding epitope is introduced into the ring of a lasso peptide by replacing natural ring residues with the epitope sequence residues, which leads to a ring that is the same size but is mutated to incorporate the epitope sequences.
  • the binding epitope Arg-Gly-Asp can be introduced into an eight-residue lasso peptide ring that lacks RGD, leading to a 117 ACTIVE 705331286v1 lasso peptide that contains an eight-residue ring containing the RGD epitope.
  • the linear or continuous binding epitope is introduced into the loop of a lasso peptide by replacing natural loop residues with the epitope sequence residues, which leads to a loop that is the same size but is mutated to incorporate the epitope sequences.
  • the linear or continuous binding epitope is introduced into the tail of a lasso peptide by replacing natural tail residues with the epitope sequence residues, which leads to a tail that is the same size but is mutated to incorporate the epitope sequences.
  • a conformational or discontinuous binding epitope is introduced into a lasso peptide by inserting into or extending the length of a natural lasso peptide sequence to include the binding epitope sequence.
  • the conformational or discontinuous binding epitope is inserted into the ring and loop of a lasso peptide, which leads to a ring and loop that are expanded by the inserted epitope sequences.
  • the conformational or discontinuous binding epitope is inserted into the loop and tail of a lasso peptide, which leads to a loop and tail that are expanded by the inserted epitope sequences.
  • the conformational or discontinuous binding epitope is inserted into the ring and tail of a lasso peptide, which leads to a ring and tail that are expanded by the inserted epitope sequences.
  • the conformational or discontinuous binding epitope is introduced into the ring and loop of a lasso peptide by replacing natural ring residues with the epitope sequence residues, which leads to a ring and loop that are the same size but are mutated to incorporate the epitope sequences.
  • the conformational or discontinuous binding epitope is introduced into the loop and tail of a lasso peptide by replacing natural loop and residues with the epitope sequence residues, which leads to a loop and tail that are the same size but is mutated to incorporate the epitope sequences.
  • the conformational or discontinuous binding epitope is introduced into the ring and tail of a lasso peptide by replacing natural ring and tail residues with the epitope sequence residues, which leads to a ring and tail that are the same size but is mutated to incorporate the epitope sequences [00335]
  • two different binding epitopes from the same or different ligands are introduced into a lasso peptide.
  • a binding epitope from ligand 1 is introduced into the loop of a lasso peptide, and the binding epitope from ligand 2, which binds to the same or a different receptor, is introduced into the tail of the same lasso peptide.
  • a binding epitope from ligand 1 is introduced into the ring of a lasso peptide, and the binding epitope from ligand 2, which binds to the same or a different receptor, is introduced into the tail of the same lasso peptide.
  • a binding epitope from ligand 1 is introduced into the loop of a lasso peptide, and the binding epitope from ligand 2, which binds to the same or a different receptor, is introduced into the ring of the same lasso peptide.
  • a set of nucleic acids encoding the desired activities of a lasso peptide biosynthesis pathway can be introduced into a host organism to produce a lasso peptide or can be introduced into a cell-free biosynthesis reaction mixture containing a cell extract or other suitable medium to produce a lasso peptide.
  • mutations can be introduced into an encoding nucleic acid molecule (e.g., a gene), which ultimately leads to a change in the amino acid sequence of a protein, enzyme, or peptide, and such mutated proteins, enzymes, or peptides can be screened for improved properties.
  • Such optimization methods can be applied, for example, to increase or improve the activity or substrate scope of an enzyme, protein, or peptide and/or to decrease an inhibitory activity.
  • Lasso peptides are derived from precursor peptides that are ribosomally produced by transcription and translation of a gene encoding the linear precursor peptide.
  • Ribosomally produced peptides such as lasso precursor peptides
  • Ribosomally produced peptides are known to be readily evolved and optimized through variation of nucleotide sequences within genes that encode for the amino acid residues that comprise the peptide.
  • Large libraries of peptide mutational variants have been produced by methods well known in the art, and some of these methods are referred to as directed or random evolution.
  • Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene, specific oligonucleotide sequences within a gene (e.g., oligonucleotides corresponding to specific amino acid residues in the peptide product), or an oligonucleotide sequence containing a gene, in order to improve and/or alter the properties or production of an enzyme, protein or peptide (e.g., a lasso peptide).
  • Improved and/or altered enzymes, proteins or peptides can be identified through the development and implementation of sensitive high-throughput assays that allow automated screening of many enzyme or peptide variants (for example, >10 4 ).
  • Enzyme and protein characteristics that have been improved and/or altered by directed and random evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K m ), including broadening of ligand or substrate binding to include non-natural substrates; inhibition (Ki), to remove inhibition by products, substrates, or key intermediates; activity (k cat ), to increase enzymatic reaction rates to achieve desired flux; isoelectric point (pI) to improve protein or peptide solubility; acid dissociation (pK a ) to vary the ionization state of the protein or peptide with repect to pH; expression levels, to increase protein or peptide yields and overall pathway flux; oxygen stability, for operation of air-sensitive enzymes or peptides under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme or peptide in the
  • a number of exemplary methods have been developed for the mutagenesis and diversification of genes and oligonucleotides to introduce desired properties into specific enzymes, proteins and peptides. Such methods are well-known to those skilled in the art and allow either single-site or multi-site mutagenesis of a gene. Any of these can be used to alter and/or optimize the activity of a lasso peptide biosynthetic pathway enzyme, protein, or peptide, including a lasso precursor peptide, a lasso core peptide, or a lasso peptide.
  • EpPCR error-prone polymerase chain reaction
  • epRCA Error-prone Rolling Circle Amplification
  • DNA, Gene, or Family Shuffling typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc. Natl. Acad. Sci.
  • Staggered Extension which entails template priming followed by repeated cycles of 2-step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol., 1998,16, 258-261); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Res.,1998, 26, 681-683).
  • Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (See: Volkov et al, Nucleic Acids Res., 1999, 27:e18; Volkov et al., Methods Enzymol., 2000, 328, 456-463); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single-stranded DNA (ssDNA) (See: Coco et al., Nat.
  • ITCHY Incremental Truncation for the Creation of Hybrid Enzymes
  • THIO-ITCHY Thio- Incremental Truncation for the Creation of Hybrid Enzymes
  • THIO-ITCHY Thio- Incremental Truncation for the Creation of Hybrid Enzymes
  • phosphothioate dNTPs are used to generate truncations
  • SCRATCHY which combines two methods for recombining genes, ITCHY and DNA Shuffling (See: Lutz et al., Proc. Natl. Acad. Sci.
  • Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothiolate nucleotide and cleavage, which is used as a template to extend in the presence of “universal” bases such as inosine, and replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (See: Wong et al., Biotechnol. J., 2008, 3, 74-82; Wong et al., Nucleic Acids Res., 2004, 32, e26; Wong et al., Anal.
  • Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (See: Sieber et al., Nat.
  • SHIPREC Sequence Homology-Independent Protein Recombination
  • GSSMTM Gene Site Saturation MutagenesisTM
  • the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations, enabling all amino acid variations to be introduced individually at each position of a protein or peptide
  • dsDNA supercoiled double stranded DNA
  • CCM Combinatorial Cassette Mutagenesis
  • CMCM Combinatorial Multiple Cassette Mutagenesis
  • LTM Look-Through Mutagenesis
  • Saturation mutagenesis at multiple sites within a protein or peptide simultaneously allows for the production of random mutagenesis libraries with the potential to identify synergistic combinations of mutations to improve performance and/or properties.
  • a rapid way to create and screen very large random libraries of diverse peptides involves the use of display technologies (For a review, see: Ullman, C.G., et al., Briefings Functional Genomics, 2011, 10, 125-134).
  • Peptide display technologies offer the benefit that specific peptide encoding information (e.g., RNA or DNA sequence information) is linked to, or otherwise associated with, each corresponding peptide in a library, and this information is accessible and readable (e.g., by amplifying and sequencing the attached DNA oligonucleotide) after a screening event, thus enabling identification of the individual peptides within a large library that exhibit desirable properties (e.g., high binding affinity).
  • the cell-free biosynthesis methods provided herein can facilitate and enable the creation of large lasso peptide libraries containing lasso peptide analogs that can be screened for 123 ACTIVE 705331286v1 favorable properties.
  • Lasso peptide mutants that exhibit the desired improved properties (hits) may be subjected to additional rounds of mutagenesis to allow creation of highly optimized lasso peptide variants.
  • the evolution of lasso peptides also can be conducted using chemical synthesis methods.
  • large combinatorial peptide libraries containing mutational variants can be synthesized by using known solution phase or solid phase peptide synthesis technologies (See review: Shin, D.-S., et al., J. Biochem. Mol. Bio., 2005, 38, 517-525).
  • Chemical peptide synthesis methods can be used to produce lasso precursor peptide variants, or alternatively, lasso core peptide variants, containing a wide range of alpha-amino acids, including the natural proteinogenic amino acids, as well as non-natural and/or non-proteinogenic amino acids, such as amino acids with non-proteinogenic side chains, or alternatively D-amino acids, or alternatively beta-amino acids.
  • Cyclization of these chemically synthesized lasso precursor peptides or lasso core peptides can provide vast lasso peptide diversity that incorporates stereochemical and functional properties not seen in natural lasso peptides.
  • Any of the aforementioned methods for lasso peptide mutagenesis and/or display can be used alone or in any combination to evolve and improve the performance of lasso peptide biosynthesis pathway enzymes, proteins, and peptides.
  • any of the aforementioned methods for mutagenesis and/or display can be used alone or in any combination to enable the creation of lasso peptide variants which may be selected for improved properties.
  • lasso peptide precursor peptides are evolved whereby mutants of a parent lasso peptide precursor peptide are created and converted by a lasso peptidase and a lasso cyclase into lasso peptide variants that are screened for improved properties.
  • multiple sites of a parent lasso peptide precursor peptide are varied simultaneously to generate a directed or random library of mutants that are screened for improved properties (FIG.12).
  • mutational variants or a mutational library of lasso core peptides is created and converted by a lasso cyclase into a library of lasso peptide variants that are screened for improved properties.
  • 124 ACTIVE 705331286v1 6.8.3 Chemical or Enzymatic Modification [00346]
  • one or more components function to modify the lasso peptide or lasso peptide analog produced by a cell-free or cell-based method or process.
  • the lasso peptides or lasso peptide analogs produced by cell-free or cell- based method or process are chemically modified.
  • the lasso peptides or lasso peptide analogs produced by cell-free or cell-based method or process are enzymatically modified.
  • the core peptides or the lasso peptides produced by cell-free or cell-based methods or processes are modified further through chemical steps.
  • the core peptides or the lasso peptides produced by cell-free or cell-based methods or processes are modified through chemical steps that allow the attachment of chemical linker units connected to small molecules to the C-terminus of the core peptide or the lasso peptide.
  • the core peptides or the lasso peptides produced by cell-free or cell-based methods or processes are modified through the attachment of chemical linkers connected to small molecules to the side chain of functionalized amino acids (e.g., the OH or serine, threonine, or tyrosine, or the N of lysine).
  • the lasso core peptides or the lasso peptides produced by cell-free or cell-based methods or processes are modified further through chemical steps.
  • the lasso core peptides or the lasso peptides produced by cell-free or cell-based methods or processes are modified by PEGylation.
  • the lasso core peptides or the lasso peptides produced by cell-free or cell-based methods or processes are modified by biotinylation.
  • the lasso core peptides or the lasso peptides produced by cell-free or cell-based methods or processes are modified through the formation of esters, sulfonyl esters, phosphonate esters, or amides by reaction with the side chain of functionalized amino acids (e.g., the OH or serine, threonine, or tyrosine, or the N of lysine).
  • the core peptides or the lasso peptides produced by cell-free or cell-based methods or processes may contain non-natural amino acids which are modified further through chemical steps.
  • the core peptides or the lasso peptides produced by cell-free or cell-based methods or processes may contain non-natural amino acids which are modified through the use of click chemistry involving amino acids with azide or alkyne functionality within the side chains (Presolski, S.I., et al., Curr Protoc Chem Biol., 2011, 3, 153–162).
  • the core peptides or the lasso peptides produced by cell-free or cell- based methods or processes may contain non-natural amino acids which are modified further 125 ACTIVE 705331286v1 through metathesis chemistry involving alkene or alkyne groups within the amino acid side chains (Cromm, P.M., et al., Nat. Comm., 2016, 7, 11300; Gleeson, E.C., et al., Tetrahedron Lett., 2016, 57, 4325–4333).
  • the lasso peptide or lasso peptide analogs generated by cell-free or cell-based methods or processes are modified chemically or by enzyme modification.
  • Exemplary modifications to the lasso peptide or lasso peptide analogs include but are not limited to halogenation, lipidation, pegylation, glycosylation, adding hydrophobic groups, myristoylation, palmitoylation, isoprenylation, prenylation, lipoylation, adding a flavin moiety (optionally comprising addition of: a flavin adenine dinucleotide (FAD) an FADH 2 , a flavin mononucleotide (FMN), an FMNH 2 ), phospho-pantetheinylation, heme C addition, phosphorylation, acylation, alkylation, butyrylation, carboxylation, malonylation, hydroxylation, adding a halide group, iodination, propionylation, S-glutathionylation, succinylation, glycation, adenylation, thiolation, condensation (optionally the “condensation” comprising addition of: an amino
  • the enzymes comprise one or more central metabolism enzyme (optionally tricarboxylic acid cycle (TCA, or Krebs cycle) enzymes, glycolysis enzymes or Pentose Phosphate Pathway enzymes), and optionally the chemical or enzyme modification comprises addition, deletion or replacement of a substituent or functional groups, optionally a hydroxyl group, an amino group, a halogen, an alkyl or a cycloalkyl group, optionally by hydration, biotinylation, hydrogenation, an aldol condensation reaction, condensation polymerization, halogenation, oxidation, dehydrogenation, or creating one or more double bonds.
  • a substituent or functional groups optionally a hydroxyl group, an amino group, a halogen, an alkyl or a cycloalkyl group, optionally by hydration, biotinylation, hydrogenation, an aldol condensation reaction, condensation polymerization, halogenation, oxidation, dehydrogenation, or creating one or more double bonds.
  • cell-free or cell-based methods or processes are used to facilitate the creation of mutational variants of lasso peptides using the above method. For example, in some embodiments, the synthesis of codon mutants of the core lasso peptide gene sequence which are used in the cell-free or cell-based methods or processes, thus enabling the creation of high-density lasso peptide diversity libraries.
  • cell-free or cell-based methods or processes are used to facilitate the creation of large mutational lasso peptide libraries using, for example, using site-saturation mutagenesis and recombination 126 ACTIVE 705331286v1 methods or in vitro display technologies (Josephson, K., et al., Drug Discov. Today,.2014, 19, 388-399; Doi, N., et al., PLoS ONE, 2012, 7, e30084, pp 1-8; Josephson, K., et al., J. Am. Chem.
  • cell-free or cell-based methods or processes are used to facilitate the creation of mutational variants of lasso peptides by introducing non-natural amino acids into the core peptide sequence, through either biological or chemical means, followed by formation of the lasso structure using the cell-free or cell-based methods or processes involving, at minimum, a lasso cyclase gene or a lasso cyclase for lasso peptide production as described above.
  • FIG.2A Design-Build-Test-Evolve iterative workflow that is used to obtain optimized lasso peptides.
  • FIG. 16 a method and iterative workflow for the generation of lasso peptides and lasso peptide analogs that are designed and optimized for specific biological and physicochemical properties.
  • a lasso peptide is designed using computational methods, produced using cell-free or cell-based methods, tested using assays that allow assessment of functional performance or other properties of interest, and evolved using peptide mutagenesis methods to optimize lasso peptides and lasso peptide analogs.
  • a lasso peptide is subjected to a workflow whereby the lasso peptide is designed using computational methods, produced using cell-free or cell-based methods, tested using assays that allow assessment of functional performance or other properties of interest, evolved using peptide mutagenesis methods, and subsequently the evolved peptide is subjected to the iterative workflow one or more additional times for optimization of functional performance or other properties of interest.
  • an iterative workflow is implemented which involves computational design, production, testing, and evolution of lasso peptides.
  • this iterative workflow is referred to as a Design-Build-Test-Evolve (DBTE) workflow.
  • DBTE Design-Build-Test-Evolve
  • this DBTE workflow is used for one cycle to obtain a lasso peptide with properties and activities that are improved or optimized for a given purpose.
  • this DBTE workflow is used for two cycles to obtain a lasso peptide with properties and activities that are improved or optimized for a given purpose.
  • this DBTE workflow is used for three or more cycles to obtain a lasso peptide with properties and activities that are improved or optimized for a given purpose.
  • a lasso peptide is designed using computational methods, predicted to bind to a specific biological target of interest, scored and ranked for binding affinity versus other lasso peptides, produced using cell-free or cell-based methods, tested using assays that allow assessment of the designed lasso peptide for potency and selectivity in binding to the biological target, and evolved using peptide mutagenesis methods and tested to optimize properties such as binding affinity, binding selectivity, ligand-induced pathway activation or deactivation, disease-associated efficacy, in vivo properties, solubility, cell penetration, etc.
  • a lasso peptide is designed using computational methods and predicted to bind to a specific biological target of interest, produced using cell- free or cell-based methods, tested using assays that allow assessment of the designed lasso peptide for potency and selectivity in binding to the biological target, and the lasso peptide is re-subjected to computational methods and tested to optimize properties such as binding affinity, binding selectivity, ligand-induced pathway activation or deactivation, disease- associated efficacy, in vivo properties, solubility, cell penetration, etc. based on the testing results.
  • a lasso peptide is designed using computational methods and predicted to bind to a specific biological target of interest, produced using cell-free or cell- based methods, tested using assays that allow assessment of the designed lasso peptide for potency and selectivity in binding to the biological target, and evolved using peptide mutagenesis methods and tested to optimize properties such as binding affinity, binding selectivity, ligand-induced pathway activation or deactivation, disease-associated efficacy, in vivo properties, solubility, cell penetration, etc, and the highest ranked binders are subjected to the iterative workflow one or more additional times for optimization of functional performance or other properties of interest.
  • a plurality of lasso peptides are designed using computational methods, predicted to bind to a specific biological target of interest, scored and 128 ACTIVE 705331286v1 ranked for binding affinity versus other lasso peptides, the top 2 up to 1000 of the highest ranked lasso peptide variants are produced using cell-free or cell-based methods, tested using assays that allow assessment of the designed lasso peptide for potency and selectivity in binding to the biological target, and lasso peptides with the highest binding affinities are evolved using peptide mutagenesis methods and tested to optimize properties such as binding affinity, binding selectivity, ligand-induced pathway activation or deactivation, disease- associated efficacy, solubility, cell penetration, etc.
  • a plurality of lasso peptides are designed using computational methods and predicted to bind to a specific biological target of interest, scored and ranked for binding affinity, the top 2 up to 1000 of the highest ranked lasso peptide variants are produced using cell-free or cell-based methods, tested using assays that allow assessment of the designed lasso peptide for potency and selectivity in binding to the biological target, and the lasso peptide is re-subjected to computational methods to optimize properties such as binding affinity, binding selectivity, ligand-induced pathway activation or deactivation, disease-associated efficacy, solubility, cell penetration, etc. based on the testing results.
  • a plurality of lasso peptides are designed using computational methods and predicted to bind to a specific biological target of interest, scored and ranked for binding affinity, the top 2 up to 1000 of the highest ranked lasso peptide variants are produced using cell-free or cell-based methods, tested using assays that allow assessment of the designed lasso peptide for potency and selectivity in binding to the biological target, and lasso peptides with the highest binding affinities are evolved using peptide mutagenesis methods to optimize properties such as binding affinity, binding selectivity, ligand-induced pathway activation or deactivation, disease-associated efficacy, solubility, cell penetration, etc., and the highest ranked binders are subjected to the iterative workflow one or more additional times for optimization of functional performance or other properties of interest.
  • a binding epitope is grafted into a lasso peptide structure. In some embodiments a binding epitope is grafted into the loop of a lasso peptide. In some embodiments a binding epitope is grafted into the ring of a lasso peptide. In some embodiments a binding epitope is grafted into the tail of a lasso peptide. In some embodiments a binding epitope is grafted into the loop and tail of a lasso peptide. In some embodiments a binding epitope is grafted into the ring and tail of a lasso peptide.
  • a binding epitope is grafted into the ring and loop of a lasso peptide. In some embodiments a binding epitope is grafted into the ring, loop and tail of a lasso peptide. In some embodiments, a conformational epitope is identified whereby two or more different, 129 ACTIVE 705331286v1 non-contiguous sections (one or more amino acid residues) of a natural or synthetic polypeptide ligand bind to a target protein, and the conformational epitope is grafted into a lasso peptide structure.
  • the grafting process involves a replacement of specific amino acids in an existing natural or synthetic lasso peptide sequence with amino acids of a binding epitope. In some embodiments, the grafting process involves the insertion of amino acids into the sequence of an existing natural or synthetic lasso peptide sequence, thereby extending the length of the sequence. In some embodiments, the grafting process involves the de novo creation of lasso peptide sequences containing the binding epitopes of interest in the loop, ring, and/or tail of the lasso peptide. [00356] In some embodiments, a binding epitope is computationally grafted into a lasso peptide structure.
  • a binding epitope is computationally grafted into the loop of a lasso peptide. In some embodiments a binding epitope is computationally grafted into the ring of a lasso peptide. In some embodiments a binding epitope is computationally grafted into the tail of a lasso peptide. In some embodiments a binding epitope is computationally grafted into the loop and tail of a lasso peptide. In some embodiments a binding epitope is computationally grafted into the ring and tail of a lasso peptide. In some embodiments a binding epitope is computationally grafted into the ring and loop of a lasso peptide.
  • a binding epitope is computationally grafted into the ring, loop and tail of a lasso peptide.
  • a conformational epitope is identified whereby two or more different, non-contiguous sections (one or more amino acid residues) of a natural or synthetic polypeptide ligand bind to a target protein, and the conformational epitope is computationally grafted into a lasso peptide structure.
  • the grafting process involves a computational replacement of specific amino acids in an existing natural or synthetic lasso peptide sequence with amino acids of a binding epitope.
  • the grafting process involves the computational insertion of amino acids into the sequence of an existing natural or synthetic lasso peptide sequence, thereby extending the length of the sequence. In some embodiments, the grafting process involves the computational de novo creation of lasso peptide sequences containing the binding epitopes of interest in the loop, ring, and/or tail of the lasso peptide. [00357] In some embodiments, a binding epitope is synthetically grafted into a lasso peptide structure. In some embodiments a binding epitope is synthetically grafted into the loop of a lasso peptide.
  • a binding epitope is synthetically grafted into the ring of a lasso peptide. In some embodiments a binding epitope is synthetically grafted into the tail of a lasso peptide. In some embodiments a binding epitope is synthetically 130 ACTIVE 705331286v1 grafted into the loop and tail of a lasso peptide. In some embodiments a binding epitope is synthetically grafted into the ring and tail of a lasso peptide. In some embodiments a binding epitope is synthetically grafted into the ring and loop of a lasso peptide.
  • a binding epitope is synthetically grafted into the ring, loop and tail of a lasso peptide.
  • a conformational epitope is identified whereby two or more different, non-contiguous sections (one or more amino acid residues) of a natural or synthetic polypeptide ligand bind to a target protein, and the conformational epitope is synthetically grafted into a lasso peptide structure.
  • the grafting process involves a synthetic replacement of specific amino acids in an existing natural or synthetic lasso peptide sequence with amino acids of a binding epitope.
  • the grafting process involves the synthetic insertion of amino acids into the sequence of an existing natural or synthetic lasso peptide sequence, thereby extending the length of the sequence. In some embodiments, the grafting process involves the synthesis of de novo created lasso peptide sequences containing the binding epitopes of interest in the loop, ring, and/or tail of the lasso peptide. [00358] In some embodiments, a plurality of target protein binding epitopes from the same or different polypeptide ligands are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of binding epitopes are computationally grafted into the loop of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the ring of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the tail of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from different polypeptide ligands are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are computationally grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the loop of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are computationally grafted into the tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are computationally grafted into the loop and ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are computationally grafted into the loop and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are computationally grafted into the ring and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are computationally grafted into the tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of binding epitopes are synthetically grafted into the loop of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the ring of a lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the tail of a lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants. In some embodiments, a plurality of target protein binding epitopes from different polypeptide ligands are synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into a lasso peptide structure to create a library of lasso epitope graft variants.
  • a plurality of target protein binding epitopes from the same or different polypeptide ligands are synthetically grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality 132 ACTIVE 705331286v1 of 3D binding epitopes are synthetically grafted into the loop of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the loop and ring of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the loop and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants. In some embodiments, a plurality of 3D binding epitopes are synthetically grafted into the ring and tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • a plurality of 3D binding epitopes are synthetically grafted into the tail of a plurality of lasso peptide structures to create a library of lasso epitope graft variants.
  • the epitope grafted lasso peptides are designed computationally, and the resulting grafted lasso peptides serve as starting sequences and structures for further in silico modeling to afford improved designs predicted to have higher affinity.
  • computationally designed lasso peptides are synthesized in a cell-free reaction mixture or a host organism that contains the enzymes of a lasso peptide biosynthetic pathway.
  • computationally designed lasso peptides are synthesized in a cell-free reaction mixture or a host organism that contains the enzymes of a lasso peptide biosynthetic pathway composed of a precursor peptide (A), and lasso peptidase (B), a lasso cyclase (C), and optionally a RiPP recognition sequence (E).
  • computationally designed lasso peptides are synthesized by treating a precursor peptide (A), with at least one of a lasso peptidase (B), a lasso cyclase (C), and a RiPP recognition sequence (E).
  • computationally designed lasso peptides are synthesized by treating an isolated precursor peptide (A), with at least one of an isolated lasso peptidase (B), an isolated lasso cyclase (C), and an isolated RiPP recognition sequence (E).
  • computationally designed lasso peptides are synthesized by adding a precursor peptide (A), with a cell-free reaction mixture or a host organism that contains at least one of a lasso peptidase (B), a lasso cyclase (C), and a RiPP recognition sequence (E).
  • computationally designed lasso peptides are experimentally produced and screened or assayed for biological activity in a testing step that involves contacting such lasso peptides with biological targets, proteins, or receptors and measuring a property, such as cell penetration, binding affinity, or inhibition of activity.
  • the testing step allows the ranking of computationally designed and experimentally produced lasso peptides for desired biological activities and/or other properties.
  • computationally designed and experimentally produced lasso peptides that are ranked highest are subjected to further optimization through iterative computational refinement based on the structure of the best ranked lasso peptides.
  • computationally designed and experimentally produced lasso peptides that are ranked highest are subjected to further optimization through directed or random peptide evolution methods.
  • computationally designed and experimentally produced lasso peptides that are ranked highest are subjected to further optimization through directed or random peptide evolution methods and lasso peptides that are ranked highest after evolution are further subjected to iterative computational modeling, production, and testing. 7.
  • lasso peptides or lasso peptide analogs are prepared following a protocol of a Scheme, it is understood that conditions may vary, for example, any of the solvents, reaction times, reagents, temperatures, supplements, work up conditions, or other reaction parameters may be varied.
  • 7.1 General Methods Reagents used for molecular biology experiments are purchased from New England BioLabs (Ipswich, MA), Thermo Fisher Scientific (Waltham, MA), or Gold Biotechnology Inc. (St. Louis, MO). Other chemicals are purchased from Sigma-Aldrich (St. Louis, MO).
  • Escherichia coli DH5 ⁇ and BL21 (DE3) strains are used for plasmid maintenance, extract production, or lasso peptide production.
  • Matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS) analysis is performed using a Bruker UltrafleXtreme mass spectrometer in reflector positive mode.
  • Electrospray ionization (ESI)- MS/MS analyses are performed using a ThermoFisher Scientific Orbitrap Fusion ESI-MS 134 ACTIVE 705331286v1 using an Advion TriVersa Nanomate 100.
  • LC-MS/MS analyses are performed on an Agilent 6530 Accurate-Mass Q-TOF MS equipped with a dual electrospray ionization source and an Agilent 1260 LC system with diode array detector or an Agilent 1290 Infinity II HPLC System interfaced with an Agilent 6460C Triple Quadrupole LC/MS system. MS and UV data are analyzed with Agilent MassHunter Qualitative Analysis version B.05.00.
  • Preparative HPLC is carried out using an Agilent 218 purification system (ChemStation software, Agilent) equipped with a ProStar 410 automatic injector, Agilent ProStar UV-Vis Dual Wavelength Detector, a 440-LC fraction collector and preparative HPLC column indicated below.
  • Semi-preparative HPLC purifications are performed on an Agilent 1200 Series Instrument with a multiple wavelength detector and Phenomenex Luna 5 ⁇ m C8(2) 250x100 mm semi preparative column. NMR data are acquired using a 600 MHz Bruker Avance III spectrometer with a 1.7 mm cryoprobe.
  • media used is either M9 minimal medium [17.1 g/L Na 2 HPO 4 ⁇ 12 H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl, 1 mL/L MgSO 4 solution (2 M), 0.2 mL/L CaCl2 solution (0.5 M), pH 7.0; after autoclaving, 10 mL/L sterilized glucose solution (40% w/v), 10 mL/L trace metals, and 10 mL/L standard vitamin mix - for trace metals solution, 27 g/L of FeCl 3 ⁇ 6H 2 O, 2 g/L of ZnCl 2 ⁇ 4H 2 O, 2 g/L of CaCl 2 ⁇ 6H 2 O, 2 g/L of Na 2 MoO 4 ⁇ 2H 2 O, 1.9 g/L of CuSO 4 ⁇ 5H 2 O, 0.5 g/L of H 3 BO 3 ,
  • E. coli BL21 StarTM (DE3) cells are grown in the minimum medium containing MM9 salts (13 g/L), calcium chloride (0.1 mM), magnesium sulfate (2 mM), trace elements (2 mM) and glucose (10 g/L), in a 10 L bioreactor (Satorius) to the mid-log growth phase. The grown cells are then harvested and pelleted.
  • the crude cell extracts are prepared as described in Kay, J., et al., Met. Eng., 2015, 32, 133–142 and Sun, Z. Z., J. Vis. Exp.2013, 79, e50762, doi:10.3791/50762.
  • a green fluorescence protein (GFP) reporter is used to determine the additional amount of Mg-glutamate, K-glutamate, and DTT that are subsequently added to each batch of the crude cell extracts to prepare the optimized cell extracts for optimal transcription-translation activities.
  • GFP green fluorescence protein
  • the optimized cell extracts Prior to cell-free biosynthesis of lasso peptide, the optimized cell extracts are pre-mixed with buffer that contains ATP, GTP, TTP, CTP, amino acids, t-RNA, magnesium glutamate, potassium glutamate, potassium phosphate, and other salts, NAD+, NADPH, glucose, 500 uM IPTG and 3 mM DTT to achieve a desirable reaction volume.
  • An exemplary cell extract comprises the ingredients, and optionally with the amounts, as set forth in the following Table 1.
  • the sample of lasso peptides fused to an affinity tag is lyophilized and resuspended in a binding buffer with respect to its affinity tag according to the manufacturer’s recommendation.
  • the resuspended lasso peptide sample is directly applied to an immobilized matrix corresponding to its fused affinity tag (Tactin for Strep-tag® II, Ni-NTA for His-tag, or amylose resin for maltose binding protein) and incubated at 4°C for an hour.
  • the matrix is then washed with at least 40X volume of washing buffer and eluted with three successive 1X volume of elution buffer containing 2.5 mM desthiobiotin for Strep-Tactin® resin, 250 mM imidzole for Ni-NTA resin or 10 mM maltose for amylose resin.
  • the eluted fractions are analyzed on a gradient (10-20%) Tris-Tricine SDS-PAGE gel (Mini-PROTEAN, BioRad) and then stained with Coomassie brilliant blue.
  • the purity of eluted lasso peptide is examined by LC-MS/MS on an Agilent 6530 Accurate-Mass Q-TOF mass spectrometer.
  • MS/MS fragmentation is used to further characterize lasso peptides based on the rule described in Fouque, K.J.D, et al., Analyst, 2018,143, 1157-1170. If impurities are observed in chromatographic spectra, preparative chromatography is performed to further enrich the purity of lasso peptides.
  • Analytical LCMS Analytical Method Column: Phenomenex Kinetex 2.6 ⁇ XB-C18100 A, 150 x 4.6 mm column.
  • Fractions 137 ACTIVE 705331286v1 containing lasso peptides are identified using the LCMS method described above, or by direct injection (bypassing the LC column in the above method) prior to combining and lyophilizing. Analytical LC-MS/MS (see method above) is then performed on the combined and concentrated lasso peptides.
  • Conformational Analysis and Molecular Dynamics Simulations were performed using MOE’s LowModeMD module. MD simulations were performed using OpenMM and NAMD simulation software and visualized through MOE’s graphical user interface. Force field parameters were generated using the ff14SB and ff19SB force fields which were implemented using a 10 Angstrom octahedral solvent box with preset parameters over 20,000 cycles of minimization for 40 picoseconds while heating to 300 o K, 1 ns for NPT equilibration where backbone atoms were restrained with a harmonic potential of 2 kcal/mol ⁇ K, 1 ns for NVT equilibration (unrestrained), and 100 ns production MD simulations.
  • a custom forcefield that does not permit cis-amide bonds was created by CCG for lasso peptide conformational sampling using LowModeMD. Each simulation used a time step of 2 fs, Langevin thermostat, and Berendsen barostat. From a 100 ns MD trajectory, 1000 snapshots are evenly extracted to form a conformational ensemble that is grouped into clusters. Positive controls for MD simulations were characterized through derivation of the conformers from known experimentally-determined lasso peptide structures by calculating the root mean square deviation (i.e., RMSD) of the 139 ACTIVE 705331286v1 backbone heavy atoms relative to either the lowest energy average structure of the NMR ensemble or the crystal structure. 7.2 Example 1.
  • RMSD root mean square deviation
  • CCR2 Zheng, Y., et al., Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists, Nature, 2016, 540, 458-461
  • CCR5 Tean, Q., et al., Structure of the CCR5 chemokine receptor – HIV entry inhibitor Maraviroc complex, Science.2013, 341, 1387- 1390
  • CXCR4 Wang, B.
  • CCL17 and CCL1 were docked into the CCR4 and CCR8 homology models, respectively, and ligand binding was energy minimized to obtain poses with predicted high binding affinity (FIG.7A and FIG.7B). From these docked CCL17-CCR4 or CCL1-CCR8 complexes, numerous ligand-target binding interactions were identified and a set of CCL17 and CCL1 linear and conformational (3D) binding epitopes were defined for computational grafting into the lasso peptide scaffolds.
  • 3D conformational
  • a binding epitope in the N-loop of CCL17 was identified as a linear 6-amino acid stretch of residues consisting of Leu12, Glu13, Tyr14, Phe15, Lys16, Gly17. 140 ACTIVE 705331286v1 7.2.2 Docking Natural Lasso Peptide Structures and Lasso Peptide Backbones. [00379] Structural information of 37 known lasso peptide was retrieved from the Protein Data Bank (Table 2), and atomic coordinates were used to generate 3D structural models using MOE 2020.09 computational modeling platform. The modeled backbone structures of these lasso peptides are shown in FIG.8.
  • Each lasso peptide in Table 2 was subjected to conformational analysis using the LowMode MD algorithm of the MOE software platform. Table 2. Thirty-seven known lasso peptide sequences and PDB codes. 141 ACTIVE 705331286v1 142 ACTIVE 705331286v1 [00380] Using the homology models of CCR4 or CCR8 chemokine receptors and the docked structures with CCL17 or CCL1, the modeled 3D structures of Lasso 1 to 37 were docked with the CCR4 or CCR8 chemokine receptor in the orthosteric binding pocket as well as potential allosteric sites within the extracellular domains of the receptor.
  • Lasso peptide backbone size and shape assessment was performed by docking and pose and conformational sampling for the 37 known lasso peptide structures using the AMBER99 and FF19SB force fields for (Hornak, V., et al. Comparison of multiple Amber force fields and development of improved protein backbone parameters, Proteins, 2006, 15;65(3): 712-725) (FIG.8).
  • Lasso peptides that are scored highly for size and shape complementarity in potential CCR4 or CCR8 binding sites are then subjected to computational design for optimizing CCR4 or CCR8 binding.
  • Two approaches are used.
  • binding epitopes of CCL17 or CCL1 predicted in Example 6.2.1 were computationally grafted into the ring, loop, and/or tail of the highly ranked lasso peptides.
  • binding epitopes of 2 to 8 amino acids were chosen to substitute for the natural amino acid residues of highly ranked lasso peptide structures.
  • amino acid residues of predicted binding epitopes are computationally inserted into the ring, loop, and/or tail of highly ranked lasso peptides. These amino acid insertions expand the size of the ring, loop, and/or tail of the lasso peptide scaffold and their impact on CCR4 or CCR8 binding is computationally examined. Optimization of epitope-grafted lasso peptide binding affinities is accomplished by varying the poses and conformational sampling using MOE’s LowModeMD algorithm, as well as MD simulations using OpenMM and NAMD simulation software through MOE’s graphical user interface. Energys are minimized and epitope-grafted lasso peptides are scored and ranked as candidates for synthesis and testing.
  • lasso peptide scaffolds with highly ranked size and shape complementarity, with or without grafted binding epitopes predicted in Example 6.2.1 are subjected to computational mutagenesis and screening whereby the amino acid residues in the loop, ring, and tail are varied in order to minimize the potential energy surface of the binding interactions between the lasso peptide residues and CCR4 or CCR8 residues in the receptor binding pocket. Binding interaction energies are minimized, and lasso peptide variant poses are scored and ranked for predicted binding affinity.
  • Top-ranked lasso peptide variants in complex with CCR4 or CCR8 are analyzed through conformational sampling using MOE’s LowModeMD algorithm, as well as MD simulations using OpenMM and NAMD simulation software through MOE’s graphical user interface. Conformations are scored and ranked, with the top 5 conformations with dE ⁇ 20 kcal being selected for docking.
  • the top 5 ranking lasso peptide variants with favorable conformations in complex with the CCR4 or CCR8 144 ACTIVE 705331286v1 receptors are transitioned to Example 6.2.4, the Build phase of the DBTE workflow (FIG. 2A). 7.2.4 Production of Highly Ranked Lasso Peptide Analogs for CCR4 or CCR8 binding.
  • the biosynthetic genes associated with lasso peptides ranked highly in Example 6.2.3 are synthesized and cloned into suitable expression vectors.
  • the vectors are transformed into E. coli or other suitable microbial host and tested for production of lasso peptide variants, which is measured by various methods such as HPLC, LCMS, gel chromatography, NMR, and/or UV vis spectrophotometry.
  • the vectors are modified by varying the gene order, ribosome binding site, and promoters to enhance lasso peptide production levels. Once sufficient yield is observed, lasso peptides are isolated and purified by chromatography methods, including preparative HPLC, and characterized by LCMS and biochemical assays.
  • Isolated lasso peptide variants are then transitioned to Test phase of the DBTE workflow (Example 1.5).
  • the biosynthesis genes of lasso peptides with SEQ ID NOS.: 1-37 are synthesized (Twist Bioscience, San Francisco, CA) and cloned into a pET28 vector for expression in E. coli BL21(DE3) cells which carry the T7 RNA polymerase in its chromosome and driven by the LacUV5 promoter.
  • Genes A, B, C, and E are cloned together as an operon with the T7lac promoter controlling gene expression through vector driven expression of LacI protein, which represses expression until the inducer IPTG is added.
  • a binding epitope in the N-loop of CCL17 is identified as a linear 6-amino acid stretch of residues consisting of Leu12, Glu13, Tyr14, Phe15, Lys16, Gly17, is introduced into the ring and into long loops of lasso peptides with ⁇ 7-amino acid loops.
  • precursor peptide genes containing overlapping sets of 4 amino acid epitopes based on the L12, E13, Y14, F15, L16, G17 are designed and introduced into the lasso core peptide sequence of SEQ ID NOS.: 1-37 by synthesizing the A gene variants and cloning into the pET28 vectors containing a kanamycin resistance marker, together with the corresponding B, C, and E genes.
  • pET28 expression vectors containing genetic variants of SEQ ID NOS.: 1-37 are transformed into E. coli BL21(DE3) cells and individual colonies are grown on lysogeny broth (LB) agar plates for 24-36 h at 37 o C.
  • OD600 optical density
  • This preculture is grown for 12 h, and then IPTG is added (0.05 mM final concentration) to induce expression of the biosynthesis genes and promote the production of lasso peptides.
  • IPTG is added (0.05 mM final concentration) to induce expression of the biosynthesis genes and promote the production of lasso peptides.
  • the cultures are centrifuged and the cell mass is extracted with 2 x 50 mL of methanol for 2 h at 25 o C.
  • the methanol is evaporated and the residue is purified by high-performance liquid chromatography (HPLC, Agilent 1200 series). Extract is injected onto a C18 column (Thermo Fisher Scientific betasil C18, 250 mm ⁇ 10 mm 5 ⁇ M particle size).
  • Lasso peptides are separated using mobile phase A [water, 0.1% trifluoroacetic acid (TFA)] and mobile phase B (acetonitrile, 0.1% TFA) over a gradient of 4 min at 10% B, 2 min 10 ⁇ 32.5% B, 8 min 32.5 ⁇ 35% B, and 2 min 35-95%, 4 min 95%, at a flow rate of 5 mL/min. UV-vis absorbance from 190 ⁇ 450 nm is recorded.
  • TFA trifluoroacetic acid
  • Lasso peptide variants produced in Example 6.2.4 are tested for CCR4 or CCR8 binding affinity using a radioligand binding assay and for functional activity by using a calcium flux assay.
  • 125 I-labelled lasso peptide is produced by reaction with 125 iodide, or alternatively 125 I-CCL17 and 125 I-CCL1 are purchased from Perkin Elmer (Cambridge, MA, USA) or Vitrax Radiochemicals (Placentia, CA, USA) and reacted with mammalian cells (HEK 293 or CHO cells, Sigma Aldrich, St. Louis, MO, USA) that overexpress the CCR4 or CCR8 receptor.
  • CCR4-expressing or CCR8-expressing HEK 293 cells (2 x 106 cells in a final volume of 50 microliters) in binding buffer (RPMI 1640, 25 mM HEPES, 0.1% BSA, 0.05% NaN3, pH 7.4) are incubated in 96-well plates with a fixed concentration of 0.1 nM 125 I- CCL17 and treated with varying concentrations of unlabeled chemokines or lasso peptides at 146 ACTIVE 705331286v1 room temperature.
  • binding buffer RPMI 1640, 25 mM HEPES, 0.1% BSA, 0.05% NaN3, pH 7.4
  • CCR4 or CCR8 functional antagonism by lasso peptide variants is determined by competitive Ca flux measurements in the presence of CCL17 or CCL1, respectively. Intracellular calcium levels are measure using calcium-sensitive indicator dyes which exhibit large fluorescence intensity increases upon binding to calcium (FLIPR Calcium 6 Assay Kit, Molecular Devices, Inc., San Jose, CA).
  • HEK-293 cells overexpressing CCR4 or CCR8 are treated with lasso peptides at different concentrations for 60 min, in the absence and presence of the natural chemokine ligands CCL17 or CCL1, respectively, and variation in i[Ca2+] is measured by treating with dyes that enter cells and fluoresce upon binding to calcium.
  • EC 50 values for lasso peptide bioactivity (antagonism) are determined by analyzing concentration dependent fluorescence data.
  • the lasso peptide variant with the best overall properties (KD, EC50) is transitioned to the Evolve phase of the DBTE workflow in Example 6.2.6.
  • Each amino acid residue of Lasso A or Lasso B is mutated to the other 19 amino acids by site saturation mutagenesis and binding affinity is measured for each variant (FIG. 11A).
  • a library of site saturation NNK mutants of the A gene of Lasso A or Lasso B is constructed using synthetic degenerate oligonucleotides purchased from Twist Biosciences (San Francisco, CA). All amino acids are varied except the acceptor residue E or D in the ring of Lasso A.
  • the libraries of mutant A genes containing up to 8 NNK-mutated sites are cloned into the pET28 plasmid containing the B, C, and E pathway genes of the natural variant of Lasso A.
  • Loaded plates are incubated for 12 h at 37 o C, induced by adding IPTG, and incubated a further 48 h.
  • Wells are passed through a short column of C18 resin, concentrated, and characterized by LCMS. Lasso peptides thus formed are screened for affinity and functional activity as described in Example 6.3.5.
  • the top 10 mutations are recombined in all possible combinations to create a multi-mutational library that is generated by known methods (See: Stemmer, W.P.C. DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Natl. Acad. Sci.
  • Example 6 The top five lasso peptide variants emerging from the Evolve stage are re-introduced into the in silico models and docked into the CCR4 binding pocket. Amino acid residues are varied to minimize predicted binding energies. The top five variants are then selected for production and screening, as described. This workflow is continued until low nanomolar or picomolar KD and EC50 values are obtained for a lasso peptide antagonist of CCR4 or CCR8. 7.3 Example 2.
  • ETBR Lasso Peptide Antagonist of Endothelin Type B Receptor
  • Atomic coordinates were obtained from the NMR structure of a one-amino acid mutant (S7A) of SEQ ID NO: 75 (Katahira, R., et al., Solution Structure of Endothelin B Receptor Selective 149 ACTIVE 705331286v1 Antagonist RES-701-1 Determined by 1H NMR Spectroscopy. Bioorg Med. Chem., 1995, 3 (9), 1273-1280) and were used to construct a structural model of SEQ ID NO: 75, which was then docked into the human ETBR model using the MOE docking program.
  • S7A one-amino acid mutant of SEQ ID NO: 75
  • Conformation sampling and MD simulations were performed on different poses to understand the highest likelihood binding configurations (e.g., loop down, tail up vs tail down, loop up configurations, as shown in FIG.6). All tail down, loop up configurations and poses showed significant clashes between the ligand and ETBR receptor.
  • the loop down configuration was used to similarly construct a docked model between SEQ ID NO: 75 and the human and murine ETBR models developed in Section 6.3.1. A model of the SEQ ID NO: 75-human ETBR is shown in FIG.11B.
  • MYM maltose yeast extract
  • TLB tryptone soya broth
  • PKS tryptone soya broth
  • R5 medium R5 medium [103 g/L sucrose, 0.25 g/L K 2 SO 4 , 10.12 g/L MgCl2.6H2O, 10 g/L glucose, 0.1 g/L Difco casamino acids, 2 mL/L trace element solution, 5 g/L Difco yeast extract, 5.73 g/L TES buffer.
  • the trace element solution used contained 40 mg/L ZnCl 2 , 200 mg/L FeCl 3 .6H 2 O, 10 mg/L CuCl 2 .2H 2 O, 10 mg/L MnCl2.4H2O, 10 mg/L Na2B4O7.10H2O, 10 mg/L (NH4)6Mo7O24.4H2O; after autoclaving, 10 mL/L KH2PO4 (0.5% w/v), 4 mL/L CaCl2.2H2O, 15 mL/L L-proline (20% w/v), and 7 mL/L NaOH (1N).
  • the oriT carrying vector was provided by and purchased from Varigen Biosciences (Middleton, WI, USA) and gBlock fragments provided by IDT Biosciences (San Diego, CA, USA) were PCR amplified with Q5® High-Fidelity DNA polymerase using primers with the appropriate homologous sequences between neighboring fragments.
  • Vector fragments were DpnI-treated and all PCR fragments were purified with the Zymo DNA Clean and Concentrator® -5 kit. Purified fragments were diluted to 30 fmols and Gibson 150 ACTIVE 705331286v1 assembled using NEBuilder® HiFi DNA Assembly Master Mix.
  • a single colony of ET12567/pUB307 was also grown in LB plus chloramphenicol (35 ⁇ g/mL) and kanamycin (50 ⁇ g/mL) for the triparental mating procedure.
  • the overnight cultures were diluted 1:100 in fresh LB plus selective antibiotics and grown at 37 °C until an OD 600 of 0.4-0.6.
  • the cells were centrifuged at 4000 x g washed twice with equal volumes of LB and resuspended in 0.1 volume of LB. On the day of conjugation, 15 ⁇ L spores (approximately 10 9 colony forming units (CFU)) of S.
  • venezuelae host strain ATCC 15439 were mixed with 100 ⁇ L of 2 x YT medium, heat shocked at 50 °C for 10 minutes and allowed to cool.
  • 100 ⁇ L of the heat shocked spores of the host were mixed with 100 ⁇ L of both resuspended E. coli strains.
  • the mixture was plated out on a MS 151 ACTIVE 705331286v1 agar plus 20 mM MgCl2 plate and incubated at 30 °C. After 20 hours, the plates were overlayed with 1 mL of filter sterilized molecular biology grade water containing 1 mg of apramycin and 1 mg of nalidixic acid and distributed evenly with a spreader.
  • TSB culture After 2 days, 400 ⁇ L of the TSB culture was used to inoculate a 10 mL R5 culture with apramycin (50 ⁇ g/mL) and ⁇ -caprolactam (0.5% w/v) in a 50 mL bio-reaction tube. The same TSB culture was also used to plate a MS plate with apramycin (50 ⁇ g/mL) and nalidixic acid (25 ⁇ g/mL). R5 cultures were allowed 7 days to grow before checking for production titers. [00399] Spore stock preparation - The 7-day-old MS plate from the colony with the highest titer was used to make a spore stock.
  • the culture was shaken at 28 °C at 200 rpm for 1 day.
  • 40 mL of the 1-day preculture was used to inoculate 1 L of R5 medium containing apramycin (50 ⁇ g/mL) and ⁇ -caprolactam (0.5% w/v).
  • the production culture was shaken at 28 °C at 200 rpm for 10 days.
  • Lasso Peptide Isolation - Lasso peptide with SEQ ID NO: 75 was extracted from the whole cell broth by first centrifuging the broth in 750 mL Nalgene bottles, then separating the supernatant from the cell pellet.
  • the pellet was first extracted by addition of a quantity of 152 ACTIVE 705331286v1 HPLC grade methanol (MeOH) equal to 3 times the volume of the pellet. This was added directly to the centrifuge bottles, which were shaken overnight on an orbital shaker. The bottles were then centrifuged again, and the methanol extracts collected, pooled, and concentrated on a rotary evaporator to ⁇ 10-20% methanol in water.
  • This cell extract concentrate was subjected to solid phase extraction (SPE) as follows: 100 g of HP20ss resin (Itochu) was packed into an empty SPE column. The column was washed with 1 L MeOH, then equilibrated with 1 L deionized water.
  • SPE solid phase extraction
  • the concentrated extract was loaded onto the SPE resin using a vacuum manifold.
  • the column was washed with 1 L deionized water, then eluted with 1 L 40% MeOH/water, 1 L 50% MeOH/water, 5 x 600 mL 75% MeOH/water, and 1 L 100% MeOH.
  • Each fraction was run on the LC-MS to determine which contained the lasso peptide and approximate the quantity.
  • Fractions containing lasso peptide were pooled and concentrated on a rotary evaporator to remove MeOH, then dried completely on a lyophilizer. Fractions originating from cell pellet material were directly purified on prep HPLC (see below).
  • the clarified cell broth via centrifugation at 5,000 x g was loaded directly to a prepare HP20 column as described above and fractionated as described. Fractions containing target lasso were then dried on a minimal amount of celite, which was loaded into an ISCO Combiflash system using the solid load option. A 50 g HP C18 Redisep column was used for the flash separation.
  • the gradient used DI water as mobile phase A, and HPLC grade methanol as mobile phase B, with the following program: 0 to 20% B over 5 minutes, 20 to 60% B over 25 minutes, 10 minutes at 60% B, 60 to 75% B over 5 minutes, 10 minutes at 65% B, 75 to 100% B over 2 minutes, and 8 minutes at 100% B.
  • Fractions were run on the LCMS quantification method to determine which contained lasso peptide, and those were pooled and concentrated to dryness on a rotary evaporator and lyophilizer. These fractions were then further purified by preparative HPLC (method below).
  • Lasso Peptide Quantification The quantity of eluted lasso peptide with SEQ ID NO: 75 was determined by LCMS on an Agilent 6460C Triple Quadrupole LC/MS system (LC/TQ) equipped with a Jet stream source (AJS), an Agilent 1290 Infinity II LC system, and a diode array detector (DAD). UV210 signals corresponding to the lasso peptide were integrated, and area under the curve was used to calculate lasso peptide concentration based on a standard curve generated from a previously generated standard (SEQ ID NO: 75). Standard concentrations of SEQ ID NO: 75 used were 0.52, 1.3, 3.2, 8, and 20 mg/L.
  • the LCMS method for quantification included the following: 153 ACTIVE 705331286v1 • Column: Phenomenex Kinetex 1.7 ⁇ m XB-C18100 A, 50 x 2.1 mm column. • Flow rate: 0.4 mL/min • Temperature: 40 °C • Mobile Phase A: 0.1% formic acid in water (LCMS grade) • Mobile Phase B: acetonitrile (LCMS grade) • Injection amount: 1 ⁇ L • HPLC Gradient: 5% B for 1.0 min, then 5 to 95% B over 3 minutes followed by 95% B for 0.9 min. 1.5-minute post run equilibration time.
  • Preparative HPLC - Preparative HPLC was carried out using an Agilent 1100 purification system (ChemStation software, Agilent) equipped with an autosampler, multiple wavelength detector, Prep-LC fraction collector and Phenomenex Luna 5 ⁇ m C18(2) 150 x 30 mm preparative column. Fractions containing lasso peptides were identified using the LCMS method described above prior to combining and lyophilizing. Product quality control (QC) was performed on the pooled and concentrated lasso fractions (see Product QC method below).
  • the preparative HPLC method included the following: • Column: Phenomenex Luna® preparative column 5 ⁇ M, C18(2) 100 ⁇ 150 x 30 mm • Flow rate: 20 mL/min • Temperature: RT • Mobile Phases: HPLC grade water, MeOH, acetonitrile, isopropyl alcohol, trifluoroacetic acid (TFA), in different percentages and used as gradients • Injection amount: variable 0.01-2.5 mL • Example method: Solvent A is water with 0.05% TFA, solvent B is acetonitrile with 0.05% TFA.35.5% B for 20.0 min, then 35.5 to 95% B over 1 minute followed by 95% B for 3 minutes.5-minute post run equilibration time.
  • MSMS fragmentation was used to further characterize lasso peptides based on the rule described in Fouque, K.J.D, et al., Analyst, 2018, 143, 1157- 1170, and to confirm amino acid sequences.
  • Proton NMR and high-resolution LCMS data were acquired as described above to further confirm the lasso peptide structure.
  • the analytical LCMS method for purity assessment included the following: • Column: Phenomenex Kinetex 1.7 ⁇ m XB-C18100 A, 50 x 2.1 mm column.
  • CHO-K1 cells were maintained in Kaighn’s F-12K medium supplemented with 10% FB Essence and 2 mM glutamate under a humidified 5% CO2-95% air atmosphere.
  • EBR-CHO Cell lines transiently expressing ETBR (ETBR-CHO) are obtained using a mammalian HA-epitope tag expression vector, pHM6 (Roche Life Sciences, Wilmington MA, USA), that carries a cDNA 155 ACTIVE 705331286v1 construct encoding human recombinant ETBR receptor (GenBank Accession Number NP_000106) or mouse recombinant ETBR receptor (GenBank Accession Number NP_031930.1).
  • Each expression vector was introduced into CHO cells by lipofection using Lipofectamine 2000 (Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer’s instructions. ETBR gene expression was confirmed in cell populations by surface staining with antibodies (anti-HA tag AlexaFluor 488 conjugated mouse IgG purchased from R&D Systems, Minneapolis, MN, USA, Cat # IC6875G) in combination with flow cytometry.
  • antibodies anti-HA tag AlexaFluor 488 conjugated mouse IgG purchased from R&D Systems, Minneapolis, MN, USA, Cat # IC6875G
  • transiently expressing CHO-K1 cells were placed under antibiotic selection for two weeks using Genistein (Millipore Sigma, Burlington MA, USA) at a concentration of 500ug/ml.
  • CHO-K1 cells stably expressing recombinant ETBR receptors selected above were cultured under standard conditions at 37 o C/5% CO2.
  • Cells were collected in ice-cold phosphate buffered saline, pH 7.4 (PBS), and subsequently centrifuged at 500 x g for 5 min at 4 o C.
  • the resulting cell pellet was then resuspended in cell lysis buffer containing 5 mM HEPES, pH 7.4 containing 10 mM EDTA and 2 mM EGTA, homogenized on ice by Dounce homogenization, and centrifuged (48,000 x g for 15 min at 4 o C).
  • the initial pellet was washed twice more by resuspending in 20 mM HEPES, pH 7.4, on ice, and centrifuged as before (48,000 x g for 15 min at 4 o C). Crude membrane pellets were aliquoted and stored at -80 o C prior to use in radioligand binding assays. [00412] Competition binding studies. The total assay volume in each well was 200 ⁇ L and used 96-well microwell plates.
  • Reagent volumes consisted of 3 ⁇ L/well of DMSO containing various lasso peptides (for example, having amino acid sequences described in Table 1) prepared at a range of concentrations, 50 ⁇ L/well of [ 125 I]-endothelin-1 diluted in Assay Buffer (20 mM HEPES, 10 mM MgCl 2 , 0.2% bovine serum albumin (BSA), pH 7.4), and 150 ul/well of diluted ETBR expressing membranes prepared in Assay Buffer. All 156 ACTIVE 705331286v1 reagents were combined and incubated for 2 hours at room temperature.
  • K d radioligand affinity constant
  • the final concentration of radioligand ranged from 0.015 to 5 nM, calculated based on the stock radioactivity concentration and the specific activity (2200Ci/mmol).
  • 10 ug/well of diluted membranes were added to initiate the assay. Quadruplicate wells were used for each concentration in the assay. Wells were incubated for 2 hours at room temperature. Assay incubations were terminated by rapid filtration through Perkin Elmer GF/C filtration plates under vacuum pressure using a 96-well Packard filtration apparatus, as described above.
  • the dissociation constant (K d ) of [ 125 I]-endothelin-1 was calculated using non-linear regression 157 ACTIVE 705331286v1 analysis of the specific amount of radioactivity bound to the membrane as a function of the radioligand concentration. 7.3.5 In Silico Mutational Analysis Aimed at Improving Mouse ETBR Binding Affinity (Design and Evolve) [00415] The computational models of human and mouse ETBR in complex with SEQ ID NO: 75 were used to predict amino acid variations that could lead to improved binding to mouse ETBR, and also ideally to human ETBR as well.
  • a series of single-site mutations around the structure of SEQ ID NO: 75 were introduced in silico and conformational sampling was performed using MOE’s LowModeMD algorithm to obtain a conformational ensemble.
  • the top 10 conformations with dE ⁇ 20 kcal/mol were docked into the ETBR model in a loop-down configuration and binding of all possible analogs to ETBR was analyzed. Docking scores were calculated and the most promising analog-ETBR complexes, based on energetic ranking, were subjected to conformational sampling using LowModeMD, as well as MD simulations using OpenMM and NAMD simulation software through MOE’s graphical user interface.
  • Binding data is shown in Table 4 for single-site mutational analogs produced. Table 4. Binding data for single-site mutants SEQ ID NOS 76-93 using stably transfected mouse ETBR CHO clone 9 158 ACTIVE 705331286v1 [00417] Double-site mutational analysis. On the basis single-site data shown in Table 4, along with further computational modeling analysis, including docking, conformational sampling and MD simulations, numerous double-site mutants were predicted and explored for synergistic binding behavior. These double-site mutants were produced as described above in section 6.3.3 and the binding affinity was measured as described in section 6.3.4. This data is shown in Table 5. Table 5.
  • Binding data for double-site mutants SEQ ID NOS 94-97 using stably transfected mouse ETBR CHO clone 9. 159 ACTIVE 705331286v1 7.3.7 Pharmacokinetic Study of the Parent Lasso Peptide and an Exemplary Evolved Lasso Peptide in CD-1 mice
  • This example shows the pharmacokinetic assessment of the parent lasso peptide (SEQ ID NO: 75) and an exemplary evolved lasso peptide (SEQ ID NO: 87).
  • Pharmacokinetic (PK) assessments of the parent lasso peptide (SEQ ID NO: 75) were performed by Inotiv (West Lafayette, IN, USA) as follows.
  • mice Male CD-1 mice, weighing 30 - 42 g, were obtained from Charles River Laboratories (Wilmington, MA, USA) and acclimated for at least 3 days. All mice were housed in separate cages and given ad libitum access to water and food pellets. The doses and routes of administrations are listed in Table 6. On the day of dosing, all mice were bled according to the PK timepoints outlined in the Table 7 below, depending on the route of administration. All timepoints, except for the last timepoint were bled via the submandibular vein, and the terminal bleed was via the vena cava. Approximately 0.2 mL of whole blood is collected per each timepoint.
  • lasso peptides SEQ ID NOS 4 and 5 containing the RGDF (PBD: 2MMT) and RGD (PDB: 2MMW) epitopes grafted into in the loop of SEQ ID NO: 14.
  • the top scoring epitope-grafted lasso peptides that were predicted to bind strongly to ⁇ v ⁇ 6 were analyzed through conformational sampling using MOE’s LowModeMD algorithm, as well as MD simulations using OpenMM and NAMD simulation software through MOE’s graphical user interface.
  • FIG.15A and FIG.15B show an example of a lasso structure of an exemplary lasso peptide having the RGDL binding epitope (e.g., SEQ ID NO: 109), which is docked into the ligand binding site of integrin ⁇ v ⁇ 6, showing the key binding interactions with the loop-grafted epitope RGDL.
  • RGDL binding epitope e.g., SEQ ID NO: 109
  • Amino acids of the epitope-grafted lasso peptide structures were varied in a manner to create appropriate complementarity between the lasso scaffold side-chains and proximal side-chains on the receptor amino acids.
  • deletions of one or more amino acids in the loops of parent scaffolds also were introduced computationally. All epitope- grafted and otherwise mutated lasso structures were docked, energy minimized, scored, and ranked for binding affinity to ⁇ v ⁇ 6.
  • the top ranked lasso peptide variants were based on natural lasso peptide scaffolds that emerged as the best suited for epitope grafting and integrin binding, namely SEQ ID NOS: 10, 14, 19, and 26 and these were transitioned to 6.4.4, the Build phase of the DBTE workflow (see FIG.16 for complete workflow).
  • SEQ ID NO.: 14 which does not contain the RGD binding epitope, is predicted to be a poor binder to the ⁇ v ⁇ 6 integrin, but the variants with RGD (SEQ ID NO.: 5) or RGDF (SEQ ID NO.: 4) grafted into the loop of SEQ ID NO.: 14 are predicted to display higher binding affinity for ⁇ v ⁇ 6.
  • the plasmids were modified by varying the gene order, ribosome binding site, and promoters to enhance lasso peptide production levels. Once sufficient lasso titer was observed, all lasso peptide analogs were produced from the same plasmid configuration (“A” and “B” plasmids, as described below). Lasso peptides and analogs were 165 ACTIVE 705331286v1 isolated by solid phase extraction methods and purified by chromatography methods, including preparative HPLC, and characterized by LCMS and biochemical assays. Isolated lasso peptide variants are then transitioned to Test phase of the DBTE workflow (FIG.2A).
  • Luria-Bertani (LB) solid 10 g/L casein peptone, 5 g/L yeast extract, 10g/L NaCl, 15 g/L agar, pH 7.0
  • liquid 10 g/L casein peptone, 5 g/L yeast extract, 10g/L NaCl, pH 7.0
  • the coli was accomplished by expressing the lasso peptide biosynthetic genes on 2 different plasmids.
  • the “A” plasmid consisted of the leader and core peptide of a given lasso peptide and changes with each variant.
  • SEQ ID NO: 14 leader and core peptide transcription was driven by mcjA promoter (monocistronic) or the P119 promoter from Anderson promoter collection (Identifier: BBa_J23119) and the sacB promoter rbs region was used to promote transcription of the sacB gene.
  • All “A” plasmids were constructed using a type-2 restriction endonuclease (BsaI) and ligase system (Golden Gate) using a plasmid containing the p15a origin of replication, a chloramphenicol resistance gene, and levansucrase from Bacillus subtilis (sacB), which is a counter-selectable expression marker which renders sucrose toxic 166 ACTIVE 705331286v1 to E. coli.
  • the sacB gene was flanked by inverted BsaI sites such that when sacB was replaced by the desired lasso peptide variant in the correct configuration, the clones were able to grow.
  • the “B” plasmid was constructed to contain the parent lasso peptide biosynthetic genes (e.g., peptidase, cyclase, and transporter if present) and does not change between any given variant of the same natural parent scaffold. [00431]
  • the workflow described remained the same for cloning and producing lasso peptide analogs across different scaffolds, whereas the genes in the “A” and “B” plasmids were different, with the “A” plasmid containing a unique precursor peptide, and “B” plasmid containing the unique biosynthetic enzyme genes for producing a respective parent scaffold, such as SEQ ID NOS: 10, 14, 19, or 26.
  • Biosynthetic genes were synthesized by Twist Bioscience (South San Francisco, CA, USA). Desired A gene fragments of range 300 bp to 350 bp were ordered as eblocks corresponding to respective parent scaffolds SEQ ID NOS: 10, 14, 19, or 26. Short double- stranded DNA fragments (eblocks) were ordered from Integrated DNA Technologies (IDT, San Diego, CA, USA) containing the entire leader and core peptide flanked by BsaI sites. Additionally, the gene fragments carried a common upstream region TTTGAGGCTTTTACGACGATGGCGGTCTCTA, and common downstream region TAACTGAAGAGACCGCATAATACTTTGAGAGAATGGCAATCCTT.
  • the common primers which we used to amplify the eblock gene fragments were 5’- GAGGCTTTTACGACGATGGC-3’ and 5’-GGATTGCCATTCTCTCAAAGTATTATGC- 3’ using an annealing temp of 60°C for 30 secs.
  • Q5 High- Fidelity 2X Master Mix polymerase which was obtained from New England Biolabs Inc. (Ipswich, MA, USA), and set up the 25 ⁇ L reactions as per Table 11 below and using thermocycling conditions in Table 12: Table 11.
  • PCR thermocycling conditions [00433] PCR products were analyzed using agarose gel electrophoresis to ascertain products were formed in the range of 200-300 bp to verify the PCR amplification proceeded correctly.
  • PCR purification The amplified PCR product was confirmed by running it on agarose gel electrophoresis to verify samples had the correct band size. The purification proceeded by either diluting the PCR products to 1:20 (1 ⁇ L of amplified DNA + 19 ⁇ L of dH 2 O) or using the ZYMO DNA Clean & Concentrator-5 kit and column (ZYMO Research, Irvine, CA, USA) to purify the amplified product following the ZYMO protocol.
  • Cells were centrifuged again at 5000 x g for 9 minutes at 4°C. The cell pellets were washed with 30 mL of 100 mM CaCl 2 . Centrifugation was repeated and supernatant was decanted. Pellets were washed with 30 mL of 85 mM CaCl2, 15% glycerol. Centrifugation was repeated and supernatant decanted.
  • Pellets were resuspended in 1.5 mL of 85 mM CaCl2, 15% glycerol and 50 ⁇ L aliquots of chemically competent BL21(DE3) E coli cells containing 169 ACTIVE 705331286v1 the Lasso “B” plasmid cells were transferred into 1.5 ml individual Eppendorf tubes which were pre-autoclaved and prechilled. Tubes were snap-frozen tubes after aliquoting using 100% ethanol and dry ice mixture.
  • Three verified colonies from each transformation above were then used to individually inoculate a 3 mL LB culture containing kanamycin (50 ⁇ g/mL) and chloramphenicol (17 ⁇ g/mL) in a 10 mL well from a sterile 24-well deep well plate. After 16- 18 hours at 37 °C and shaking at 200 rpm, 400 ⁇ L of the LB culture was used to inoculate a 3 mL supplemented M9 culture with kanamycin (50 ⁇ g/mL) and chloramphenicol (17 ⁇ g/mL) in a 10 mL well from a sterile 24-well deep well plate.
  • Glycerol stock preparation The overnight LB culture was used to make a glycerol stock. This was done by mixing 500 ⁇ L of the LB culture from a single colony with 500 ⁇ L of a 50% glycerol solution in a 2.0 m: cryogenic vial and storing it at -80 °C.
  • Small scale production of lasso peptides and analogs A preculture was generated by adding 3 mL of LB broth + 50 mg/mL Kanamycin + 17mg/mL Chloramphenicol to each well of 24 well cell culture plates for each lasso analogs.
  • a colony of each analog was added in triplicate into the wells containing media and the same colony was patched onto a patch plate (LB agar + 50 mg/mL Kanamycin + 17mg/mL Chloramphenicol). Pre-cultures were grown overnight in shakers at 37°C, 200 rpm. After 14- 16 hrs, a small-scale production cultures were generated from the pre-cultures using supplemented M9 media. M9 supplemented media was prepared according to Table 15. 170 ACTIVE 705331286v1 Supplemented M9 media (3 mL) was added to each well (24-well plates) which were then inoculated with 400 ⁇ L of preculture. Production cultures were grown for 3 days at 30°C, 200 rpm.
  • M9 supplemented media was prepared from 5X M9 salts (Teknova, Hollister, CA, USA) as outlined in Table 15 by first autoclaving the 1X M9 media and then adding the remaining components
  • Precultures were generated by inoculating individual sequence verified E coli colonies containing the “A” and “B” plasmids for each analog from either a patch plate or a premade glycerol stock into 10 mL of LB broth in culture tubes with 50 mg/mL of kanamycin and 17 mg/mL of chloramphenicol and shaking overnight at 37°C, 200 rpm.
  • Precultures should reach about OD600 of 2 to 3 after 16 hours incubation.
  • the starter culture was shaken at 37 °C at 200 rpm for 16-18 hours, then 20 mL of the culture was used to inoculate a 500 mL LB preculture containing kanamycin (50 ⁇ g/mL) and chloramphenicol (17 ⁇ g/mL) in a 2 L baffled flask.
  • the preculture was shaken at 200 rpm and 37 °C for 4.5 hours, until optical density at wavelength 600 nm (OD600) reached 2.
  • Lasso peptides and analogs were extracted from the whole cell broth by first centrifuging the broth in 750 mL Nalgene bottles, then separating the supernatant from the cell pellet.
  • the pellet was first extracted by addition of a quantity of HPLC grade methanol (MeOH) equal to 3 times the volume of the pellet. This was added directly to the centrifuge bottles, which were shaken overnight on an orbital shaker. The bottles were then centrifuged again, and the methanol extracts collected, pooled, and concentrated on a rotary evaporator to ⁇ 10-20% methanol in water.
  • MeOH HPLC grade methanol
  • This cell extract concentrate was subjected to solid phase extraction (SPE) as follows: For every liter of culture volume, 12.5 g of HP20ss resin (Itochu) was packed into an empty SPE column. The column was washed with 5 column volumes of MeOH, then equilibrated with 5 column volumes of deionized water. The concentrated extract was loaded onto the SPE resin using a vacuum manifold. The column was washed with 125 mL deionized water, then eluted with 125 mL 30% MeOH/water, 125 mL 50% MeOH/water, 125 mL 75% MeOH/water, and 125 mL 100% MeOH.
  • SPE solid phase extraction
  • UV210 signals corresponding to the lasso peptide were integrated, and area under the curve was used to calculate lasso peptide concentration based on a standard curve generated from a previously generated standard (SEQ ID NO: 98). Standard concentrations of SEQ ID NO: 98 used were 0.52, 1.3, 3.2, 8, and 20 mg/L.
  • the LCMS method for quantification included the following: Column: Phenomenex Kinetex 1.7 ⁇ m XB-C18100 A, 50 x 2.1 mm column.
  • Preparative HPLC Preparative HPLC was carried out using an Agilent 1100 or 1260 purification system (ChemStation software, Agilent) equipped with an autosampler, multiple wavelength detector, Prep-LC fraction collector and Phenomenex Luna 5 ⁇ m C18(2) 150 x 30 mm preparative column.
  • the 1260 system additionally included an Agilent MSD. Fractions containing lasso peptides were identified using the LCMS method described above prior to combining and lyophilizing. Product quality control (QC) was performed on the pooled and concentrated lasso fractions (see Product QC method below).
  • the preparative HPLC method included the following: Column: Phenomenex Luna® preparative column 5 ⁇ M, C18(2) 100 ⁇ 150 x 30 mm Flow rate: 20 mL/min Temperature: RT 173 ACTIVE 705331286v1
  • Mobile Phases HPLC grade water, MeOH, acetonitrile, isopropyl alcohol, trifluoroacetic acid (TFA), in different percentages and used as gradients
  • Injection amount variable 5-200 ⁇ L
  • Solvent A is water with 0.05% TFA
  • solvent B is acetonitrile with 0.05% TFA.24% - 26% B over 20.0 min, then 20% to 95% B over 1 minute followed by 95% B for 3 minutes.5-minute post run equilibration time.
  • MSMS fragmentation was used to further characterize lasso peptides based on the rule described in Fouque, K.J.D, et al., Analyst, 2018, 143, 1157-1170, and to confirm amino acid sequences. Proton NMR and high-resolution LCMS data were acquired as described above to further confirm peptide structures.
  • the analytical LCMS method for purity assessment included the following: Column: Phenomenex Kinetex 1.7 ⁇ m XB-C18100 A, 50 x 2.1 mm column.
  • ActivIVE 705331286v1 7.4.5 Measuring Lasso Peptide Variants for Integrin Binding (Test).
  • the inhibiting activity and selectivity of the tested antagonists are determined by using ELISA assays.
  • Human integrins ⁇ v ⁇ 6, ⁇ v ⁇ 3, and ⁇ v ⁇ 8 were purchased from R&D Systems (Minneapolis, MN).
  • Vitronectin, fibrinogen, and fibronectin were purchased from Sigma Aldrich (St. Louis, MO), and LAP protein from R&D Systems.
  • integrins ⁇ v ⁇ 6, ⁇ v ⁇ 3 and ⁇ v ⁇ 8 integrins the binding is visualized using a mouse anti-human integrin ⁇ -V monoclonal antibody for the ⁇ v subunit (MM0421-3H31 was purchased from Thermo Fisher Scientific).
  • MM0421-3H31 mouse anti-human integrin ⁇ -V monoclonal antibody for the ⁇ v subunit
  • anti-mouse IgG-peroxidase from Sigma Aldrich is used as secondary antibody, containing a peroxidase conjugate that is employed for the visualization and quantification.
  • Peroxidase development is performed by using the supplier’s recommended methods with the substrate 3,3,5,5’-tetramethylethylenediamine (TMEDA) purchased from Sigma Aldrich and by adding 3M H2SO4 to stop the reaction.
  • TEDA 3,3,5,5’-tetramethylethylenediamine
  • the absorbance (450, 492 nm) is recorded with a Victor Nivo plate reader (Perkin Elmer, San Jose, CA). Every concentration is analyzed in duplicate and the resulting inhibition curves are analyzed using GraphPad Prism software. The inflection point of these curves describes the IC50 value.
  • Each plate also contains either cilengitide or MK-0429 (purchased from MedChemExpress, Monmouth Junction, NJ) as reference compounds. Blocking and binding steps are performed with TS buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2) containing 1% BSA.
  • PBST buffer (10 mM Na 2 HPO 4 , pH 7.5, 150 mM NaCl, and 0.01% Tween 20).
  • PBST buffer 10 mM Na 2 HPO 4 , pH 7.5, 150 mM NaCl, and 0.01% Tween 20.
  • a clear flat-bottom 96-well ELISA plate (from Sigma Aldrich) was coated overnight with 100 ⁇ L of vitronectin (2 ⁇ g/mL) in carbonate buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , pH 9.6) at a temperature of 4°C. After removing the solutions from the plate, the wells are blocked for 1 h at room temperature with 150 ⁇ L TSB buffer per well.
  • the plate is subsequently washed three times with 200 ⁇ L PBST buffer per well. Afterward, 4.0 ⁇ g/mL of the soluble integrin ⁇ v ⁇ 3 and a serial dilution of the different compounds and a cilengitide control are incubated in the coated wells for 2 h at room temperature. After washing three times, the plate is treated with each 100 ⁇ L of the primary antibody (MM0421-3H31, diluted 1:500 in TSB buffer, 1.0 ⁇ g/mL) and 100 ⁇ L of the secondary antibody (anti-mouse IgG-peroxidase, diluted 1:385 in TSB buffer, 2.0 ⁇ g/mL) per well for 1 h at room temperature.
  • the primary antibody MM0421-3H31, diluted 1:500 in TSB buffer, 1.0 ⁇ g/mL
  • the secondary antibody anti-mouse IgG-peroxidase, diluted 1:385 in TSB buffer, 2.0 ⁇ g
  • the plate is washed three times and the 175 ACTIVE 705331286v1 binding is then visualized using TMEDA.
  • the oxidation reaction is performed for only 5 min and the absorbance is measured at 450 nm. IC50 is determined as explained below.
  • clear flat-bottom immune-nonsterile 96-well plates were coated with 50 ⁇ L 0.4 ⁇ g/ml LAP (R&D Systems, cat. no.246-LP) in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) overnight at 4 o C.
  • PBST PBS, 0.05% Tween 20
  • TSB buffer 20 mM Tris HCl [pH 7.5], 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 1% BSA
  • the plates were washed three times with 200 ⁇ L PBST containing 1 mM MgCl 2 .
  • 50 ⁇ L of integrin 0.5 ⁇ g/ml ⁇ v ⁇ 6 [R&D Systems, cat.
  • the plates were then washed three times in 200 ⁇ L PBST containing 1 mM MgCl2.
  • 50 ⁇ L of goat anti-mouse IgG peroxidase conjugate (Sigma, cat. no. DC02L) at concentrations of 0.0266 ⁇ g/ml for ⁇ v ⁇ 6 or 0.0665 ⁇ g/ml for ⁇ v ⁇ 8 was added and incubated for 1 hour at room temperature.
  • the plates were then washed in three times in 200 ⁇ L PBST containing 1 mM MgCl2.
  • 50 ⁇ L TMB substrate (Thermo Fisher, cat. no. 34021) was added and incubated for 10-30 minutes at room temperature.
  • RGD and RGDX epitopes were computationally introduced in the loop and ring of a range of different parent scaffolds, including SEQ ID NOS 10, 14, 19, and 26, and epitope-grafted analogs that docked with acceptable rankings and were able to be produced, were then tested for integrin binding affinity.
  • IC50 values for ⁇ v ⁇ 8 and ⁇ v ⁇ 3 were measured 176 ACTIVE 705331286v1 and are shown for comparison to provide an indication of the selectivity of predicted integrin inhibitors.
  • RGD-grafted analogs for 4 different natural scaffolds were produced and integrin binding was measured. Table 16.
  • 177 ACTIVE 705331286v1 “****” indicates an IC 50 of ⁇ 10 nM; “***” indicates an IC 50 of >10 nM and ⁇ 50 nM; “**” indicates an IC 50 of > 50 nM and ⁇ 500 nM; “*” indicates an IC 50 of > 500 nM and ⁇ 10,000 nM; “--” indicates data not available; N/A indicate information not applicable 7.4.6 Mutating Parent Lasso Peptides to Improve ⁇ v ⁇ 6 Integrin Binding (Evolve).
  • SEQ ID NOS 109 and 110 Two lasso peptide variants from section 6.4.5 (SEQ ID NOS 109 and 110) demonstrated good ⁇ v ⁇ 6 binding (IC50 ⁇ 100 nM) and 3-6 x selectivity vs ⁇ v ⁇ 3 inhibition. These two epitope-grafted analogs emerged as top parent scaffolds and were selected for further optimization of affinity and other properties. In order to optimize binding affinity and bioactivity, SEQ ID NOS 109 and 110 were subjected to molecular evolution methods.
  • SEQ ID NOS 109 and 110 Evolution of SEQ ID NOS 109 and 110 was conducted by one of two methods (1) computationally predicting analogs to test by docking variants with amino acid mutations and identifying those that rank highly when bound to the model of ⁇ v ⁇ 6, as described in sections 6.4.2 and 6.4.3, or (2) randomly introducing mutations into parent scaffolds containing the desired RGD or RGDX epitope.
  • a description of a random NNK mutagenesis process for SEQ ID NO: 109 is provided below.
  • Random NNK lasso peptide mutagenesis Lasso analog libraries were generated by random mutagenesis using NNK codons for key loop positions around the RGDL epitope as well as ring positions.
  • Other codon design schemes also can be used, including NNB, NNS, and MAX (see Sieber, T., et al. Biomathematical Description of Synthetic Peptide Libraries, PLoS ONE, 2015, 10(6): e0129200. doi:10.1371/journal.pone.0129200; Nov, Y. When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis. Appl. Environ. Microbiol.2012, 78, 258 –262).
  • This example illustrates a 2-site NNK mutagenesis procedure using SEQ ID NO: 109 as the parent lasso peptide.
  • a similar procedure was used for other parent lasso peptides and for a larger number of mutated sites (e.g., 3-site, 4-site, 5- site NNK, etc.).
  • 178 ACTIVE 705331286v1 Design of degenerate codon primers. Cloning of each mutagenesis library depended on (i) a parent template, made of an “A” plasmid with a lasso precursor peptide sequence inserted into it, and (ii) a forward and reverse degenerate oligo primer pair.
  • degenerate codon NNK encodes all 20 amino acids, but limits the number of codons to 32, including one stop codon.
  • the amino acid site to be mutated that sits closest to the N-terminal of the peptide will be further referred as “degenerate site 1”, while the randomized amino acid site that sits closest to the C-terminal of the peptide will be referred as “degenerate site 2”.
  • the binding region of the forward primer was directly downstream of the degenerate site 2.
  • the forward primer contained an overhang that, reading in the direction of 5’ to 3’, consisted of the BsaI recognition sequence GGTCTC, a single nucleotide, a four-base overhang, followed by the degenerate codon NNK at the position of the degenerate site 2.
  • the binding region of the reverse primer was directly upstream of the degenerate site 1.
  • the reverse primer binds to the reverse complement DNA strand of the parent template.
  • the reverse primer contained an overhang that, reading in the direction of 5’ to 3’, consisted of the BsaI recognition sequence, a single nucleotide, a BsaI overhang that is the reverse complement to the overhang sequence used in the forward primer, followed by the sequence MNN - the degenerate codon complementary to NN - at the position of degenerate site 1. Depending on the position of this site within the core peptide, more nucleotides were added between the BsaI overhang the MNN site, matching the reverse complement sequence directly downstream of degenerate site 1. In the cases where the two degenerate sites sat next to each other, only one of the primers would have the degenerate codons.
  • the other primer’s overhang would only contain the BsaI recognition site and the BsaI overhang.
  • Oligos containing degenerate codons were ordered and synthesized by standard mixing, where a synthesizer dispenses activated nucleotides in ratios enabling an equal probability of incorporating A, T, G, or C at each of the N-sites.
  • Random 2-site NNK libraries of SEQ ID NO: 109 were generated by designing and synthesizing (Integrated DNA Technologies (IDT), San Diego, CA, USA) specific degenerate primers containing the NNK codons for amino acids at 2 positions that are to be 179 ACTIVE 705331286v1 mutated can theoretically generate a library size of 400 variants with all possible amino acid substitutions.
  • two primers no longer than 60 bp were designed and synthesized by IDT with a high-fidelity overhang at the tails and BsaI restriction site with overlaps between them and NNK position to afford 25 nmol of the corresponding DNA oligos.
  • primers were used to generate 2 NNK sites required to produce mutants of SEQ ID NO: 109: Table 17.
  • PCR amplification of parent template with degenerate codon prime rs The parent “A” plasmid template containing the precursor peptide sequence corresponding to SEQ ID NO: 109 was PCR amplified with the forward and reverse primers that allow the introduction of degenerate codons at positions A3 and I16 (Table 17). The template concentration was kept between 50 and 100 ng/ ⁇ L. Four PCR reactions were performed through serial dilutions of the template.
  • each subsequent reaction had a 1:50 template dilution. All PCR reactions were run on an agarose gel. The two reactions with the lowest concentration of template that gave a clear band at the expected size were pooled together and either (i) DpnI-treated and purified using a Zymo® column, or (ii) run on an agarose gel to check for quality, PCR product bands of the correct size were then extracted and purified. [00459] Golden Gate reactions to generate degenerate “A” plasmid libraries.
  • the two BsaI sites present in the template plasmid were first digested and then ligated with PCR fragments to forming a new “A” plasmid with two degenerate codons in the core region of SEQ ID NO: 109.
  • the Golden Gate reaction was performed using 0.75 ⁇ L NEB® CutSmart Buffer, 0.75 ⁇ L T4 ligase buffer, 1 ⁇ L BsaI and 1 ⁇ L T4 ligase, which were mixed with parent template plasmid and the pooled DNA fragments purified above.
  • the maximum DNA was used plus molecular biology-grade water for a total reaction volume of 15 ⁇ L Table 18). If more than 1000 ng of DNA fragment was purified, the reaction was scaled up to larger volumes.
  • Transformations were performed as many times as required to use all of the DNA from the Golden Gate reactions above. The transformations were pooled together and recovered in 12.5 mL LB media in a 125 mL flask at 200 rpm and 37 °C. After one hour, 200 ⁇ L of the culture was removed from the flask and reserved. Chloramphenicol at 17 ⁇ g/mL was added to the flask and it was returned to the shaker overnight.
  • the 12.5 mL recovery culture was harvested and miniprep purified using the PureYieldTM Plasmid Miniprep System purchased from Promega, Inc. (Madison, WI, USA).
  • the maximum recovered degenerate “A” plasmid DNA was used for five transformations into the “B” plasmid-containing E coli BL21 chemically competent cells generated in section 6.4.4.
  • the DNA was mixed into 50 ⁇ L of competent cells and incubated on ice for 30 minutes, followed by a heat shock at 42 °C for 30 seconds, and then incubated on ice for another five minutes.
  • the fully transformed cells were then recovered by culturing in LB media containing kanamycin (50 ⁇ g/mL) for one hour, wherein 200 ⁇ L of the culture was removed for plating serial dilutions as described in the section above. The rest of the culture is allowed to grow overnight at 37 °C and 200 rpm. [00463] Production cultures of saturated mutagenesis libraries.
  • the recovery culture obtained from transformation of degenerate “A” plasmids into E coli BL21 was used in its entirety as the pre-culture to inoculate a supplemented M9 with kanamycin (50 ⁇ g/mL) and chloramphenicol (17 ⁇ g/mL) culture in a 2 L baffled flask, at 12% inoculum. Because all the recovery culture was used to inoculate the production culture, the volume of the recovery culture varied depending on the volume of that library’s scale-up, but final volume typically is in the range of 1-1.5 L. The production culture was grown for 3 days at 37 °C and 200 rpm. [00464] Analysis of mutagenesis libraries.
  • the production culture produced above theoretically contained up to 400 different variants and the two-site NNK mutagenesis libraries were analyzed and validated in two ways.
  • Table 19 provides integrin binding data for representative 1-site, 2-site, and 3-site mutational analogs, based on parent lasso peptides with SEQ ID NOS: 109 and 110. Table 19. IC 50 values for evolved variants epitope-grafted variants of SEQ ID NOS: 109 and 110 in competitive ELISA binding assays using integrins ⁇ v ⁇ 6, ⁇ v ⁇ 8, and ⁇ v ⁇ 3.
  • mice Female Balb/c mice, weighing 20 - 25 g, were obtained from Envigo (Indianapolis, IN, USA) and acclimated for at least 3 days. All mice were housed in separate cages and given ad libitum access to water and food pellets. The doses and routes of administration are listed in Table 20. On the day of dosing, all mice were bled according to the PK timepoints outlined in the Table 21 below, depending on the route of administration, via microsampling. All timepoints, except for the last timepoint were bled via the tail vein, and the terminal bleed was via the vena cava or by cardiac puncture. Approximately 0.2 mL of whole blood is collected per each timepoint.

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Abstract

L'invention concerne des procédés de découverte et d'optimisation de peptides lasso à l'aide d'un flux de travail intégré et itératif impliquant des principes de conception à base de structure, une modélisation in silico, une greffe d'épitope, un criblage virtuel, une ingénierie génétique, une évolution peptidique et un test expérimental. L'invention concerne également des peptides lasso qui ont été découverts et optimisés pour la sélectivité de liaison, l'affinité de liaison, l'activité biologique et d'autres propriétés à l'aide des procédés décrits ici.
PCT/US2024/061377 2023-12-20 2024-12-20 Procédés intégrés et itératifs de conception, d'identification et d'optimisation de peptides lasso Pending WO2025137511A1 (fr)

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