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WO2025106597A2 - Compositions et procédés pour modulateurs à base de peptides de papp-a - Google Patents

Compositions et procédés pour modulateurs à base de peptides de papp-a Download PDF

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Publication number
WO2025106597A2
WO2025106597A2 PCT/US2024/055798 US2024055798W WO2025106597A2 WO 2025106597 A2 WO2025106597 A2 WO 2025106597A2 US 2024055798 W US2024055798 W US 2024055798W WO 2025106597 A2 WO2025106597 A2 WO 2025106597A2
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WO
WIPO (PCT)
Prior art keywords
synthetic peptide
optionally substituted
peptide
group
alkyl
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.)
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PCT/US2024/055798
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English (en)
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WO2025106597A3 (fr
Inventor
Qi Hao
Dan L. Eaton
Janani SRIDAR
Matthew Alan BRATKOWSKI
Andrei LOAS
Bradley L. Pentelute
Vladimir AKHMETOV
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.)
Massachusetts Institute of Technology
Calico Life Sciences LLC
Original Assignee
Massachusetts Institute of Technology
Calico Life Sciences LLC
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Publication of WO2025106597A2 publication Critical patent/WO2025106597A2/fr
Publication of WO2025106597A3 publication Critical patent/WO2025106597A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24079—Pappalysin-1 (3.4.24.79)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2

Definitions

  • Pregnancy-associated plasma protein-A is a secreted zinc- metalloproteinase that increases insulin-like growth factor (IGF) availability through cleavage of IGF-binding proteins (IGFBPs).
  • IGF insulin-like growth factor
  • IGFBPs IGF-binding proteins
  • the present disclosure provides synthetic peptide inhibitors for PAPP-A.
  • the synthetic peptides of the disclosure can be used in a variety of in vitro and in vivo methods, as research reagents, for diagnostic purposes, and for therapeutic uses.
  • the present disclosure provides a synthetic peptide comprising the formula L 1 Z 1 L 2 Z 2 R 10 or a pharmaceutically acceptable salt thereof, wherein
  • L 1 is 7 to 10 amino acids in length
  • L 2 is an amino acid sequence having a distance of about 20-35 angstroms
  • Z 1 is a basic amino acid
  • Z 2 is a bond or serine
  • the distance between Z 1 and Z 2 is 14 amino acids in length.
  • Z 1 is selected from the group consisting of arginine, lysine, and histidine.
  • Z 1 is lysine.
  • Z 1 is arginine or homo-Arginine.
  • Z 1 is histidine.
  • Z 2 is serine.
  • the synthetic peptide comprises an amino acid sequence from Insulin Growth Factor Binding Protein 5 (IGFBP5).
  • L 1 comprises the amino acid sequence of PKHTRISEL.
  • L 2 comprises the amino acid sequence of AEAVKKDRRKKLT, optionally AEAVKKDRRKKLTQ.
  • R 10 is X- R 10b , wherein X is and R 10b is selected from the group consisting of:
  • R 10 is X-R 10b , wherein
  • R 10 is X-R 10b , X is a bond and R 10b is: [0009]
  • the N-terminus of the peptide is modified with a lipidated tag, optionally an albumin-binding tag.
  • the present disclosure provides a synthetic peptide comprising formula I or a pharmaceutically acceptable salt thereof:
  • R 1 is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)R la , and -C(O)OR la ;
  • R la is optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, wherein R la optionally comprises a fluorophore;
  • R 3a is optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl
  • R 4 is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)R 4a , -C(O)OR 4a and a peptide comprising 2-10 amino acids;
  • R 4a is optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl optionally substituted with a peptide comprising 2-10 amino acids;
  • R 6a is optionally substituted Ci-Ce alkyl substituted or optionally substituted phenyl, wherein R 6a is substituted with -SO2F or -OSO2NH2;
  • R 8 is a warhead
  • R 9a is optionally substituted Ci-Ce alkyl or optionally substituted phenyl, wherein R 9a is substituted with -SO2F or -OSO2NH2;
  • X is a bond, -C(O)-, or
  • R 1 is -C(O)R la and R 4 is -C(O)R 4a
  • R la is optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, optionally comprising a fluorophore
  • R 4a is optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl.
  • Ri is selected from the group consisting of:
  • R 4 is selected from the group consisting of:
  • R 5 is Ci-Ce alkyl substituted with -NH2
  • R 7 is Ci-Ce alkyl substituted with -NH2.
  • R 5 and R 7 are taken together to form a staple comprising 1-10 carbon atoms.
  • each of R 6 and R 9 is independently selected from the group consisting of: [0018]
  • R 6 is
  • R 8 is a warhead, and wherein a warhead is L-R 8a , wherein
  • L is selected from the group consisting of a bond, optionally substituted C1-C10 alkyl and optionally substituted C2-C10 alkenyl chain wherein 1-7 methylene units of the optionally substituted C1-C10 alkyl and optionally substituted C2-C10 alkenyl chain is each independently replaced with -C(O)NH-, -O- or -S-; and
  • R 8a is optionally substituted phenyl substituted with -N(H)C(O)-R 8b or -N(H)S(O)2- R 8b , wherein R 8b is optionally substituted C2-C6 alkenyl.
  • R 9 is Ci-Ce alkyl.
  • R 9 i is optionally substituted C2-C6 alkenyl.
  • R 8a is selected from the group consisting of: [0022]
  • X is
  • R 10 is selected from the group consisting of: [0023]
  • R 10 is:
  • X is and R 10 is selected from the group consisting of:
  • X is a bond and Rio is:
  • the synthetic peptide comprises formula II or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula III or a pharmaceutically acceptable salt thereof: [0029] In some embodiments, the synthetic peptide comprises formula IV or a pharmaceutically acceptable salt thereof:
  • the present disclosure provides a synthetic peptide comprising the amino acid sequence of PKHTRISELKAEAVKKDRRKKLTQS, PKHTRISELKAEAVKKDRRKKLTQ, PKHTRISELKAEAVKKDRRKKLT, ISELKAEAVKKDRRKKLTQS, ISELKAEAVKKDRRKKLTQ, ISELKAEAVKKDRRKKLT, or a pharmaceutically acceptable salt thereof, wherein the C- terminus of the peptide is modified with a zinc-binding chemical moiety.
  • the modification increases the synthetic peptide’s inhibition of pregnancy- associated plasma protein A (PAPP-A) by at least 1-3 fold relative to comparators.
  • the present disclosure provides a synthetic peptide comprising the amino acid sequence of PKHTRISELKAEAVKKDRRKKLTQS, PKHTRISELKAEAVKKDRRKKLTQ, PKHTRISELKAEAVKKDRRKKLT, or a pharmaceutically acceptable salt thereof, wherein the C- terminus of the peptide is modified with a zinc -binding chemical moiety.
  • a synthetic peptide as disclosed herein is truncated by 1-5 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 2-5 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 3-5 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 4 or 5 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 1-4 amino acid at the N-terminus.
  • a synthetic peptide as disclosed herein is truncated by 2-4 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 3 or 4 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 1-3 amino acid at the N-terminus In some embodiments, a synthetic peptide as disclosed herein is truncated by 2 or 3 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 1 amino acid at the N-terminus.
  • a synthetic peptide as disclosed herein is truncated by 2 amino acids at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 3 amino acids at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 4 amino acids at the N-terminus. In some embodiments, a synthetic peptide as disclosed herein is truncated by 5 amino acids at the N- terminus. Modified or unmodified amino acids are contemplated for truncation.
  • a synthetic peptide of the present disclosure comprises an amino acid sequence at least 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 99.5% identity, about 99.9% identity to the amino acid sequence of PKHTRISELKAEAVKKDRRKKLTQS.
  • a synthetic peptide of the present disclosure comprises an amino acid sequence at least 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 99.5% identity, about 99.9% identity to the amino acid sequence of PKHTRISELKAEAVKKDRRKKLTQ.
  • a synthetic peptide of the present disclosure comprises an amino acid sequence at least 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 99.5% identity, about 99.9% identity to the amino acid sequence of PKHTRISELKAEAVKKDRRKKLT.
  • a synthetic peptide of the present disclosure comprises an amino acid sequence at least 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 99.5% identity, about 99.9% identity to the amino acid sequence of ISELKAEAVKKDRRKKLTQS.
  • a synthetic peptide of the present disclosure comprises an amino acid sequence at least 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 99.5% identity, about 99.9% identity to the amino acid sequence of ISELKAEAVKKDRRKKLTQ.
  • a synthetic peptide of the present disclosure comprises an amino acid sequence at least 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 99.5% identity, about 99.9% identity to the amino acid sequence of ISELKAEAVKKDRRKKLT.
  • the zinc -binding chemical moiety is a secondary amine optionally substituted with at least one substituent selected from the group consisting of a carbonyl, sulfonamide, phosphate, phosphonate, carboxylic acid, amide, hydroxyamide, hydroxyalkyl, thioalkoxy, and alkyoxy.
  • the zinc-binding chemical moiety is hydroxamate
  • the N-terminus of the peptide is modified with a lipid, optionally a lipidated tag, optionally an albumin-binding tag.
  • the Pro in position 1 or the Lys in position 15 of the peptide is modified with a lipid, optionally a lipidated tag, optionally an albumin-binding tag.
  • the synthetic peptide shows no or a negligible amount of inhibition of pregnancy-associated plasma protein-A2 and metalloproteinases other than PAPP-A.
  • the synthetic peptide shows an increased half-life relative to comparators, some embodiments, the synthetic peptide has a half-life of about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, or about 20 hours in human serum, some embodiments, the synthetic peptide has a half-life of up to about 20 hours in human serum.
  • the synthetic peptide inhibits PAPP-A with an IC50 of 500 nM or less. In some embodiments, the synthetic peptide inhibits PAPP-A with an IC50 of 100 nM or less.
  • the present disclosure provides a synthetic peptide, wherein the synthetic peptide is any one of the peptides disclosed in Tables 7-10.
  • a synthetic peptide of the present disclosure comprises an amino acid sequence at least 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 99.5% identity, about 99.9% identity to an amino acid sequence disclosed in Tables 7-10.
  • the present disclosure provides a pharmaceutical comprising a synthetic peptide as disclosed herein and a pharmaceutically acceptable excipient.
  • the present disclosure provides a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a synthetic peptide as disclosed herein.
  • the disease or condition is an age-related disease.
  • the disease or condition is atherosclerosis.
  • administering the synthetic peptide intravenously, subcutaneously or intraperitoneally.
  • FIGS. 1A-1E show a schematic for the screening of various zinc-binding groups at the C-terminus of IGFBP5 -anchor peptide.
  • FIG. 2 shows a schematic and results from L-Alanine scanning of the peptide 13.
  • FIGS. 3A-3B show plots depicting results from the enzymatic degradation of the indicated peptides.
  • FIG. 4 shows the potency of IGFBP5-hydroxamate measured for PAPP -A inhibition, determined by the percentage of IGFBP4 and IGFBP5 cleavage.
  • FIG. 6 shows a plot depicting the selectivity of IGFBP5-hydroxamate, tested by measuring percentage of IGFBP5 cleavage by PAPP-A2.
  • FIG. 7A shows stability of IGFBP5-hydroxamate, measured by enzymatic cleavage and renal clearance in human serum for compounds AV-196PK, AV-196HT, AV-21a, and AV-164.
  • FIG. 7B shows inhibition of PAPP -A in human serum for AV-21a.
  • FIG. 8 shows the stability of IGFBP5-hydroxamate, measured by percentage of remaining peptide over 24 hours, with 50 pM of peptide in 100% human serum for compounds AV-221, AV-245PK, AV-245HT, and AV-233.
  • FIG. 9 shows the inhibition for recombinant PAPP-A for the indicated compounds.
  • FIG. 10 shows results from an A549-based assay that involved measuring the percentage of cleavage of IGFBP4/IGF1 for AV-21a and AV-245PK for secreted PAPP-A, and the percentage of cleavage of IGFBP4/IGF1 for AV-21a and AV-245PK for membranebound PAPP-A.
  • FIG. 11 shows the potency of lipidated IGFBP5-hydroxamate, tested by measuring inhibition with pregnancy serum PAPP-A for AV-245PK, and intact and cleaved IGFBP4.
  • FIG. 12 shows the PAPP-A/IGFBP5 complex and rationale for picking the modification positions.
  • FIG. 13 shows a strategy to incorporate covalent warheads.
  • FIG. 14 shows MALDI-MS of PAPP-A using different matrices.
  • FIG. 15 shows a schematic representation of reactides.
  • FIG. 16 shows the initial screening of the SO2F and OSO2NH2 reactides.
  • FIG. 17 shows a control experiment showing importance of covalent binders for PAPP-A staining.
  • FIG. 18 shows MALDI-MS of PAPP-A (grey) and mixture obtained after incubation of PAPP-A with reactide 1.
  • FIG. 19 shows selective PAPP-A staining in human serum.
  • FIG. 20 shows paralog specificity of the screened reactides.
  • FIG. 21 shows an experiment demonstrating that PAPP-A staining is sensitive to the catalytic groove availability.
  • FIG. 22 shows the inhibitory activity against PAPP-A for the indicated reactides.
  • FIG. 23 shows IGFBP5 -based reactides with acrylamide (AM) warheads.
  • FIG. 24 shows the inhibitory activity against PAPP-A of 147-cys mutants with AM warheads.
  • FIG. 25 shows the binding activity of reactides with AM warheads against PAPP-A 1 in presence of PAPP-A2.
  • FIG. 26 shows a potential experiment to target Cys with acrylamides.
  • FIG. 27 shows the binding activity of an IGFBP5-based fluorescent probe.
  • FIG. 28 shows labeling ability of NHOH-Zn interactions.
  • FIG. 29 shows the bioavailability from administering an exemplary peptide inhibitor to mice at various doses and via the indicated routes of administration.
  • FIG. 30 shows inhibition of IGFBP5 -anchor peptide featuring amide or carboxylic acid at the C-terminus.
  • FIGS. 31A-31C show a schematic and inhibition results from screening various zinc binding groups (ZBGs).
  • FIG. 32 shows the gel-based IC50 assay results for the indicated IGFBP5 sequence and alternative binding moieties based on IGFBP4 cleavage and PAPP-A autocleavage sites, all having hydroxamate at their C-termini.
  • FIG. 34 shows He 124 and Lys 128 hotspot interactions.
  • FIG. 35 shows a 2D-map for the identified hotspots in the PAPP-A inhibitory sequence.
  • FIG. 36 shows the gel-based IC50 results for the indicated C-terminus hydroxamate peptide variations.
  • FIGS. 37A-37E show a schematic and gel-based IC50 results for the IGFBP5-anchor peptide optimization.
  • FIGS. 38A-38B show flexible oligoethyleneglycol linkers capable of connecting two IGFBP5 -anchor peptide fragments coordinated to the PAPP-A catalytic grooves of two different subunits.
  • FIG. 39 shows the flexible oligoethyleneglycol linkers’ effects on affinity.
  • FIG. 40 illustrates the interaction between PAPP-A and the peptide inhibitor.
  • FIG. 41 shows the results from a fluorescence polarization study of the FITC-labelled peptides.
  • FIG. 42 shows the results from a fluorescence polarization study of the labelled peptides.
  • FIG. 43 shows the serum stability results for a mutated variation of the peptide.
  • FIG. 44 shows the serum stability results for a mutated variation of the peptide.
  • FIGS. 45A-45B show results from optimization conducted on the zinc-binding chemical moiety.
  • FIG. 46 shows a 25-30 angstrom distance between two hotspots on the peptide.
  • FIG. 47 shows IC50 range results from L-Alanine scanning.
  • FIG. 48 shows results from an A549-based assay from measuring the percentage of cleavage for AV-245PK for secreted PAPP -A.
  • FIG. 49 shows the A549 assay workflow.
  • FIG. 50 shows results from an A549 free PAPP -A conditioned media-based assay from measuring the percentage of cleavage of IGFBP4/IGF1 for AV-245PK for secreted PAPP-A.
  • FIG. 51 shows results from an A549 cell-based assay involved in measuring the percentage cleavage of IGFBP4/IGF 1 for AV-245PK for secreted PAPP-A.
  • FIG. 52 shows lipid-modified peptides inhibit PAPP-A in pregnancy serum.
  • the present disclosure is based, in part, upon synthetic peptide inhibitors of pregnancy-associated plasma protein-A (PAPP-A). These synthetic peptide inhibitors exhibit pronounced selectivity towards PAPP-A and over other members of metzincin family. Also disclosed herein are methods of making such synthetic peptides and methods of using such synthetic peptides for active site-directed inhibition of PAPP-A.
  • PAPP-A pregnancy-associated plasma protein-A
  • IGFBP5-anchor peptide Functionalization of the Insulin Growth Factor Binding Protein 5 (IGFBP5)-anchor peptide with zinc -binding groups (ZBGs) (also referred to herein as “zinc-binding chemical moieties”) allows for potent inhibitory activity against PAPP-A, whereas incorporation of lipidated-binding tags (e.g., albumin-binding tag) improves the pharmacokinetic properties of the peptide.
  • ZBGs zinc -binding groups
  • compositions and kits are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions and kits of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “is,” “are” or any other variation thereof, are intended to cover a non-exclusive inclusion. They are to be interpreted synonymously with the phrases “having at least” or “including at least”.
  • the term “consisting of’ refers to including, and being limited to, whatever follows the phrase “consisting of.”
  • the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise. Similarly, the term “consisting essentially of’ is intended to include embodiments encompassed by the term “consisting of’. [0108] As used herein, “about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, “about” will mean up to plus or minus 10% of the particular value.
  • variable or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges.
  • an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40
  • an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • residue refers to a position in a peptide and its associated amino acid identity.
  • modulation refers to an increase or decrease in the level of a target molecule or the function of a target molecule.
  • modulator refers to modulation of (e.g., an increase or decrease in) the level of a target molecule or the function of a target molecule.
  • peptide refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins but is commonly shorter in length.
  • the shortest peptide is a “dipeptide” consisting of two amino acids joined by a peptide bond.
  • a peptide has an amino end and a carboxyl end, unless it is a cyclic peptide.
  • polypeptide refers to a single linear chain of amino acids bonded together by peptide bonds and preferably comprises at least five amino acids.
  • a polypeptide can be one chain or may be composed of more than one chain, held together by covalent bonds, e.g. disulfide bonds and/or non-covalent bonds.
  • the peptides or polypeptides are suspended in a liquid solution.
  • a liquid solution include water, aqueous buffer mixtures, acidic or basic solutions, organic solvents such as alcohol or acetonitrile, or any combination thereof.
  • Suitable amino acids include, without limitation, natural alpha-amino acids such as D- and L-isomers of the 20 common naturally occurring alpha-amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as provided in Table A depicted below), non-canonical alpha-amino acids (as depicted in Table B below), natural beta-amino acids (e.g., beta-alanine), and unnatural beta-amino acids.
  • natural alpha-amino acids such as D- and L-isomers of the 20 common naturally occurring alpha-amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as provided in Table A depicted below), non-can
  • Amino acids used in the construction of peptides of the present disclosure may be prepared by organic synthesis, or obtained by other means, including, but not limited to, degradation of or isolation from a natural source, and automated peptide synthesis. Additional examples of amino acids and methods for synthesis are described in U.S. Patent No.
  • Percent “identity” between a peptide sequence and a reference sequence is defined as the percentage of amino acid residues in the peptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent 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, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE 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.
  • biotinylation refers to the addition of a biotin.
  • glycosylation refers to the addition of a glycosyl group, usually to, but not limited to an arginine, an asparagine, a cysteine, a hydroxy lysine, a serine, a threonine, a tyrosine, or a tryptophan residue, resulting in a glycoprotein.
  • glycosyl group refers to a substituent structure obtained by removing the hemiacetal hydroxyl group from the cyclic form of a monosaccharide and, by extension, of a lower oligosaccharide.
  • sulfation refers to the addition of a sulfo group usually to a tyrosine residue.
  • a sulfo group refers to group SO3H-, derived from sulfuric acid.
  • phosphorylation refers to the addition of a phosphate group. Phosphorylation commonly occurs at the serine, threonine, tyrosine, arginine, lysine, or cysteine residues. It can alter the structural conformation of a protein, causing it to become activated, deactivated, or modifying its function.
  • methylation refers to the addition of a methyl group.
  • a methyl group refers to an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms (-CH3). Methylation can commonly occur at the arginine or lysine amino acid residues.
  • hydroxylation refers to the addition of a hydroxyl group (- OH).
  • acetylation refers to the addition of an acetyl group.
  • An acetyl group contains a methyl group single-bonded to a carbonyl.
  • warhead refers to a moiety of an inhibitor which participates, either reversibly or irreversibly, with the reaction of a donor, e.g., a protein, with a substrate. Warheads may, for example, form covalent bonds with the protein, or may create stable transition states, or be a reversible or an irreversible alkylating agent.
  • a covalent warhead as disclosed herein, can be a functional group on a peptide that can participate in a covalent bond-forming reaction, wherein a new covalent bond is formed between a portion of the warhead and a donor, for example, an amino acid residue of a protein.
  • the warhead is an electrophile and the “donor” is a nucleophile, such as the side chain of a cysteine residue.
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC), and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • HPLC high pressure liquid chromatography
  • a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess).
  • an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form.
  • enantiomerically pure or “pure enantiomer” denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the enantiomer.
  • the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
  • an enantiomerically pure compound can be present with other active or inactive ingredients.
  • a pharmaceutical composition comprising an enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound.
  • the enantiomerically pure R-compound in such compositions can, for example, comprise, at least about 95% by weight R-compound and at most about 5% by weight S-compound, by total weight of the compound.
  • a pharmaceutical composition comprising enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound.
  • the enantiomerically pure S- compound in such compositions can, for example, comprise, at least about 95% by weight S- compound and at most about 5% by weight R-compound, by total weight of the compound.
  • the active ingredient can be formulated with little or no excipient or carrier.
  • the term “kd” (s' 1 ) refers to the dissociation rate constant between a given entity and a target (e.g., of a particular peptide-target interaction). This value is also referred to as the kofi value.
  • k a (M' 1 x s' 1 ) refers to the association rate constant of a given entity and a target (e.g., a particular peptide-target interaction). This value is also referred to as the k on value.
  • KD kd/ka.
  • KA ka/kd.
  • the affinity of a molecule X for its target Y can be represented by the dissociation equilibrium constant (KD).
  • KD dissociation equilibrium constant
  • the kinetic components that contribute to the dissociation equilibrium constant are as described above.
  • Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).
  • SPR surface plasmon resonance
  • BIACORE® BIACORE®
  • FORTEBIO® biolayer interferometry
  • C1-C6 alkyl is intended to encompass, Cl, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4- C5, and C5-C6 alkyl.
  • aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 p electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”).
  • aromatic ring system e.g., having 6, 10, or 14 p electrons shared in a cyclic array
  • an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl).
  • an aryl group has ten ring carbon atoms (“CIO aryl”; e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl).
  • An aryl group may be described as, e.g., a C6-C 10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
  • Aryl groups include, but are not limited to, phenyl, naphthyl, indenyl, and tetrahydronaphthyl.
  • Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is unsubstituted C6-C14 aryl.
  • the aryl group is substituted C6-C14 aryl.
  • Halo or “halogen,” independently or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.
  • halide by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom. In certain embodiments, the halo group is either fluorine or chlorine.
  • aliphatic or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule.
  • aliphatic groups can contain 1-6 aliphatic carbon atoms.
  • aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-26 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms.
  • cycloaliphatic refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
  • Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • haloaliphatic refers to an aliphatic group that is substituted with one or more halogen atoms.
  • alkyl refers to a straight or branched alkyl group.
  • exemplary alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.
  • haloalkyl refers to a straight or branched alkyl group that is substituted with one or more halogen atoms.
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized forms of nitrogen or sulfur, and any quatemized form of a basic nitrogen.
  • Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
  • heteroaryl and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
  • Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin- 3(4H)-one.
  • heteroaryl group may be mono- or bicyclic.
  • heteroaryl may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.
  • heteroarylkyl refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
  • heterocycle As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
  • nitrogen includes a substituted nitrogen.
  • the nitrogen may be N (as in 3,4- dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in TV-substituted pyrrolidinyl).
  • a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted.
  • saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
  • heterocycle refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
  • heteroalkyl refers to an alkyl group as described herein which further includes at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus within the parent chain (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • heteroatom e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms
  • a heteroalkyl as disclosed herein is a C1-C33 heteroalkyl (e.g., a Cl heteroalkyl, C2 heteroalkyl, C3 heteroalkyl, C4 heteroalkyl, C5 heteroalkyl, C6 heteroalkyl, C7 heteroalkyl, C8 heteroalkyl, C9 heteroalkyl, CIO heteroalkyl, CI I heteroalkyl, C12 heteroalkyl. Cl 3 heteroalkyl, C14 heteroalkyl. Cl 5 heteroalkyl, C16 heteroalkyl, C17 heteroalkyl. Cl 8 heteroalkyl, C19 heteroalkyl, C20 heteroalkyl, C21 heteroalkyl.
  • a Cl heteroalkyl e.g., a Cl heteroalkyl, C2 heteroalkyl, C3 heteroalkyl, C4 heteroalkyl, C5 heteroalkyl, C6 heteroalkyl, C7 heteroalkyl, C8 heteroalkyl, C9 heteroalky
  • partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
  • partially unsaturated is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • compounds of the invention may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • Suitable monovalent substituents on R° are independently halogen, — (CH 2 )O- 2 R*, -(haloR*), — (CH 2 )o- 2 OH, — (CH 2 )o- 2 OR*, — (CH 2 )o- 2 CH(OR*) 2 ; — O(haloR’), — CN, — N3, — (CH 2 )O- 2 C(0)R*, — (CH 2 )O- 2 C(0)OH, — (CH 2 )O- 2 C(0)OR*, — (CH 2 )O- 2 SR*, — (CH 2 )o- 2 SH, — (CH 2 )O- 2 NH 2 , — (CH 2 )O- 2 NHR*, — (CH 2 )O- 2 NR* 2, — N0 2 , — SiR* 3,
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: — O(CR* 2 ) 2 -3O — , wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R* include halogen, — R*, -(haloR*), —OH, —OR*, — O(haloR’), — CN, — C(O)OH, — C(O)OR*, — NH 2 , — NHR*, —NR* 2, or — NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, — CH 2 Ph, — 0(CH 2 )o-iPh, or a 5-6-membered saturated, partially unsaturated, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include — R T , — NR T 2 , — C(O)R T , — C(O)OR T , — C(O)C(O)R T , — C(O)CH 2 C(O)R T , — S(O) 2 R T , — S(O) 2 NR : 2 , — C(S)NR : 2 , — C(NH)NR : 2 , or — N(R T )S(O) 2 R T ; wherein each R' is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted — OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R'.
  • Suitable substituents on the aliphatic group of R' are independently halogen, — R*, - (haloR*), —OH, —OR*, — O(haloR’), — CN, — C(O)OH, — C(O)OR*, — NH 2 , — NHR*, — NR* 2, or — NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, — CH2PI1, — 0(CH 2 )o- iPh, or a 5-6-membered saturated, partially unsaturated, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • lipidation refers to the covalent attachment of a lipid group, or a modified lipid group, to an amino acid in a peptide disclosed herein. As used herein, it may also be referred to as the addition of a “lipidated tag”. Without being limited to the mechanism of action, the lipidated tags can be used herein to increase the pharmacokinetic properties including, but not limited to, half-life relative to baseline and/or comparators.
  • a presently disclosed synthetic peptide is provided in the form of a pharmaceutical salt.
  • the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.
  • Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N(Ci-4alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, boronate, and aryl sulfonate.
  • the term “stapling” refers to a synthetic methodology wherein two olefin-containing sidechains present in a polypeptide chain are covalently joined (e.g., “stapled together”) using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, the cover art for J Org. Chem. (2001) vol. 66, issue 16 describing metathesis-based crosslinking of alpha-helical peptides; Blackwell et al.; Angew Chem. Int. Ed. (1994) 37:3281).
  • RCM ring-closing metathesis
  • peptide stapling encompasses the joining of two double bond-containing sidechains, two triple bond-containing sidechains, or one double bond-containing and one triple bond-containing side chain, which may be present in a polypeptide chain, using any number of reaction conditions and/or catalysts to facilitate such a reaction, to provide a singly “stapled” polypeptide.
  • hydrocarbon stapling is contemplated.
  • the staples also comprise nitrogen.
  • one-component stapling is contemplated in the disclosed synthetic peptides.
  • two-component stapling is disclosed.
  • One- component stapling refers to direct cyclisation between two side-chains, whilst two- component stapling refers to utilizing a separate bifunctional linker to bridge the two side- chains together.
  • peptide stapling using an all-hydrocarbon cross-link can help maintain the peptides native conformation and/or secondary structure, particularly under physiologically relevant conditions (see Schafmiester, et al., J. Am. Chem. Soc. (2000) 122:5891-5892; Walensky et al., Science (2004) 305: 1466- 1470).
  • stapling a polypeptide by an all-hydrocarbon crosslink predisposed to have an alpha-helical secondary structure can constrain the polypeptide to its native alpha-helical conformation, which could, for example, increase the peptide’s resistance to proteolytic cleavage, may increase the peptide’s hydrophobicity, allow for better penetration of the peptide into the target cell’s membrane (e.g., through an energy-dependent transport mechanism such as pinocytosis), and/or may lead to an improvement in the peptide’s biological activity relative to a corresponding uncrosslinked peptide.
  • sample refers to any fluid, cell, tissue, organ or a portion thereof. It also includes, but is not limited to, whole blood, plasma, serum, urine, saliva, tears, spinal fluid, synovial fluid, cell lysate, tissue lysate, exosomes, individual cell organelles, or any combination thereof.
  • the sample can be from any organism. It includes, but is not limited to human, mouse, yeast, worm, fish, bacteria, etc.
  • biological sample includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.
  • Examples of such purposes include, but are not limited to, blood transfusion, organ transplantation, biological specimen storage, and biological assays.
  • a “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered as part of a dosing regimen to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
  • the effective amount of a provided compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • treatment refers to partially or completely alleviating, inhibiting, delaying onset of, preventing, ameliorating and/or relieving a disorder or condition, or one or more symptoms of the disorder or condition, as described herein.
  • treatment may be administered after one or more symptoms have developed.
  • the term “treating” includes preventing or halting the progression of a disease or disorder.
  • treatment may be administered in the absence of symptoms.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • the term “treating” includes preventing relapse or recurrence of a disease or disorder.
  • administering or “administration of’ a peptide or composition thereof to a subject can be carried out using a variety of methods known to those skilled in the art.
  • a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct).
  • a peptide can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow, or controlled release of the compound or agent.
  • the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug or therapeutic.
  • subject refers to an animal, preferably a mammal, and most preferably a human.
  • compositions of the compounds disclosed herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene
  • a “pharmaceutically acceptable derivative” means any non-toxic salt, ester, salt of an ester or other derivative of a compound of this disclosure that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this disclosure or an inhibitorily active metabolite or residue thereof.
  • age-related disease refers to disorders or diseases in which aging is a major risk factor.
  • age-related disease refers to atherosclerosis. See, e.g., Conover CA, Exp Gerontol. 2013 Jul;48(7):612-3.
  • the term “comparators” refers to, without limitation, natural PAPP -A inhibitors, anti-PAPP-A antibodies, or unmodified IGFBP5 anchor sequences as described herein.
  • a list of natural PAPP -A inhibitors, which can be used as comparators, includes but is not limited to pro-MBP, stanniocalcin- 1 (STC1), stanniocalcin-2 (STC2).
  • a list of anti- PAPP-A antibodies which can be used as comparators, includes but is not limited to metalloprotease inhibitors (e.g., 1,10-phenanthroline), polyclonal antibodies, monoclonal antibodies, and pappalysin-1 (Botkjser, et al., Scientific Reports , 2019; Monget, et al., Ann. Endocrinol (Paris), 2016; AU2009200138B2, ThermoFisher).
  • metalloprotease inhibitors e.g., 1,10-phenanthroline
  • polyclonal antibodies e.g., 1,10-phenanthroline
  • monoclonal antibodies e.g., monoclonal antibodies
  • pappalysin-1 e.g., pappalysin-1
  • PAPP-A Pregnancy-associated plasma protein-A
  • IGF insulin-like growth factor
  • IGFBPs IGF-binding proteins
  • IGF-signaling at later stages of life has been associated with various physiological changes and age-related diseases. While PAPP-A has emerged as a potential target for interventions aimed at modulating IGF-signaling, there is a lack of active site directed inhibitors of PAPP-A.
  • IGF signaling plays a crucial role in regulating cell proliferation, differentiation, and survival (Roith, N. Engl. J. Med., 1997).
  • the IGF pathway is regulated by the IGFBP family that binds IGF1 and IGF2, preventing them from interactions with the cell surface receptor IGF1R (Allard, et al., Front. Endocrinol., 2018). Release and bioavailability of IGFs are mediated by PAPP -A and PAPP-A2 , which cleave IGFBP2,4,5 and IGFBP3,5, respectively (Judge, et al., Nature Communications, 2022).
  • PAPP-A inhibition also improves health span by reducing age-related pathology progression (Conover, et al., The Journals of Gerontology, 2010; Vallejo, et al., PNAS, 2009; Tanner, et al., Journal of Bone and Mineral Research, 2009; Conover, et al., Journal of Cardiovascular Translational Research, 2016; Conover, et al., American Journal of Physiology - Endocrinology and Metabolism,' Harrington, et al., Circulation Research, 2WU; Heitzeneder, et al., Journal of the National Cancer Institute, 2019; Kashyap, et al., JCI Insight, 2020; Torres, et al., PLoS ONE, 2019).
  • Inhibitors can also be developed by targeting the pockets adjacent to the active site(S3-S3’). However, in some circumstances, access to more distant recognition (allosteric) sites can provide more selective inhibition.
  • Peptides represent a great binding modality having major advantages such as the ability to cover a large area of PPI and higher specificity compared to small molecules, and lower production costs together with lower immunogenicity compared to biologies.
  • linker domain of IGFBP5 contains an anchor sequence recognizing the catalytic groove of PAPP-A and providing direct access to the active site (Judge, et al., Nature Communications, 2022).
  • the inventors of the instant disclosure identified and developed synthetic peptides that provide potent and selective inhibition of PAPP-A. These peptides are a great modality to gain a facile access to those allosteric sites. These peptides include a modified IGFBP5 anchor sequence bound to a zinc-binding chemical moiety.
  • a Lys (16 positions away from the cleavage site) is crucial for the PAPP-A mediated peptide probe cleavage.
  • the synthetic peptide disclosed herein provide a dual binding mode via the Lys 16 positions away from the cleavage site and the zinc binding moiety.
  • the functionalization of the IGFBP5-anchor peptide with ZBGs, as disclosed herein, allows for gaining potent inhibitory activity against PAPP-A, whereas incorporation of albumin-binding tags improve pharmacokinetic properties of the peptide.
  • IGFBP5 anchor sequence is PKHTRISELKAEAVKKDRRKKLTQS .
  • the lysine residue in position 10 and terminal serine residue are two major points of interactions. That lysine residue may be substituted with any residue having an aliphatic chain or charged group. That serine residue is modified with a zinc -binding chemical moiety, e.g. a hydroxamate, , or a thiol group.
  • a zinc -binding chemical moiety e.g. a hydroxamate, , or a thiol group.
  • the distance between these two positions is about 14 amino acids in length. In some embodiments, the distance between these two hotspot positions is 20-35 angstrom. See e.g. FIG. 46.
  • a synthetic peptide as disclosed herein comprises the formula L 1 Z 1 L 2 Z 2 R 10 or a pharmaceutically acceptable salt thereof, wherein
  • L 1 is 7 to 10 amino acids in length
  • L 2 is an amino acid sequence having a distance of about 20-35 angstroms
  • Z 1 is a basic amino acid
  • Z 2 is a bond or serine
  • R 10 is selected from the group consisting of -R 10a , -OH, - NH 2 , -N(H)0H, -N(H)R 10a , -
  • each R 10a is independently selected from the group consisting of optionally substituted
  • PAPP-A pregnancy-associated plasma protein A
  • the distance between Z 1 and Z 2 is 14 amino acids in length.
  • Z 1 is selected from the group consisting of arginine, lysine, and histidine In some embodiments, Z 1 is lysine. In some embodiments, Z 1 is arginine or homo-Arginine. In some embodiments, Z 1 is histidine. In some embodiments, Z 2 is serine.
  • the synthetic peptide comprises an amino acid sequence from Insulin Growth Factor Binding Protein 5 (IGFBP5).
  • L 1 comprises the amino acid sequence of PKHTRISEL.
  • L 2 comprises the amino acid sequence of AEAVKKDRRKKLT, optionally AEAVKKDRRKKLTQ.
  • R 10 is X- R 10b , wherein X is and R 10b is selected from the group consisting of:
  • R 10 is X-R 10b , wherein and
  • R 10 is X-R 10b , X is a bond and R 10b is:
  • the synthetic peptide comprises formula II or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula II- 1 or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula II-2 or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula Ila or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula lib or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula lie or a pharmaceutically acceptable salt thereof: [0198] In some embodiments, the synthetic peptide comprises formula lid or a pharmaceutically acceptable salt thereof: [0199] In some embodiments, the synthetic peptide comprises one the R 1 , R 2 , R 3 , R 4 , R 5 , R 6 ,
  • the synthetic peptide comprises formula III or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula Illa or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula Illb or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula IIIc or a pharmaceutically acceptable salt thereof: [0204] In some embodiments, the synthetic peptide comprises formula Illd or a pharmaceutically acceptable salt thereof: [0205] In some embodiments, the synthetic peptide comprises one the R 1 , R 2 , R 3 , R 4 , R 5 , R 6 ,
  • the synthetic peptide comprises formula IV or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula IVa or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula IVb or a pharmaceutically acceptable salt thereof:
  • the synthetic peptide comprises formula IVc or a pharmaceutically acceptable salt thereof: [0210] In some embodiments, the synthetic peptide comprises formula IVd or a pharmaceutically acceptable salt thereof: [0211] In some embodiments, the synthetic peptide comprises one the R 1 , R 2 , R 3 , R 4 , R 5 , R 6 ,
  • R 4 is a lipid. In some embodiments, R 4 is a lipidated tag, optionally an albumin-binding tag. In some embodiments, R 1 is a lipid. In some embodiments, R 1 is a lipidated tag, optionally an albumin-binding tag. [0213] In some embodiments, (e.g., formulae Ila, lie, lid, Illa, IIIc, Illd, IVa, IVc, or Ivd), as used herein, R 4 is or
  • R 1 is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)R la , and -C(O)OR la ;
  • R la is optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, wherein R la optionally comprises a fluorophore.
  • R 3a is optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl;
  • R 4 is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)R 4a , -C(O)OR 4a and a peptide comprising 2-10 amino acids; wherein R 4a is optionally substituted C1-C33 alkyl or optionally substituted Ci- C33 heteroalkyl optionally substituted with a peptide comprising 2-10 amino acids.
  • R 5 and R 7 are taken together to form a staple comprising 1-10 carbon atoms.
  • R 6a is optionally substituted Ci-Ce alkyl substituted or optionally substituted phenyl, wherein R 6a is substituted with -SO2F or -OSO2NH2;
  • R 8 is a warhead
  • R 9a is optionally substituted Ci-Ce alkyl or optionally substituted phenyl, wherein R 9a is substituted with -SO2F or -OSO2NH2.
  • R 1 is -C(O)R la and R 4 is -C(O)R 4a
  • R la is optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, optionally comprising a fluorophore
  • R 4a is optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl.
  • Ri is selected from the group consisting of:
  • R 1 is
  • R 3 is selected from the group consisting of:
  • R 4 is selected from the group consisting of:
  • R 5 is Ci-Ce alkyl substituted with -NH2
  • R 7 is Ci-Ce alkyl substituted with -NH2.
  • R 5 and R 7 are taken together to form a staple comprising 1-10 carbon atoms.
  • each of R 6 and R 9 is independently selected from the group consisting of:
  • R 8 is a warhead, and wherein a warhead is L-R 8a , wherein L is selected from the group consisting of a bond, optionally substituted Ci-Cio alkyl and optionally substituted C2-C10 alkenyl chain wherein 1-7 methylene units of the optionally substituted C1-C10 alkyl and optionally substituted C2-C10 alkenyl chain is each independently replaced with -C(O)NH-, -O- or -S-; and R 8a is optionally substituted phenyl substituted with -N(H)C(O)-R 8b or -N(H)S(O)2-
  • R 8b wherein R 8b is optionally substituted C2-C6 alkenyl.
  • R 9 is Ci-Ce alkyl. In some embodiments, R 9 is . In some embodiments, L is: [0235] In some embodiments, R 8a is selected from the group consisting of:
  • the present disclosure provides a synthetic peptide comprising the amino acid sequence of PKHTRISELKAEAVKKDRRKKLTQS, PKHTRISELKAEAVKKDRRKKLTQ, PKHTRISELKAEAVKKDRRKKLT, ISELKAEAVKKDRRKKLTQS, ISELKAEAVKKDRRKKLTQ, ISELKAEAVKKDRRKKLT, or a pharmaceutically acceptable salt thereof, wherein the C- terminus of the peptide is modified with a zinc -binding chemical moiety.
  • zinc-binding chemical moiety As used herein, the term “zinc-binding chemical moiety,” “zinc-binding group” or “ZBG” refers to refers a moiety with the ability to coordinate with zinc ions (Zn 2+ ).
  • a zinc -binding chemical moiety includes a secondary amine optionally substituted with at least one substituent selected from the group consisting of a carbonyl, sulfonamide, phosphate, phosphonate, carboxylic acid, amide, hydroxyamide, hydroxyalkyl, thioalkoxy, and alkyoxy.
  • the zinc-binding chemical moiety is hydroxamate, , or a thiol group.
  • a zinc -binding chemical moiety includes X-R 10b , wherein X is A NC
  • a zinc -binding chemical moiety includes X-R 10b , wherein X is and R 10b is ;
  • a zinc -binding chemical moiety includes X-R 10b , X is a bond and R 10b is:
  • One approach to modulate pharmacokinetic profiles and improve the potency and selectivity of a potential drug is the exploitation of covalent binding. Stability issues in peptides can be addressed via various strategies such as cyclization, incorporation of D- and non-canonical amino acids, and backbone modifications. Irreversible covalent inhibition of an interaction can results in increased potency, selectivity, sustained pharmacodynamics, and could alleviate the effects of fast renal elimination. Therapeutic peptides may benefit from a covalent binding mode of action and alleviate pharmacokinetic limitations of this class of therapeutics.
  • the synthetic peptide disclosed herein has one or more modifications to the IGFBP5 anchor sequence as disclosed herein.
  • the present disclosure contemplates any combination of these modifications.
  • the N- or C- terminus of the peptide is modified with a chemical moiety.
  • a synthetic peptide of the present disclosure comprises a lipidated tag.
  • a lipidated tag increases the half life of synthetic peptide, e.g. in human serum.
  • Non-limiting examples of lipidation include Myristoylation, Palmitoylation, Glycosylphosphatidylinositol (GPI)-anchor addition, and Prenylation.
  • a synthetic peptide as disclosed herein has a half-life of between about 1-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 2-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 3-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 4-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 5-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 6- 20 hours in human serum.
  • a synthetic peptide as disclosed herein has a half-life of between about 7-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 8-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 9- 20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 10-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 11-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 12-20 hours in human serum.
  • a synthetic peptide as disclosed herein has a half-life of between about 13-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 14-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 15-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 16-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 17-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 18-20 hours in human serum.
  • a synthetic peptide as disclosed herein has a half-life of between about 19-20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 8- 12 hours, about 9-12 hours, or about 10-12 hours in human serum.
  • a synthetic peptide as disclosed herein has a half-life of about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, or about 20 hours in human serum. In some embodiments, a synthetic peptide as disclosed herein has a half-life of up to about 20 hours in human serum.
  • a synthetic peptide disclosed herein has one or more modifications to its sequence.
  • the modification is at the N- or C- terminus of the peptide.
  • the modification is on a side chain of an amino acid in the peptide.
  • the modification is a substitution of one or more L-amino acid with a D-amino acid.
  • the modification is a substitution of an a-amino acid with a [3-amino acids.
  • the peptide comprises a substituted amino acid.
  • the substituted amino acid is a canonical amino acid.
  • Canonical amino acids are known to those of ordinary skill in the art. Non-limiting examples of canonical amino acids for use in substitutions are listed in TABLE A.
  • the canonical substituted amino acids are an Ala, a Ser, a Gin, or an Arg.
  • the synthetic peptide comprises one or more non- canonical amino acids (also referred to as ncAAs or unnatural amino acids).
  • Non-canonical amino acids are known to those of ordinary skill in the art.
  • Non-limiting examples of non- canonical amino acids that can be used for substitution are shown in TABLE B.
  • the amino acids of the synthetic peptide are mixed canonical and non-canonical amino acids.
  • a “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution of an amino acid with a chemically or functionally similar amino acid.
  • Table 1 Selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.
  • Table 2 Additional selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.
  • Table 3 Further selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.
  • Methods for producing synthetic peptide or peptidomimetic of the disclosure are known in the art such as solid phase peptide synthesis (SPPS), Fmoc-based synthesis, and Boc-based synthesis by an automatic peptide synthesizer.
  • SPPS solid phase peptide synthesis
  • Fmoc-based synthesis Fmoc-based synthesis
  • Boc-based synthesis by an automatic peptide synthesizer.
  • peptides can be chemically synthesized using the sequence information provided herein and using peptide synthesis methods known in the art.
  • the produced synthetic peptide or peptidomimetic can be modified during or after peptide synthesis with several modifications, for example with a lipidated tag, a protective group, or pegylation.
  • the peptide may be modified at its amino terminus or carboxy terminus or protected by various organic groups for protecting the peptide from protein-cleaving enzymes in vivo while increasing its stability.
  • the produced synthetic peptide can then be purified further. Purification strategies for peptides are known in the art, and include FPLC and HPLC based methods.
  • a synthetic peptide or peptidomimetic disclosed herein preferably is combined with a pharmaceutically acceptable carrier and/or an excipient.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • compositions refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • compositions containing a synthetic peptide disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration, e.g., oral administration.
  • the pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form will depend upon the intended mode of administration and therapeutic application.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration.
  • Sterile solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization.
  • dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent, described herein, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • pharmaceutically acceptable excipient refers to a non-toxic carrier, adjuvant, diluent, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
  • Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical compositions of the invention are any of those that are well known in the art of pharmaceutical formulation and include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils.
  • compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial
  • any of the synthetic peptides disclosed herein is assembled into a pharmaceutical or diagnostic or research kit to facilitate their use in therapeutic, diagnostic or research applications.
  • a kit may include one or more containers housing any of the systems or vectors disclosed herein and instructions for use.
  • the kit may be designed to facilitate use of the methods described herein by researchers and can take many forms.
  • Each of the compositions of the kit may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure.
  • Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.
  • H-Rink Amide-ChemMatrix resin was purchased from PCAS BioMatrix Inc.
  • Amino acids Fmoc-Ala-OH, Fmoc-[3-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(/Bu)-OH.
  • Fmoc-Trp(Boc)-OH, Fmoc- Tyr(/Bu)-OH. and Fmoc-Val-OH were purchased from Novabiochem (Billerica, MA).
  • Fmoc- Lys(Alloc)-OH, _OEG_C 18_Om_TAMRA_fitc_Bodipy_aliphatic linkers, ([2-[2-(Fmoc- amino)ethoxy] ethoxy] acetic acid), IGFl_IGFBP4_IGFBP5_PAPPA_pappa2_ were purchased from Combi-blocks (Billerica, MA).
  • Palladium tetrakistriphenylphosphine(O) (Pd(PPh3)4) was purchased from Sigma Aldrich.
  • Diisopropylethylamine (DIEA; biotech, grade; 99.5%) was purchased from Millipore Sigma and purified by a Seca Solvent Purification system from Pure Process Technology (Nashua, NH).
  • Trifluoroacetic acid Trifluoroacetic acid (TFA; for HPLC, >99%), triisopropylsilane (TIPS; 98%) were purchased from Sigma-Aldrich (St. Louis, MO).
  • Reagents used in peptide post-synthesis modifications Acetic anhydride (> 98%) was purchased from Sigma-Aldrich (St. Louis, MO), 5-TAMRA (5- carboxytetramethylrhodamine) and Biotin-PEG4-carboxylic acid from ChemPep.
  • Bovine serum albumin BSA
  • BSA Bovine serum albumin
  • THRA Human Thyroid Hormone Receptor alpha 1 protein
  • AFPS Automated flow peptide synthesis
  • the first two pumps are activated for 8 pumping strokes to prime the coupling agent and amino acid before the DIEA pump is activated.
  • the three pumps are then actuated together for a period of 7 pumping strokes, after which the activating agent pump and amino acid pump are switched using a rotary valve to select DMF.
  • the three pumps are actuated together for a final 8 pumping strokes, after which the DIEA pump is shut off, and the other two pumps continue to wash the resin for another 40 pump strokes.
  • two HPLC pumps are used. Using a rotary valve, one HPLC pump selects deprotection stock solution and DMF. The pumps are activated for 13 pump strokes. Both solutions are mixed in a 1: 1 ratio.
  • the rotary valves select DMF for both HPLC pumps, and the resin is washed for an additional 40 pump strokes.
  • the coupling-deprotection cycle is repeated for all additional monomers.
  • the reaction vessel was sealed tightly, removed from the glovebox and allowed to stir at room temperature for 4 h during which time a precipitate formed.
  • the reaction mixture was opened to atmosphere, and cold pentane (2 mL) was added.
  • the vial was centrifuged, and the supernatant decanted. Additional cold pentane (2 mL) was added, the vial was centrifuged, and the supernatant decanted. Drying under high vacuum afforded the desired product as a solid.
  • the resulting mixture was sonicated briefly, and then transferred to the fritted syringe containing the resin. Coupling was performed for 30 minutes. The resin was washed with DMF (3 x 5 mL). Fmoc deprotection was done by treating the resin with 20% piperidine in DMF (3 mL) for 5 minutes and this step was repeated twice. The resulting resin was then washed with DMF (3 x 5 mL). The synthetic cycle was repeated to completion of the peptide sequence.
  • Fmoc- Lys(Aloc)-OH is incorporated manually to enable lipidation during the later steps.
  • the N- terminal amine was protected by incubating the resin with 5 equivalents of BOC2O (20 min x2).
  • peptidyl resin was washed with DCM (5 mL x3) and then treated with Pd(PPh3)4 (3 equivalents) and phenylsilane (50 equivalents) in DCM (for 30 minutes at room temperature).
  • the resin was then drained and washed with DCM (5 mL x3), DMF (5 mL x3), 20 mM DMF solution of sodium diethyldithiocarbamate trihydrate (5 mL x3) and again DMF (5 mL x3).
  • the peptidyl resin was then manually coupled with Fmoc-PEG2-CH2COOH, Fmoc-PEG2-CH2COOH, Fmoc-Glu(OH)-OtBu, and 18-(tert-Butoxy)-18-oxooctadecanoic acid].
  • the resin was washed with DCM (5mL x3) and reacted with 5 equivalents 4-Nitrophenyl chloroformate in DCM (20 min x2). The resin was then incubated with 0.5 M DIPEA in DMF for 20 min and washed with DMF (5 mL x3). After the activation, the peptides were cleaved by mixing the resin with 10 equivalents of the corresponding nucleophile and 15 equivalents of DIPEA at room temperature overnight. The liquid fraction was collected, and the resin was additionally washed with DCM (3 mL).
  • Flash chromatography was done using Biotage Selekt flash purification system. Water with 0.1% TFA (solvent A) and MeCN with 0.1% TFA (solvent B) was utilized as mobile phases for purifications. The crude peptide was dissolved in a minimal amount of 10% MeCN in water with 0.1% TFA and then loaded onto a 25 g Biotage Bio C18D column. The purification was performed using a gradient as follows: 10% B for 2 column volume (CV), the linear ramp from 10% B to 50% B for 20 CV, 25 mL/min flow rate.
  • CV column volume
  • Preparative Separation The purification column was equilibrated to 5% B, and the remainder of the filtered protein sample was injected onto the column. The column rinsed with 5% B until the denaturing buffer components were rinsed from the column, and the absorbance at 214 nm retuned to baseline. The B percentage was then linearly raised from 5% B to (C - 10) % B at a rate of 1% B / min. The B percentage was then linearly raised from (C - 10) % B to (C + 10) % B over 100 minutes, with 1 -minute fractions. The column was then washed with a gradient from (C + 10) % B to 65% B at a rate of 1% B / min.
  • LC-MS characterizations were carried out using an Agilent 6550 quadrupole time-of- flight LC-MS. Total ion current (TIC) chromatograms were plotted. Mass spectra were integrated over the principal TIC peaks. High-performance liquid chromatography was done by the following methods: (solvent A: water with 0.1% formic acid; solvent B: MeCN with 0.1% formic acid).
  • Method A Column: Phenomenex Jupiter C4 column (1.0 x 150 mm, 5 pm particle size, 300 A pore size) Gradient: 1% B (0-2 min), linearly ramp from l% B to 91% B (2-8 min). The flow rate is 100 pL/min. MS acquisition is from 2 to 8 min.
  • Method B Column: Phenomenex Jupiter C4 column (1.0 x 150 mm, 5 pm particle size, 300 A pore size) Gradient: 1% B (0-2 min), linearly ramp from 1% B to 61% B (2-12 min), 61% B to 95% B (11-16 min). The flow rate is 100 pL/min. MS acquisition is from 4 to 12 min.
  • Method C Column: Agilent Zorbax 300SB C3 column (2.1 x 150 mm, 5 pm particle size, 300 A pore size) Gradient: 1% B (0-2 min), linearly ramp from 1% B to 91% B (2-12 min), 91% B to 91% B (12-13 min). The flow rate is 500 pL/min. MS acquisition is from 4 to 12 min.
  • IGFBP5 is specific substrates of PAPP -A
  • the recognition mechanisms of these proteins by PAPP-A are fundamentally different.
  • IGFBP5 cleavage depends exclusively on availability of the PAPP-A catalytic site: the IGFBP5 linker domain fragment 119Pro-143Ser is responsible for PAPP-A recognition binds catalytic groove, whereas PAPP-A hydrolyzes 143Ser-144Lys amide bond cutting IGFBP5 in two fragments having substantially lower affinity for IGF1 (FIGS. 1A-1C).
  • the location of the cleavage site supports the idea that 143 Ser has close interactions with the Zn-site and can be therefore used to introduce ZBGs.
  • IGFBP4-PAPP-A recognition appears to be more sophisticated and remains opaque.
  • IGFBP4 cleavage requires an exosite located close to the PAPP-A C-terminus (Weyer, et al., Journal of Biological Chemistry, 2007: Boldt, et al., Journal of Biological Chemistry, 2004).
  • the process is IGF 1 -dependent, meaning that the presence of IGF 1 is necessary to allow the cleavage .
  • IGFBP4 is believed to be the principal PAPP-A substrate, serving as the final regulator of free IGF1 concentration (Oxvig, J. Cell. Commun. Signal, 2015).
  • the screening strategy was focused on the ability of the peptides to inhibit PAPP-A mediated cleavage of IFGBP4 in the presence of IGF1.
  • IGFBP5-anchor peptide featuring amide or carboxylic acid at the C-terminus did not show substantial inhibition below 10 pM (FIG. 30).
  • a set of peptides were prepared using MeDbz-linker to modify C-terminus with some common ZBGs e.g., hydroxamate, sulfonamides, Zn-fmger moiety (CXXC), etc. (FIG. ID).
  • ZBGs e.g., hydroxamate, sulfonamides, Zn-fmger moiety (CXXC), etc.
  • CXXC Zn-fmger moiety
  • peptides 3 and 10 (bearing hydroxamate and Zn-fmger moiety, respectively) demonstrate nanomolar range of IC50, which were evaluated as 92 and 209 nM using Simple Western Assay (also known as WES assay in the literature) (not shown).
  • AV-21a showed inhibition, while the alternative tested IGFBP5-ZBG conjugates did not demonstrate PAPP-A inhibition in the screened range of concentrations (FIGS. 31A-31C).
  • IGFBP4 and PAPP-A derived peptides were prepared containing 20-mer sequence featuring hydroxamate at the C-terminus of the cleavage site.
  • the IGFBP4- and PAPP-A-based peptides have shown substantially lower or no inhibitory activity against PAPP-A (FIG. 32).
  • IGFBP5 -anchor peptide equipped with hydroxamate was further used to investigate how peptide’s composition affects the inhibitory functions.
  • FIGS. 45A and 45B further illustrate the optimization conducted on the zinc binding moiety. Potential extensions of the initial hits (i.e. peptides 3 and 10) was studied.
  • Hydroxamate moiety has to contain free hydroxyl group, so the only room for design included N-H fragment.
  • CH3-group was incorporated — the introduction of which resulted in substantially higher IC50.
  • Variations in the zinc hook moiety (peptide 10) have showed sensitivity for the composition of spacer between two Cys, more interestingly that single thiol group showed even more potent PAPP-A inhibition.
  • SAR of peptides containing a single free thiol was focused on.
  • the C-terminal extension with the natural sequence of IGFBP5 was screened, which resulted in gradual loss of activity upon elongation of the C-terminus.
  • modifications of the Cys residue including aliphatic chain extension (homoCys), changing stereoconfiguration of the terminal amino acid and sateliting substituents were studied. The study revealed that D-Cys amide was an optimal termination.
  • IGFBP5 -anchor peptide contains putative heparin binding motif (132VKKDRRKK139) that that has a pronounced basic character and is responsible for nonspecific interactions with extra-cellular components including heparin sulfate of extra-cellular matrix (ECM) (Twigg, et al., Endocrinology, 2000). While these interactions regulate bioavailability and half-life time of IGFBP, it may result in off-target activity of the peptide. Moreover, the region is likely to be susceptible to rapid degradation by natural peptidases owing to crowded positively charged residues. Envisioning these aspects variations of this region were also studied to determine how they will affect inhibitory activity of the peptide- ZBG conjugate.
  • ECM extra-cellular matrix
  • Ring-closing olefin metathesis reaction providing stapled peptides at positions i and i+4 is a common strategy to improve enzymatic stability and confer a-helicity of a peptide (Moiola, et al., Molecules, 2019; Walensky, et al., J. Med. Chem., 2014). Ala-scanning together supported by the structural data identifies the region of 132Val-138Lys as an appropriate site to introduce an aliphatic bridge.
  • PAPP-A that is catalytic active for IGFBP4 Catalytically active towards IGFBP4 cleavage PAPP-A is believed to feature a trans homodimer with an intermolecular disulfide bond between 1130Cys of both subunits. Since catalytic sites of both units can bind IGFBP5- anchor peptide simultaneously, an investigation was conducted to determine whether covalent dimerization of the peptide hydroxamate via N-terminus would result in a tandem binding and improved inhibitory activity.
  • 119Pro-120Lys was crucial for N-terminus modified peptides and also demonstrated improve inhibition with the middle region lipidated peptides, where the latter was showing similar IC50 range as unmodified peptides.
  • 119Pro-120Lys fragment is likely responsible for an appropriate exit of the peptide from the catalytic groove, where a-C-H interactions and hydrogen bonding with 689His of PAPP-A contribute to the overall inhibitory activity (FIG. 40).
  • EXAMPLE 4 Fluorescence polarization study
  • the potency of IGFBP5 -hydroxamate was measured for PAPP-A inhibition, determined by the percentage of IGFBP4 and IGFBP5 cleavage as determined by Simple Western assay for the data that follows.
  • the IC50 value of the IGFBP5 -hydroxamate compound for IGFBP4 cleavage was calculated to be approximately 19 nM (FIG. 4).
  • the IC50 value of the IGFBP5 -hydroxamate compound for IGFBP5 cleavage was calculated to be approximately 363 nM (FIG. 4).
  • PCR tubes setup 5 pM -0.0015 pM 1.5-fold series (varies based on the IC50 range)
  • Peptides were incubated for 1 hour at RT with WT-PAPP-A/Pregnancy serum/A549 cells/A549 Conditioned media, prior to adding the substrate.
  • reaction solution 8 pL was mixed with 2 pL of 5X Fluorescent Master Mix (part of EZ Standard pack, Biotechne product #PS-ST01EZ-8).
  • the plate was loaded with 4 pL of boiled/SDS- reduced samples onto the plate and run on the JESS instrument for 3 hours.
  • the A549-based assay involved measuring the percentage of cleavage of IGFBP4/IGF1 for AV-21a and AV-245PK for secreted PAPP-A, and the percentage of cleavage of IGFBP4/IGF1 for AV-21a and AV-245PK for membrane-bound PAPP-A using Simple Western assay (FIG. 10).
  • the potency of lipidated IGFBP5-hydroxamate was tested by measuring inhibition with pregnancy serum PAPP-A for AV-245PK, and intact and cleaved IGFBP4 was observed using Simple Western assay (FIG. 11).
  • the potency of lipidated IGFBP5-hydroxamate was tested by measuring inhibition with pregnancy serum PAPP -A for AV-245PK, and intact and cleaved IGFBP5 was observed using Simple Western assay (FIG. 52).
  • IV intravenous
  • SC subcutaneous
  • IP intraperitoneal
  • IGFBP5-hydroxamate The stability of IGFBP5-hydroxamate was measured by two major elimination mechanisms: enzymatic cleavage and renal clearance in human serum, for compounds AV- 196PK, AV-196HT, AV-21a, and AV-164 (FIG. 7A).
  • IGFBP5-hydroxamate was also tested by measuring percentage of remaining peptide over 24 hours, with 50 pM of peptide in 100% human serum for compounds AV-221, AV-245PK, AV-245HT, and AV-233 (FIG. 8). Results were measured by LC-MS (EIC)-Monitoring.
  • results showed that initial peptide almost completely transforms into the N-terminally truncated fragment (ELKAEAVKxDRRxKLTQS-NHOH, x-stapling positions) (FIG. 44), which is not expected to show any activity against PAPP-A based on the truncation study.
  • lipidated peptide (AV-245PK) have shown superior stability without significant degradation after 24 hour incubation.
  • this peptide demonstrated not only preserved in vitro activity against PAPP-A but also pronounced serum stability, which renders this peptide an attractive candidate for in vivo studies.
  • all lipidated peptides have shown comparable serum stability, however lower in vitro activity together with synthetic aspects underscore AV-245PK as a matter of further focus.
  • the obtained mixtures were incubated at 37 C for 3.5-4 hours before 2 pL of EDTA (0.1 mM) was added to quench the reaction and then mixed with 7 pL of 4x Laemmli sample buffer containing 10 % (v/v) of P-mercaptoethanol.
  • the obtained samples were heated at 95 C for 10 min, cooled down and loaded on NuPAGE gel (4 to 12%, Bis-Tris, 1.0-1.5 mm).
  • IC50 ranges were estimated based on disappearances of the band corresponding to intact IGFBP4 or appearance of the corresponding fragments.
  • HiBit-IGFBP4-8X His fusion protein produced recombinantly) IGF1 Abeam IGF1 (ab270062)
  • Anti-His antibody Primary to capture HiBit-IGFBP4-8X His fusion protein
  • Antimg/ml Genscript; Cat.#A00186-THE_His_Tag_Antibody_mAb_Mouse
  • IGFBP4/IGF1 Prior incubation for complex formation to a final stock concentration of 400/600 nM
  • IGFBP4/IGF1 is added to the reaction and incubated for 3.5 hours at 37°C
  • the fluorescent master mix is prepared along with other reagents as provided in the protocol provided by Protein simple. Simple Western protocol and plate set-up (see steps below).
  • the studied reactides may be inherently unstable in human serum due to the presence of reactive covalent warheads, which may be a serious drawback of these modalities to be used in vivo.
  • Cl 8 is also referred to as “SG,” e.g., “KSG”.

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Abstract

L'invention concerne des peptides synthétiques anti-PAPP-A présentant une puissante activité inhibitrice, avec une sélectivité prononcée envers la PAPP-A et d'autres membres de la famille des metzincines, des procédés de production de tels peptides synthétiques, et des procédés d'utilisation de tels peptides synthétiques pour l'inhibition dirigée sur site actif de PAPP-A.
PCT/US2024/055798 2023-11-13 2024-11-13 Compositions et procédés pour modulateurs à base de peptides de papp-a Pending WO2025106597A2 (fr)

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CA2818654A1 (fr) * 2000-10-20 2002-04-25 Como Biotech Aps Proteine plasmatique a2 associee a la grossesse (papp-a2)
EP2890816B1 (fr) * 2012-08-30 2019-06-05 Ansh Labs LLC Papp-a2 en tant que marqueur pour la surveillance, la prédiction et le diagnostic de la prééclampsie
KR101482708B1 (ko) * 2012-10-09 2015-01-14 성균관대학교산학협력단 헤파린 결합 도메인을 포함하는 ｉｇｆｂｐ-5의 ｃ-말단 도메인의 신생 혈관 생성 억제제로서의 신규한 용도

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