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WO2024151615A1 - Bibliothèques d'affichage d'arnm et procédés d'utilisation - Google Patents

Bibliothèques d'affichage d'arnm et procédés d'utilisation Download PDF

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Publication number
WO2024151615A1
WO2024151615A1 PCT/US2024/010851 US2024010851W WO2024151615A1 WO 2024151615 A1 WO2024151615 A1 WO 2024151615A1 US 2024010851 W US2024010851 W US 2024010851W WO 2024151615 A1 WO2024151615 A1 WO 2024151615A1
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Prior art keywords
peptide
mrna
molecules
phe
mrna display
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Inventor
Albert Bowers
Sabrina ISKANDAR
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University of North Carolina at Chapel Hill
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University of North Carolina at Chapel Hill
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

Definitions

  • the invention relates to mRNA display libraries comprising non-canonical amino acids and methods of making same.
  • the invention further relates to methods of using orthogonal aminoacyl-tRNA synthetases to incorporate non-canonical amino acids in mRNA display libraries.
  • mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets.
  • mRNA display is uniquely poised for peptide library diversification due to the enhanced tunability of in vitro translation (IVT).
  • IVT in vitro translation
  • a large portion of mRNA display library expansion thus far has been achieved via flexizymes, ribozymes that aminoacylate non-canonical amino acids (ncAAs) onto optimized tRNAs.
  • Flexizymes enable perhaps the most used cyclization method in mRNA display by facilitating translation of an N-terminal alkyl chloride for reaction with downstream cysteine (Cys) thiols.
  • an mRNA display library comprising a plurality of mRNA display molecules, each mRNA display molecule comprising a peptide according to the formula (I): fMet-Cys-Xn-ncAA (I) wherein fMet is formyl-Methionine, X is any amino acid, n is an integer from 1 to 30, and ncAA is non-canonical amino acid para-cyanopyridylalanine.
  • a further embodiment of the invention is an mRNA display library comprising a plurality of mRNA display molecules each comprising a cyclized peptide according to formula (II): Cys-X n -ncAA (II) Attorney Docket No.5470.943.WO wherein each X is any amino acid, n is an integer from 1 to 30, ncAA is a non-canonical amino acid para-cyanopyridylalanine, and wherein each cyclized peptide comprises a pyridine- thiazoline bridge formed via condensation between the N-terminal cysteine and the para- cyanopyridylalanine.
  • Another embodiment of the invention is a method of preparing an mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules, the method comprising the steps of: providing a library of mRNA molecules comprising a plurality of molecules according to formula (III): fMet-Cys-(N 1 N 2 N 3 ) n -TAG-[linker] (III) wherein N 1, N 2 , and N 3 are each independently any nucleotide, and n is an integer from 1 to 30; performing in vitro translation of each of the plurality of molecules in the mRNA library with a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS) in the presence of para- cyanopyridylalanine to thereby produce a plurality of fMet-Cys-[linear peptide]-[linker]- [mRNA] molecules, the ORS incorporating a para-cyanopyridylalanine at
  • FIGS.1A-1C Promiscuous ORS para-cyanophenylalanine tRNA synthetase (p-CNF- RS) incorporates multiple ncAAs at an amber codon in IVT.
  • FIG.1A Promiscuous ORSs can translate multiple ncAAs in IVT, allowing for expanded chemical diversity and novel macrocyclization.
  • FIG.1B Sequence of a model peptide with amber codon for translation of a given ncAA.
  • FIG.1C MALDI-MS spectra of model IVT peptide translated with p-CNF-RS Attorney Docket No.5470.943.WO and various para-substituted Phe ncAAs. “E” and “O” indicate expected and observed m/z values, respectively. Asterisks indicate desired product peaks. [0015] FIGS.2A-2B.
  • FIGS.3A-3H Proposed strategy to generate pyridine-thiazoline macrocycles in IVT.
  • FIG.2B Model peptide substrates and corresponding MALDI-MS spectra for p-CNpyrA translation (top), addition of peptide deformylase (PDF) and methionine aminopeptidase (MAP) (middle), and oxidation by Cyanothece Oxidase (ThcOxi) (bottom).
  • PDF peptide deformylase
  • MAP methionine aminopeptidase
  • ThcOxi oxidation by Cyanothece Oxidase
  • FIG.3A Schematic for mRNA display of pyrdine- thiazoline (pyr-thn) cyclized libraries and selection against USP15.
  • FIG.3C Percent NGS composition of top sequences from each sub-family.
  • FIG.3F Di-ubiquitin cleavage by USP15 over time in the absence or presence of 100 ⁇ M SEI144 or SEI149.
  • FIG.3G Surface plasmon resonance (SPR) single cycle kinetics fit of SEI144 (2.5-200 nM) and SEI149 (0.033-2.7 ⁇ M) with D1D2 catalytic domain of human USP15 and USP9x.
  • FIG.5. SDS-PAGE gels of exemplary recombinant proteins used in Example 1.
  • FIG.7 USP15 cleavage of Ub-AMC over time with increasing concentrations of SEI144. Attorney Docket No.5470.943.WO [0021]
  • FIG.8 Raw response units of SPR of SEI144 with USP15 D1D2 and USP9x catalytic domain at 2.47, 7.4, 22.2, 66.6, and 200 nM, and SEI149 with D1D2 USP15 at 0.033, 0.1, 0.3, 0.9, and 2.7 ⁇ M.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • consists essentially of (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and/or C-terminal ends of the recited sequence or additional nucleotides on the 5’ and/or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), guide RNA (gRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized), synthetic RNA of any sequence (e.g., chemically synthesized), nucleic acid probes and primers.
  • mRNA messenger RNA
  • gRNA guide RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides branched polynucleot
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base-pairing abilities or increased resistance to nucleases.
  • modulate refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.
  • the term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and/or can be expressed in the enhancement and/or increase of a specified level and/or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.
  • inhibit or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1, 5, 10, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).
  • contact or grammatical variations thereof refers to bringing two or more substances in sufficiently close proximity to each other for one to exert a biological effect on the other.
  • Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like.
  • parenteral e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intra
  • Constant administration means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.
  • Systemic administration refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems.
  • local administration refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of Attorney Docket No.5470.943.WO administration and does not introduce the agent systemically in a therapeutically significant amount.
  • locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body.
  • Administration includes self-administration and the administration by another.
  • a “subject” may be any vertebrate organism in various embodiments.
  • a subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and/or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed.
  • a subject is a mammal, e.g., a human, non-human primate, lagomorph (e.g., rabbit), or rodent (e.g., mouse, rat).
  • a human subject is a neonate, child, adult or geriatric subject.
  • a human subject is at least 50, 60, 70, 80, or 90 years old.
  • Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject.
  • Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject.
  • the effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and/or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease.
  • a therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease.
  • the agent may be administered e.g., to reduce the likelihood of recurrence of evident disease.
  • a therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease.
  • “Prophylactic treatment” refers to providing medical and/or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur.
  • the subject may have been identified as being at risk Attorney Docket No.5470.943.WO of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.
  • any variable e.g., Ri
  • its definition at each occurrence is independent of its definition at every other occurrence.
  • Ri e.g., Ri
  • the definition at each occurrence is independent of its definition at every other occurrence.
  • Ri at each occurrence is selected independently from the Markush group recited for Ri.
  • combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds within a designated atom’s normal valency.
  • the present invention relates to mRNA display libraries and methods of preparing mRNA display libraries that comprise cyclized peptides wherein each of the cyclized peptides comprise a non-canonical amino acid.
  • mRNA display refers to a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target.
  • An mRNA display library may contain a plurality of mRNA display molecules, e.g., at least 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 members, or from 10 10 to 10 15 , 10 10 to 10 14 , 10 10 to 10 12 , 10 11 to 10 13 , 10 12 to 10 14 , or from 10 12 to 10 13 members.
  • at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 rounds of mRNA display screening is carried out, or from 2-10, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-8, 3-6, 3-5, 3-4, 4-10, 4-8, or 4-6 rounds of mRNA display screening is conducted.
  • a round of mRNA display screening refers to contacting an immobilized target with the library, washing the mixture to remove unbound library members, and isolating the bound mRNA-protein fusion library members.
  • the result of the mRNA display screening is a less diverse library that has been enriched for peptide variants that bind to the desired target.
  • An amino acid includes both naturally occurring and/or non- canonical occurring amino acids.
  • Naturally occurring amino acids include those encoded by the genetic code and those amino acids that are later modified, for example, carboxyglutamate and hydroxyproline.
  • Amino acid analogs comprise a structure similar to a naturally occurring amino acid but have modified R groups, such as norleucine or norvaline, or modified peptide backbones.
  • Such non-canonical amino acids may include amino acid analogs and amino acid mimetics that function similar to naturally occurring amino acids.
  • Exemplary amino acids, Attorney Docket No.5470.943.WO analogs and mimetics include, without limitation, ⁇ -alanine ( ⁇ -Ala), N- ⁇ -methyl-alanine (Me- Ala), aminobutyric acid (Abu), ⁇ -aminobutyric acid ( ⁇ -Abu), aminohexanoic acid ( ⁇ -Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid, aminopiperidinecarboxylic acid, aminoserine (Ams), aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N- methyl amide, ⁇ -aspartic acid ( ⁇ -Asp), azetidine carboxylic acid, 3-(2-benzothiazolyl)alanine, ⁇ - tert-butylglycine, 2-a
  • the amino acid is a phenylalanine derivative or other electrophilic non-canonical amino acid.
  • the phenylalanine derivative is para-functionalized, which may be, for example, p-CN-pyridylalanine, p-CN-Phe, p-N 3 -PHe, p-CCH-Phe, p-biphenyl-Ala, p-NH 2 -Phe, p-CH-Phe, p-iPr-Phe, p-F-Phe, or p-SO 2 NH 2 Phe.
  • D and L isomers of the naturally occurring or non- naturally occurring amino acids are contemplated within the term amino acid.
  • a charged amino acid can be substituted with another charged amino acid, a neutral amino acid with another neutral amino acid.
  • an amino acid can be substituted with an amino acid of a different charge to modify the interactions of the peptide targeting ligand.
  • Molecules of the invention including peptides and nucleic acid/peptide conjugates, may comprise a cap on the N-terminus of an amino acid, including formyl, pyroglutamyl, acetyl, fatty acids, urea, alkylamine and sulfonamide.
  • an mRNA display library comprising a plurality of mRNA display molecules each comprising a peptide according to the formula (I): fMet-Cys-X n -ncAA (I) wherein fMet is formyl-Methionine; X is any amino acid; e.g., any naturally occurring amino acid; n is an integer from 1 to 30; and ncAA is non-canonical amino acid para- cyanopyridylalanine. In some embodiments, X is any amino acid, and n is an integer from 4 to 20.
  • an mRNA display library comprising a plurality of mRNA display molecules each comprising a cyclized peptide according to formula (II): C-X n -ncAA (II) wherein each X is any amino acid (e.g., any naturally occurring amino acid), n is an integer from 1 to 30, ncAA is a non-canonical amino acid para-cyanopyridylalanine, and wherein each cyclized peptide comprises a pyridine-thiazoline bridge formed via condensation between the N- terminal cysteine and the para-cyanopyridylalanine.
  • the cyclized peptide can be further oxidized to comprise a pyridine-thiazole bridge. Substitutions of the amino acids can be made, and may be according to desired function, including, for example, charge, aromaticity, and R group functionality, as detailed elsewhere herein.
  • the mRNA display libraries can be used to identify one or more targeting ligands, e.g., cyclic peptides, useful for binding a target of interest.
  • the targeting ligand can, in some embodiments, be provided as a cyclized molecule, which can be according to formula (II).
  • the targeting ligand is provided as a linear molecule according to formula (I), and the linear molecule can be exposed to one or more proteases, which may remove the fMet, exposing an N-terminal cysteine, and allow for the spontaneous condensation of the N- terminal cysteine with the non-canonical amino acid, e.g., para-cyanopyridylalanine.
  • proteases which may remove the fMet, exposing an N-terminal cysteine, and allow for the spontaneous condensation of the N- terminal cysteine with the non-canonical amino acid, e.g., para-cyanopyridylalanine.
  • Another embodiment of the invention provides a method of preparing an mRNA display library comprising a plurality of mRNA-linker-macrocyclic peptide molecules, the method comprising the steps of: providing a library of mRNA molecules comprising a plurality of molecules according to formula (III) fMet-Cys-(N1N2N3)n-TAG-[linker] (III) wherein N 1, N 2 , and N 3 are each independently any nucleotide, and n is an integer from 1 to 30; performing in vitro translation of each of the plurality of molecules in the mRNA library with a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS) in the presence of para- cyanopyridylalanine to thereby produce a plurality of fMet-Cys-[linear peptide]-[linker]- [mRNA] molecules, the ORS incorporating a para-cyanopyr
  • ORS promiscuous orthogonal aminoacyl-t
  • N3 of (N1N2N3)n in formula (III) is G or T.
  • the linker according to formula (III) is a Gly-Ser linker which may comprise from two to six repeats, e.g., GSGSGS (SEQ ID NO:23), GSGSGSGS (SEQ ID NO:24), GSGSGSGSGS (SEQ ID NO:25) and GSGSGSGSGSGSGS (SEQ ID NO:26).
  • the Gly-Ser linker is GSGSGS (SEQ ID NO:23).
  • removing the N-terminal fMet is performed by one or more proteases.
  • proteases can comprise peptide deformylase and/or methionine aminopeptidase.
  • Other proteases can be utilized, with proteases and their recognition sequences known in the art, see, e.g., Barrett A., et al. Handbook of proteolytic enzymes Academic Press (1998); see also the PeptideCutter program available at Expasy, identifying enzymes and their cleavage sites within peptides.
  • a recognition sequence may be included between the N-terminal formyl methionine and the cysteine to provide a cleavage site for a particular protease.
  • a linker may also be provided between the formyl- methionine and the recognition sequence when a recognition sequence is present.
  • the method further comprises the step of converting the pyridine thaizoline-bridged macrocycle peptide to a pyridine thiazole bridged molecule.
  • the converting comprises contacting the pyridine thaizoline-bridged macrocycle peptide with thiazoline oxidase.
  • the thiazoline oxidase is from Cyanothece PCC 7425. Attorney Docket No.5470.943.WO [0057]
  • at least one (N1N2N3) is an amber codon.
  • a para-substituted phenylalanine derivative is incorporated in translation at one or more of the amber codons, providing additional non-canonical amino acids in one or more peptides of the mRNA display library.
  • the para-substituted phenylalanine derivative is selected from p-CN-pyridylalanine, p-CN-Phe, p-N3-PHe, p-CCH-Phe, p-biphenyl-Ala, p-NH2- Phe, p-CH-Phe, p-iPr-Phe, p-F-Phe, and p-SO 2 NH 2 Phe.
  • Incorporation of the non-canonical amino acids can be performed by a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS).
  • the ORS is p-CNF-RS.
  • the cell-free methods detailed herein can utilize a promiscuous ORS to incorporate non- canonical amino acids, expanding the genetic code in IVT.
  • Other exemplary promiscuous ORSs include Methanococcus jannaschii Tyr-RS and Pyl-RS, and those described in, for example, Krahn et al., Enzymes.2020; 48: 351-395, incorporated herein by reference in its entirety.
  • At least one (N 1 N 2 N 3 ) of formula (III) encodes a cysteine.
  • Methods of making the mRNA display library can further comprise the step of reacting the encoded cysteine with an alkylating agent to incorporate a non-canonical amino acid side chain.
  • the alkylating agent is an alkyl halide. Alkylating agents for the addition of side chains to cysteine are known in the art, see, e.g., Deming, Chem. Rev.2016, 116, 3, 786– 808, doi: 10.1021/acs.chemrev.5b00292.
  • a method for conjugation comprises reacting exogenous cysteine-containing chemical reagents with linear peptides comprising a non-canonical amino acid, e.g., p-cyanopyridylalanine for conjugation to mRNA display libraries.
  • Methods for screening for a targeting cyclic peptide also referred to herein as a targeting ligand, are provided. Methods comprise producing an mRNA display library according to the methods described herein or providing an mRNA display library as described herein, bringing the mRNA display library molecules into contact with the target substance, incubating the library molecules and target substance; and selecting cyclic peptides that bind the target substance.
  • the targeting ligand is a ubiquitin specific peptidase 15 (USP15) targeting ligand peptide.
  • the targeting ligand is Attorney Docket No.5470.943.WO provided as a cyclized molecule.
  • the cyclized targeting ligand may comprise a bond between an N-terminal cysteine and a C-terminal non-canonical amino acid.
  • the cyclized targeting ligand comprises a pyridine-thiazoline bridge between a cysteine and a cyano functionalized phenylalanine derivative.
  • the cyclized targeting ligand may comprise a pyridine- thiazole bridge between a cysteine and the non-canonical amino acid.
  • the targeting ligand is according to formula (IV): CX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 (IV) wherein X1 is I, L, V, P, or H; X2 is N, Q, R, S, T, K, or H, X3 is R, H, S, L, K, A, Y, M, or T; X4 is A, T, N, S, V, T, R, or L; X 5 is W, Y, F, L, or C; X 6 is Y, F, W, or S; X 7 is P or H; X 8 is N, Q, T, R, S, K, N, H, F, or M; X 9 is L, I, V, or P, and X 10 is a para-substituted phenylalanine derivative, wherein the peptide comprises a bridge between the N-terminal cysteine and X10 of the
  • the para-substituted phenylalanine derivative at X10 can be p-CN-pyridylalanine.
  • the bridge is a pyridyl-thiazoline bond or a pyridyl-thiazole bond.
  • the USP15 targeting ligand peptide can comprise one or more para-substituted phenylalanine derivatives, which may be selected from p-CN-pyridylalanine, p-CN-Phe, p-N3- PHe, p-CCH-Phe, p-biphenyl-Ala, p-NH 2 -Phe, p-CH-Phe, p-iPr-Phe, p-F-Phe, and p- SO 2 NH 2 Phe.
  • the targeting ligands can further comprise a cargo associated with the ligand.
  • the cargo can be associated at the N-terminus or C-terminus of the targeting ligand, or on a side chain of an amino acid, for example a cysteine, for example conjugated via covalent bonding.
  • the cargo is associated with the targeting ligand via a cleavable linker, the linker covalently bonded to the targeting ligand at the N-terminus or C-terminus or side chain of an amino acid.
  • the cargo can comprise a detectable label, biologically active agent, imaging agent, and/or therapeutic agent.
  • the cargo comprises a nucleic acid, a protein, a complex of a nucleic acid and a protein, a carbohydrate, a lipid, or a small molecule.
  • Kits may comprise an mRNA display library comprising a plurality of mRNA display molecules, each mRNA display molecule comprising one or more amber codons, a promiscuous orthogonal aminoacyl-tRNA synthetase (ORS), one or more non-canonical amino acids, or a combination Attorney Docket No.5470.943.WO thereof. Kits may comprise one or more targeting ligands; and/or instructions for use, in any combination.
  • ORS promiscuous orthogonal aminoacyl-tRNA synthetase
  • Kits can further comprise cargo, carriers, buffers, containers, devices for administration of the components, and the like.
  • the kit can further comprise labels and/or instructions for assay selection and execution.
  • Such labeling and/or instructions can include, for example, information concerning the amount, and methods of administration, detection and quantification for assays detailed herein.
  • ORSs have been relatively underutilized within IVT to date and Applicant could find no previous report of common ORSs (e.g., M. jannaschii Tyr-RS, Pyl-RSs) used in mRNA display (Seki, et al. ACS Synth. Biol.2020, 9, 718–732; Gerrits, et al. ACS Synth. Biol.2019, 8, 381–390; Ranji Charna, et al. Biotechnol. J.2022, 17, 2200096).
  • common ORSs e.g., M. jannaschii Tyr-RS, Pyl-RSs
  • p-CNF-RS tolerates many para-substituted Phe derivatives. Applicant hypothesized this promiscuity might be further expanded in IVT, allowing for the identification of novel ncAA substrates. Specifically, it was reasoned that p- CNF-RS might tolerate electrophilic ncAAs, such as para- and/or meta-cyanopyridylalanine (p- Attorney Docket No.5470.943.WO CNpyrA and m-CNpyrA, respectively).
  • ncAAs have recently been employed for protein functionalization in cells and macrocyclization of synthetic peptides through condensation with N-terminal Cys residues for the formation of bridged pyrdine-thiazolines (pyr-thn)(Abdelkader, et al. Angew. Chemie - Int. Ed.2022, 61 (13), e202114154; Iskandar & Bowers, ACS Med. Chem. Lett.2022, 13 (9), 1379-1383).
  • this pyr-thn cyclization provides both (1) increased rigidity, which can drastically improve affinity by reducing entropic cost to access the correct binding conformation (Iskandar & Bowers, ACS Med.
  • Applicant anticipates other ncAAs could serve as additional substrates for this versatile synthetase in IVT.
  • Applicant Having identified p-CNpyrA as a substrate of p-CNF-RS, Applicant next sought to incorporate the pyr-thn macrocyclization into mRNA display (FIG.2A).
  • Applicant designed several model peptides, each containing a Cys after the N-terminal formyl-Met (fMet) initiator and a downstream amber codon to encode p-CNpyrA.
  • Model peptides were designed to test effects of: (1) varied ring size, (2) sterically bulky adjacent residues, and (3) cross-reactivity with internal Cys thiols. Attack by an internal thiol should be reversible without a proximal amino group to complete the formal condensation for thiazoline formation.
  • Model peptides were translated from the nucleotide sequences of SEQ ID NOs:47-51 with p-CNF-RS, treated with PDF/MAP, and purified by a C-terminal HA tag for analysis by MALDI-MS.
  • p-CNF-RS facilitated translation of p-CNpyrA in all peptides.
  • Subsequent treatment with PDF/MAP furnished the desired macrocycles set forth in SEQ ID NOs:52-56 as predominant products throughout and, as expected, the internal Cys peptide did not yield a macrocycle mass (FIG.2B).
  • NNK9 randomized mRNA display library flanked by an N-terminal Cys and C-terminal amber Attorney Docket No.5470.943.WO codon (SEQ ID NO:57).
  • This library was translated with p-CNF-RS and p-CNpyrA, cyclized via addition of PDF/MAP, then panned against the D1D2 catalytic domain of human USP15 (FIG.3A)( Ward, et al. J. Biol. Chem.2018, 293 (45), 17362–17374).
  • next generation sequencing indicated strong enrichment of a single sequence (FIG.3B).
  • SEI144 (1) was synthesized via solid-phase peptide synthesis alongside a linear counterpart, SEI149 (2), that replaced the N-terminal Cys and C-terminal p-CNpyrA with Ac- Ala and Phe, respectively (FIG.3D).
  • SEI149 (2) was synthesized via solid-phase peptide synthesis alongside a linear counterpart, SEI149 (2), that replaced the N-terminal Cys and C-terminal p-CNpyrA with Ac- Ala and Phe, respectively (FIG.3D).
  • SEI149 significantly inhibited USP15 activity in both a ubiquitin-AMC and di-ubiquitin cleavage assay (FIGS.3E-3F and FIG.7).
  • SPR surface plasmon resonance
  • SEI144 proved to be a low nanomolar binder of D1D2 USP15 with a Kd of 5.51 nM (FIG.3G and FIG.8).
  • linear SEI149 showed no detectable binding to D1D2 USP15, demonstrating the necessity of the pyr-thn cyclization.
  • SEI144 also did not bind to the catalytic domain of another USP, USP9x, via SPR, suggesting potential selectivity for USP15.
  • Applicant investigated whether SEI144 stabilized melting temperatures (T m ) of several other USPs in a thermal shift assay. First, it was confirmed that USP15 was thermally stabilized upon SEI144 binding.
  • p-CNpyrA pyr-thn bridged cyclic mRNA display libraries
  • p-CNpyrA pyr-thn bridged cyclic mRNA display libraries
  • pyridine-conjugated thiazolines can be converted to thiazoles via treatment with a thiazoline oxidase, which Applicant anticipates can also be imported into mRNA display.
  • this methodology does not require leader peptides or recognition motifs, which are typically needed for enzymatic approaches to thiopeptide functionality (Fleming, et al.
  • T4 RNA ligase I (PR-M1051), RQ1 DNase (PR-M6101), and M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant (PRM3683) were purchased from Promega through Fisher Scientific.
  • Anti-HA magnetic beads (88836), HA synthetic peptide (26184), and M-280 Streptavidin DynabeadsTM (11205D) were purchased from Thermo Fisher Scientific. Bulk solvents were purchased from Fisher Scientific. Fmoc-amino acids and materials for peptide synthesis were purchased from ChemImpex unless otherwise mentioned.
  • Free amino acids for p-CNF-RS translation were purchased from ChemImpex, ChemScene, TCI America, or Thermo Fisher Scientific. DNA gene fragments and all purchased plasmids were from Twist Bioscience. Deubiquitinating enzymes (DUBs) that were not expressed in-house were purchased from R&D Systems. Primers (Table 1) were purchased from Integrated DNA Technologies. Next Generation Sequencing (NGS) was performed by Azenta using their Amplicon-EZ (150-500 bp) service. NMR spectra were recorded at room temperature with Varian Inova 400 (400 MHz).
  • the culture was shaken at 37°C until reaching an OD600 of 0.5-0.8, at which point the medium was supplemented with IPTG (1 mM).
  • the culture was grown at 18°C for 19 h.
  • Cells were pelleted and stored at -80°C until purification.
  • Pellets were suspended in lysis buffer [50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and 10 mM BME (pH 8.0), supplemented with 1 mg/mL Attorney Docket No.5470.943.WO lysozyme, 100 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor tablet and lysed by sonication.
  • PMSF phenylmethylsulfonyl fluoride
  • the sample was then centrifuged (20000 rpm for 30 min), and the supernatant was filtered through a 0.45 ⁇ m sterile syringe filter and incubated with Ni-NTA resin (Qiagen) for 1 h at 4°C.
  • the resin was washed [100 mL of 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, and 10 mM BME (pH 8.0)] and eluted [5 mL of 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, and 10 mM BME (pH 8.0)].
  • a pET28a+ expression vector with the sequence for PDF was purchased from Twist Biosience. The protein was expressed and purified from BL-21 DE3 OneShot E. coli as previously reported. 2 A 5 mL saturated overnight culture was used to inoculate 1 L of Luria-Bertani (LB) medium supplemented with kanamycin (50 ⁇ g/mL). The culture was incubated at 37 °C and shaken at 220 rpm until reaching an OD600 of 0.6-0.8 at which point the medium was supplemented with 1 mM IPTG. Culture was then cooled to 25 °C and grown for 3 hr. Cells were pelleted and stored at -80 °C until purification.
  • LB Luria-Bertani
  • Pellets were resuspended in 25 mL of lysis buffer (20 mM HEPES pH 7.6, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 5% glycerol) supplemented with 1.5 mM PMSF and an EDTA-free protease inhibitor tablet (ThermoFisher catalog number: 88666).
  • the cells were lysed by French Press (Glen Mills Model 11, 5500-000011) and the soluble protein was recovered by pelleting the cell debris by centrifugation at 4°C, 15,000 rpm for 20 mins.
  • the supernatant was filtered through a 0.45 ⁇ m sterile syringe filter, loaded onto a 5 mL HisTrap® (Ni 2+ ) IMAC column, and washed with ⁇ 5 column volumes (CV) of wash buffer (20 mM HEPES pH 7.6, 300 mM NaCl, 30 mM imidazole). Protein was eluted with elution buffer (20 mM HEPES pH 7.6, 300 mM NaCl, 250 mM imidazole) over a stepwise gradient 0-100%. Fractions containing purified protein were collected and concentrated to 2.5 mL using a Centricon® (10,000 Da MWCO) concentrator (EMD Millipore®).
  • MAP Methionine Aminopeptidase
  • SEQ ID NO:42 A pET28a+ expression vector with the sequence for MAP was purchased from Twist Biosience. The protein was expressed and purified from BL-21 DE3 OneShotTM E. coli according to known methods.
  • a 5 mL saturated culture was used to inoculate 1 L of Luria-Bertani (LB) medium supplemented with kanamycin (50 ⁇ g/mL).
  • the culture was incubated at 37°C and shaken at 220 rpm until reaching an OD600 of 1.0 at which point the medium was supplemented with 100 ⁇ M CoCl2 and 1 mM IPTG.
  • Culture was then cooled to 25°C and grown for 3 hr. Cells were pelleted and stored at -80°C until purification.
  • Pellets were resuspended in 25 mL of lysis buffer (20 mM Tris pH 7.6, 150 mM NaCl, 10 mM imidazole, 1 mM DTT, 5% glycerol). The cells were lysed by French Press (Glen Mills Model 11, 5500-000011) and the soluble protein was recovered by pelleting the cell debris by centrifugation at 4°C, 15,000 rpm for 20 mins.
  • lysis buffer 20 mM Tris pH 7.6, 150 mM NaCl, 10 mM imidazole, 1 mM DTT, 5% glycerol.
  • the supernatant was filtered through a 0.45 ⁇ m sterile syringe filter, loaded onto a 5-mL HisTrap® (Ni 2+ ) IMAC column, and washed with ⁇ 5 column volumes (CV) of wash buffer (20 mM Tris pH 7.6, 300 mM NaCl, 30 mM imidazole, 1 mM DTT). Protein was eluted with elution buffer (20 mM HEPES pH 7.6, 300 mM NaCl, 250 mM imidazole, 1 mM DTT) over a stepwise gradient 0-100%.
  • Fractions containing purified protein were collected and concentrated to 2.5 mL using a Centricon® (10,000 Da MWCO) concentrator (EMD Millipore®). The concentrated protein was then buffer-exchanged using a PD-10 column (GE Healthcare Life Sciences®) into storage buffer (10 mM Tris pH 7.6, 50 mM KCl, 1 mM DTT, 10% glycerol) and stored at -80°C. A 1 L culture yielded ⁇ 20 mg of protein. [0084] Cyanothece Oxidase (ThcOxi; SEQ ID NO:43). A pET28a+ expression vector with the sequence for ThcOxi was purchased from Twist Biosience.
  • the plasmid was expressed and purified from BL-21 DE3 OneShotTM E. coli as previously reported (Houssen, et al. Angew. Chemie - Int. Ed.2014, 53, 14171–14174).
  • a 5 mL saturated culture was used to inoculate 1 L of LB medium supplemented with kanamycin (50 ⁇ g/mL).
  • the culture was incubated at 37°C and shaken at 220 rpm until reaching an OD600 of 0.6-0.8 at which point the medium was supplemented with 0.2 mM IPTG and 50 ⁇ M riboflavin. Culture was then cooled to 20°C and grown overnight ( ⁇ 20 hours). Cells were pelleted and stored at -80°C until purification.
  • Pellets were resuspended in 25 mL of lysis buffer (25 mM HEPES pH 8.0, 500 mM NaCl, 20 mM Attorney Docket No.5470.943.WO imidazole, 3 mM BME, 50 ⁇ M flavin mononucleotide (FMN)) supplemented with 1.5 mM PMSF and an EDTA-free protease inhibitor tablet (ThermoFisher catalog number: 88666).
  • the cells were lysed by French Press (Glen Mills Model 11, 5500-000011) and the soluble protein was recovered by pelleting the cell debris by centrifugation at 4°C, 15,000 rpm for 20 mins.
  • the supernatant was filtered through a 0.45 ⁇ m sterile syringe filter, loaded onto a 5 mL HisTrap® (Ni 2+ ) IMAC column, and washed with ⁇ 5 CV of wash buffer (25 mM HEPES pH 8.0, 500 mM NaCl, 20 mM imidazole, 50 ⁇ M FMN). Protein was eluted with elution buffer (25 mM HEPES pH 8.0, 500 mM NaCl, 250 mM imidazole, 50 ⁇ M FMN)) over a stepwise gradient 0-100%.
  • GST-tagged USP15 D1D2 was cleaved by TEV protease to yield the Avi-D1D2 USP15 protein (SEQ ID NO:44).
  • the Avi-D1D2 USP15 protein was then biotinylated on an N-terminal AviTag using the biotin ligase BirA.
  • Biotinylated USP15 D1D2 was then separated from BirA and free GST through anion-exchange and size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • GST-tagged USP9x and USP21 were cleaved by TEV protease and subjected to SEC.
  • GST-tagged, full-length USP37 and USP15 were expressed in baculovirus- infected insect cells.
  • Gene fragments were first resuspended to a final concentration of 10 ng/ ⁇ L. Then a 100 ⁇ L PCR amplification was carried out under standard Q5 DNA polymerase (NEB M0491) protocols. Amplification was performed under conditions listed in Table 4 and confirmed by DNA gel (3% agarose gel supplemented with 1.5% ethidium bromide). The DNA was then purified by a PCR purification kit and isolated in MQ-H 2 O. The concentration was determined by nanodrop and then stored at -20oC until further use. Table 4. Gene fragment PCR conditions: Step Temperature (oC) Time (min) Initial Denature 95 2 [0087] Mj-tRNA PCR.
  • a 1 mL PCR amplification was set up containing all the initial extension reaction, 1x Q5 Reaction buffer, 2.5 mM MgCl 2 , 0.25 mM dNTPs, 0.5 ⁇ M forward primer (P5), 0.5 ⁇ M reverse primer (P6), 15 units Q5 DNA Polymerase (NEB M0491), and diluted in MQ-H2O.
  • Amplification was performed under conditions listed in Table 6. Amplification was confirmed by DNA gel. Then, 1x volume of phenol/chloroform/isoamyl alcohol (PCI) was added to the solution, mixed vigorously, and centrifuged.
  • PCI phenol/chloroform/isoamyl alcohol
  • the top layer was recovered, transferred to a new tube, and PCI extraction was Attorney Docket No.5470.943.WO performed once more.
  • 1/10 volume of 3M NaCl and 2x volume of 100% ethanol was added and mixed vigorously. This mixture was centrifuged for 15 mins at 13,000 rpm to pellet the DNA. Once pelleted, the DNA was washed with excess 70% ethanol, then allowed to dry at RT. The DNA was then solubilized in a 1/10 volume (100 ⁇ L) of the PCR reaction. Table 5.
  • Step Temperature (oC) Time (sec) Initial Denature 95 60
  • Step Temperature (oC) Time (sec) Initial Denature 95 60
  • RNA transcription for Mj-tRNA and NNK library.
  • a 1 mL transcription reaction (adapted from NEB T7 RNA polymerase protocols) containing amplified DNA (100 ⁇ L), 1x T7 RNA Polymerase Buffer, 1 mM Dithiothreitol (DTT), 16.5 mM MgCl2, 5 mM rNTPs, and 5 units/ ⁇ L T7 RNA Polymerase (NEB M0251) was incubated overnight at 37oC. Upon successful transcription, magnesium pyrophosphate precipitates and the solution appears cloudy.
  • RNA pellet was then solubilized in 100 ⁇ L of MQ-H 2 O and an equal volume (100 ⁇ L) of 2x RNA loading dye.
  • the sample heated at 95oC for 2 mins, then run on a large scale 8% Urea- PAGE gel at 230 V for 1.5 h in a Tris-Borate-EDTA (TBE) buffer.
  • TBE Tris-Borate-EDTA
  • the desired RNA band was Attorney Docket No.5470.943.WO then visualized under UV illumination at 254 nm on a silica-coated thin-layer chromatography plate and excised from the gel. The excised band was crushed into fine pieces and the RNA was extracted with 0.3 M NaCl (2 x 1 h incubation at RT or 1x overnight at 4oC).
  • RNA was solubilized in MQ-H2O and the concentration was determined by nanodrop and adjusted as necessary with MQ-H 2 O. Purity was confirmed via an analytical scale 8% Urea-PAGE gel. RNA was stored at -80oC until use.
  • ThcOxi Oxidation of Pyr-Thn IVT peptides were created as described above.1 volume of 2x ThcOxi buffer (40 mM Tris pH 8, 1 M NaCl, 100 ⁇ M FMN) was added, followed by ThcOxi at a final concentration of 20 ⁇ M. Samples were incubated at 37oC overnight, purified via anti-HA magnetic beads, then spotted onto a MALDI plate via Zip C18 spin column purification. mRNA Display of Pyr-Thn Libraries and Selection against USP15 [0092] Puromycin Linking.
  • RNA of the NNK library was covalently linked to puromycin via an adapted Y-ligation method (Sakai, et al. Nat. Chem. Biol.2019, 15 (6), 598–606).
  • a solution with 1 ⁇ M RNA, 20% DMSO, 1.5 ⁇ M puromycin linker (P10), 1x T4 RNA ligase buffer, and 1 unit/ ⁇ L T4 RNA ligase I (PR-M1051) in MQ-H2O was incubated at 37oC for 0.5 h. Then, 1x volume of precipitation solution (0.6M NaCl, and 50mM EDTA), 0.02x volume of 5 mg/mL glycogen, and 2x volume 100% ethanol were added.
  • Translation of the puromycin-linked NNK library was performed using a custom NEB PURExpress® kit (-aa, -tRNA, -RF123) - E6850Z, supplemented with 10 ⁇ M Mj-tRNA, 50 ⁇ M p-CNF-RS, and 20 mM p-CNpyrA.
  • a 40 ⁇ L translation was incubated at 37 oC for 30 mins, followed by a 12 min incubation at RT to facilitate fusion of peptide to its mRNA strand.
  • EDTA was then added to a final concentration of 17 mM to dissociate the ribosome, and the mixture was incubated at 37oC for 30mins.
  • PDF was added to a final concentration of 10 ⁇ M and incubated at 37oC for 15 mins, followed by addition of CoCl2 to 100 ⁇ M and MAP to 30 ⁇ M, and incubation at 37oC for 1 h.
  • complementary DNA was appended by a reverse transcription reaction containing all the translation product, 0.6 mM dNTPs, 5 ⁇ M reverse primer (P9), 62.5 mM Tris-HCl pH 8.3, 37.5 mM Mg(OAc) 2 , 25 mM KOH, 2.5 x M-MLV reverse transcriptase H (-) point mutant (Promega, Attorney Docket No.5470.943.WO M3681, supplied at 40x), and MQ-H2O (to reach final volume).
  • HA Purification The displayed library was diluted by 10x, then added to anti-HA magnetic beads (ThermoFisher catalog number: 88836) at a 4:1 ratio of bead slurry to initial IVT volume (40 ⁇ L). The bead slurry rotated at room temperature for 30 min, after which beads were washed 3x with TBS-T (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween® 20).2 mg/mL HA peptide in TBS-T was then added to the beads, and fusions were eluted by rotation at room temperature for 1 h.
  • TBS-T 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween® 20
  • the HA elution volume should be calculated such that immobilized protein is at 200 nM during the selection.
  • USP15 Streptavidin Immobilization Biotinylated D1D2 USP15 was immobilized onto DynabeadsTM M-280 Streptavidin beads (ThermoFisher catalog number: 11205D). Excess USP15 in selection buffer (25 mM HEPES pH 8, 125mM NaCl, 0.05% Tween®) was added streptavidin beads such that, when combined with the volume of HA eluted fusions, enough pmol USP15 was immobilized to arrive at a final concentration of 200 nM.
  • a separate streptavidin immobilization assay should be performed prior to the selection to determine the bead loading capacity of a given protein. Immobilization occurred at 4oC for 30 mins. Then, free biotin in H 2 O was added to final concentration of 25 ⁇ M to occupy any remaining biotin binding sites. The solution rotated for an additional 15 mins at 4oC. Once complete, USP15-immobilized SA beads were washed 3x with selection buffer. [0096] Selection and cDNA Elution. HA eluted fusions were added to the washed USP15-SA beads.
  • Each qPCR sample contained 0.25 ⁇ M forward and reverse primers (P17, P9) and 1 ⁇ L of experimental sample in 1x SsoAdvancedTM Universal SYBR® Green Supermix (Bio-Rad, 172-5271).
  • qPCR standards were prepared by reverse transcription of a known quantity of RNA into cDNA (assuming 100% yield) and prepared in 10-fold serial dilutions to 2e 9 , 2e 8 , 2e 7 , 2e 6 , 2e 5 molecules.
  • initial heating began at 50oC for 2mins followed by 10 min Attorney Docket No.5470.943.WO 95oC incubation.
  • NGS PCR Conditions Step Temperature (oC) Time (sec) Initial Denature 95 120 (S)-2-amino-3-(2-cyanopyridin-4-yl)propanoic acid (m-CNpyrA) (S1) O (Nitsche, et al. Org. Lett. 2019, 21 (12), 4709–4712).
  • Fmoc-AA-OH (5.0 equiv., 0.5 M in DMF)
  • WO Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) (5.0 equiv., 0.5 M in DMF)
  • DIPEA n,n-diisoproplyethylamine
  • the activated amino acid was then added to the vial and shaken at RT for 15 mins, after which the vial was drained and thoroughly washed with DCM (4 x 5 mL) and DMF (4 x 5 mL). This process was repeated until the full peptide sequence is prepared. All amino acids were protected with standard acid labile protecting groups. [0115] After linear synthesis, the resin was dried and cleaved with a cleavage cocktail (TFA/TIPS/H2O 95:2.5:2.5) at 37°C for 1 h. The resin was then filtered from the cleavage cocktail solution and the cleavage cocktail was evaporated under a stream of nitrogen.
  • a cleavage cocktail (TFA/TIPS/H2O 95:2.5:2.5)
  • SEI149 (Compound 2). SEI149 was acetylated on resin through a final coupling with excess acetic anhydride (40 equiv., 200 ⁇ L) and triethylamine (30 equiv., 200 ⁇ L) in a 1:1 mixture of DCM and DMF (2 mL) shaken at RT for 10 minutes.
  • acetylated peptide was cleaved and pelleted following the general method, then dissolved in DMSO (500 ⁇ L) and diluted into 1:1 MeCN/H 2 O (5 mL total) for purification by reverse phase preparatory HPLC following the general gradient.
  • SEI144 Compound 1
  • crude SEI144 was dissolved in DMSO (500 ⁇ L), diluted into 10 mM HEPES pH 7.5, 1 mM TCEP (5 mL total), and stirred at RT for 1 h. Once complete, the mixture was purified by reverse phase preparatory HPLC following the general gradient.
  • SEI144 was injected at 2.47, 7.40, 22.2, 66.6, and 200 nM.
  • SEI149 was injected at 0.033, 0.1, 0.3, 0.9, and 2.7 ⁇ M.
  • Binding sonograms were generated using a standard 1:1 binding kinetics model and kinetics constants determined using standard Biacore evaluation software.
  • General Procedure for Thermal Shift Assays [0124] Thermal melting curves were measured with 4 ⁇ M DUB, 10x SYPROTM Orange dye (ThermoFisher catalog number: S6650) and 0 – 100 ⁇ M peptide in 25 mM HEPES, 150 mM NaCl, pH 8.

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Abstract

Sont divulguées des bibliothèques d'affichage d'ARNm comprenant des peptides avec des acides aminés non canoniques et des procédés de fabrication des bibliothèques d'affichage d'ARNm qui peuvent comprendre une pluralité de molécules d'affichage d'ARNm comprenant chacune un peptide cyclisé selon la formule C-Xn-ncAA (II), chaque X étant un acide aminé quelconque, n étant un nombre entier de 1 à 30, ncAA étant une para-cyanopyridylalanine d'acide aminé non canonique, et chaque peptide cyclisé comprenant un pont pyridine-thiazoline formé par condensation entre la cystéine N-terminale et la para-cyanopyridylalanine.
PCT/US2024/010851 2023-01-10 2024-01-09 Bibliothèques d'affichage d'arnm et procédés d'utilisation Ceased WO2024151615A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140163201A1 (en) * 2009-02-04 2014-06-12 Bicycle Therapeutics Limited Multispecific peptides
US20210061860A1 (en) * 2011-12-28 2021-03-04 Chugai Seiyaku Kabushiki Kaisha Peptide-compound cyclization method
US20210079043A1 (en) * 2013-04-11 2021-03-18 Bicyclerd Limited Modulation of structured polypeptide specificity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140163201A1 (en) * 2009-02-04 2014-06-12 Bicycle Therapeutics Limited Multispecific peptides
US20210061860A1 (en) * 2011-12-28 2021-03-04 Chugai Seiyaku Kabushiki Kaisha Peptide-compound cyclization method
US20210079043A1 (en) * 2013-04-11 2021-03-18 Bicyclerd Limited Modulation of structured polypeptide specificity

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABDELKADER ELWY H., QIANZHU HAOCHENG, GEORGE JOSEMON, FRKIC REBECCA L, QIANZHU ] H, NITSCHE C, HUBER T, ABDELKADER E H, JACKSON J,: "Genetic Encoding of Cyanopyridylalanine for In-Cell Protein Macrocyclization by the Nitrile-Aminothiol Click Reaction", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, VERLAG CHEMIE, HOBOKEN, USA, vol. 61, no. 13, Hoboken, USA, XP093197269, ISSN: 1433-7851, DOI: .org/10.1002/anie.202114154 *
ISKANDAR SABRINA E, PELTON JARRETT M, WICK ELIZAVETA T, BOLHUIS DEREK L, BALDWIN ALBERT S, EMANUELE MICHAEL J, BROWN NICHOLAS G, B: "Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 145, no. 3, 25 January 2023 (2023-01-25), pages 1512 - 1517, XP093197273, DOI: 10.1021/jacs.2c11294 *

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