Compositions and methods for peptide-based modulators of PAPP-a

Abstract

Disclosed herein are anti-PAPP-A synthetic peptides showing potent inhibitory activity with pronounced selectivity towards PAPP-A and over other members of metzincin family, methods of making such synthetic peptides, and methods of using such synthetic peptides for active site-directed inhibition of PAPP-A.

Description

COMPOSITIONS AND METHODS FOR PEPTIDE-BASED MODULATORS OF PAPP-ACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 598,405, filed on November 13, 2023, the disclosure of which is incorporated by reference herein in its entirety for all purposes.SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on XXX , is named XXX, and is XXX bytes in size.BACKGROUND

[0003] Pregnancy-associated plasma protein-A (PAPP-A) is a secreted zinc- metalloproteinase that increases insulin-like growth factor (IGF) availability through cleavage of IGF-binding proteins (IGFBPs). Although activation of the IGF pathway is essential for growth and development during the early stages of life, IGF-signaling at later stages of life has been associated with various physiological changes and age-related diseases. A reduction in IGF signaling has been shown to extend lifespan in diverse species, but the exact intervention point for precluding the IGF signaling pathway appears to be critical. Previous attempts to inhibit IGF1R as potential cancer therapy have failed in clinical trials due to induced endocrine compensation from other growth factors. While PAPP-A has emerged as a potential target for interventions aimed at modulating IGF-signaling, there is a lack of selective active site-directed inhibitors of PAPP-A, particularly over other members of metzincin family including paralog PAPP-A2. Recent structural elucidation of a PAPP-A complex with IGFBP5 -fragment has demonstrated that linker domain of IGFBP5 contains an anchor sequence recognizing the catalytic groove of PAPP-A and providing direct access to the active site. Thus, there is currently a need for potent active site-directed inhibitors with pronounced selectivity towards PAPP-A.SUMMARY

[0004] 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.

[0005] The present disclosure provides a synthetic peptide comprising the formula L1Z1L2Z2R10or a pharmaceutically acceptable salt thereof, whereinL1is 7 to 10 amino acids in length;L2is an amino acid sequence having a distance of about 20-35 angstroms;Z1is a basic amino acid;Z2is a bond or serine,R10is selected from the group consisting of -R10a, -OH, - NH2, -N(H)0H, -N(H)R10a, - N(R10a)2, -N(R10a)OH, -SR10a, and -CH2R10a; and each R10ais independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted 3-6 membered heterocyclyl, optionally substituted -N(H)(Ci-Ce alkyl), andwherein R10ais optionally substituted with 1-3 instances of a group independently selected from oxo, - CH=CH2, -CO2H, -C(O)NH2, -C(O)N(H)OH, -OH, -SH, -OPO3H2, -SO2NH2, - PO3H2, -NH2, -NHCH2CH2CH3, cyclohexyl, optionally substituted 5-6 membered heteroaryl and a peptide comprising 2-10 amino acids; and wherein the synthetic peptide comprises a helical structure when bound to PAPP-A and inhibits pregnancy-associated plasma protein A (PAPP-A).

[0006] In some embodiments, the distance between Z1and Z2is 14 amino acids in length. In some embodiments, Z1is selected from the group consisting of arginine, lysine, and histidine. In some embodiments, Z1is lysine. In some embodiments, Z1is arginine or homo-Arginine. In some embodiments, Z1is histidine. In some embodiments, Z2is serine. In some embodiments, the synthetic peptide comprises an amino acid sequence from Insulin Growth Factor Binding Protein 5 (IGFBP5). In some embodiments, L1comprises the amino acid sequence of PKHTRISEL. In some embodiments, L2comprises the amino acid sequence of AEAVKKDRRKKLT, optionally AEAVKKDRRKKLTQ. In some embodiments, R10is X- R10b, wherein X isand R10bis selected from the group consisting of:Phe.

[0007] In some embodiments, R10is X-R10b, wherein

[0008] In some embodiments, R10is X-R10b, X is a bond and R10bis:

[0009] In some embodiments, the N-terminus of the peptide is modified with a lipidated tag, optionally an albumin-binding tag.

[0010] In another aspect, the present disclosure provides a synthetic peptide comprising formula I or a pharmaceutically acceptable salt thereof:whereinR1is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)Rla, and -C(O)ORla;Rlais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, wherein Rlaoptionally comprises a fluorophore;R2is selected the from the group consisting of -NH2, -N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or -N(H)C(=NH)NH2;R3is selected the from the group consisting of -CH2R3a, -NH2, -N(H)C(=NH)NH2, optionally substituted Ci-Ce alkyl, optionally substituted phenyl, and optionally substituted 5-10 membered heteroaryl, wherein R3is optionally substituted with - (CH2)O-3NH2or -(CH2)O-3N(H)C(=NH)NH2;R3ais optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl;R4is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)R4a, -C(O)OR4aand a peptide comprising 2-10 amino acids;R4ais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl optionally substituted with a peptide comprising 2-10 amino acids;R5is selected the from the group consisting of -NH2, -N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or -N(H)C(=NH)NH2;R7is selected the from the group consisting of -NH2, -N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or -N(H)C(=NH)NH2; alternatively, wherein R5and R7are taken together to form a staple comprising 1- 10 carbon atoms;R6is selected the from the group consisting of optionally substituted Ci-Ce alkyl substituted with 0-3 instances of optionally substituted 5-6 membered heteroaryl, -NH2or -N(H)C(=NH)NH2; optionally substituted 5-6 heteroaryl substituted with 1-3 instances of R6a; and -SR6a;R6ais optionally substituted Ci-Ce alkyl substituted or optionally substituted phenyl, wherein R6ais substituted with -SO2F or -OSO2NH2;R8is a warhead;R9is selected the from the group consisting of optionally substituted Ci-Ce alkyl substituted with 0-3 instances of optionally substituted 5-6 membered heteroaryl, - NH2 or -N(H)C(=NH)NH2; optionally substituted 5-6 heteroaryl substituted with 1-3 instances of R9a; and -SR9a;R9ais optionally substituted Ci-Ce alkyl or optionally substituted phenyl, wherein R9ais substituted with -SO2F or -OSO2NH2;X is a bond, -C(O)-,orR10is selected from the group consisting of -R10a, -CO2H, -C(O)OH, -C(O)NH2, - C(O)N(H)OH, -C(O)N(H)R10a, -C(O)N(R10a)2, -C(O)N(R10a)OH, -C(O)SR10aand - C(O)CH2R10a; and each R10ais independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted 3-6 membered heterocyclyl, ' optionally substituted -N(H)(Ci-Ce alkyl), andwherein R10ais optionally substituted with 1-3 instances of a group independently selected from oxo, -CH=CH2, -CO2H, -C(O)NH2, -C(O)N(H)OH, -OH, -SH, - OPO3H2, -SO2NH2, -PO3H2, -NH2, -NHCH2CH2CH3, cyclohexyl, optionally substituted 5-6 membered heteroaryl and a peptide comprising 2-10 amino acids.

[0011] In some embodiments, R1is -C(O)Rlaand R4is -C(O)R4a, Rlais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, optionally comprising a fluorophore, and R4ais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl.

[0012] In some embodiments, Ri is selected from the group consisting of:

[0013] In some embodiments, R2is Ci-Ce alkyl substituted with -NH2. In some embodiments, R3is Ci-Ce alkyl substituted with -NH2. In some embodiments, R2is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2. In some embodiments, R3is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2. In some embodiments, R2is:or

[0014] In some embodiments, R3is -CH2R3awherein R3ais optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl, wherein R3is optionally substituted with - (CH2)O-3NH2 or -(CH2)O-3N(H)C(=NH)NH2. In some embodiments, R3is selected from the group consisting of phenyl, pyridyl, thiophenyl, pyrazolyl, imidazolyl, 1,2,4-triazolyl, 1,2,3- triazolyl, tetrazolyl, and pyrrolyl, wherein R3is optionally substituted with -(CH2)o-3NH2 or - (CH2)O-3N(H)C(=NH)NH2. In some embodiments, R3is optionally substituted Ci-Ce alkyl, substituted with -NH2 or -N(H)C(=NH)NH2. In some embodiments, R3ais optionally substituted indole.

[0015] In some embodiments, R3is selected from the group consisting of:

[0016] In some embodiments, R4is selected from the group consisting of:and

[0017] In some embodiments, R5is Ci-Ce alkyl substituted with -NH2, and R7is Ci-Ce alkyl substituted with -NH2. In some embodiments, R5and R7are taken together to form a staple comprising 1-10 carbon atoms. In some embodiments, each of R6and R9is independently selected from the group consisting of:

[0018] In some embodiments, R6is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2. In some embodiments, R6is

[0019] In some embodiments, R8is a warhead, and wherein a warhead is L-R8a, whereinL 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-; andR8ais optionally substituted phenyl substituted with -N(H)C(O)-R8bor -N(H)S(O)2- R8b, wherein R8bis optionally substituted C2-C6 alkenyl.

[0020] In some embodiments, R9is Ci-Ce alkyl. In some embodiments, R9i. In some embodiments, L is:

[0021] In some embodiments, R8ais selected from the group consisting of:

[0022] In some embodiments, X is, and R10is selected from the group consisting of:

[0023] In some embodiments, R10is:

[0024] In some embodiments, X isand R10is selected from the group consisting of:

[0026] In some embodiments, X is a bond and Rio is:

[0027] In some embodiments, the synthetic peptide comprises formula II or a pharmaceutically acceptable salt thereof:

[0028] In some embodiments, 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:

[0030] In another aspect, 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. In some embodiments, 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.

[0031] In another aspect, 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.

[0032] In some embodiments, 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. In some embodiments, a synthetic peptide as disclosed herein is truncated by 2-4 amino acid at the N-terminus. In some embodiments, a synthetic peptide as disclosedherein 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. In some embodiments, 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.

[0033] In some embodiments, 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.

[0034] In some embodiments, 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.

[0035] In some embodiments, 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.

[0036] In some embodiments, 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.

[0037] In some embodiments, 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.

[0038] In some embodiments, 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.

[0039] In some embodiments, 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.

[0040] In some embodiments, the zinc-binding chemical moiety is hydroxamate, or a thiol group.

[0041] In some embodiments, the N-terminus of the peptide is modified with a lipid, optionally a lipidated tag, optionally an albumin-binding tag. In some embodiments, the Proin 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.

[0042] In some embodiments, the synthetic peptide shows no or a negligible amount of inhibition of pregnancy-associated plasma protein-A2 and metalloproteinases other than PAPP-A. In some embodiments, 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.

[0043] In some embodiments, 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.

[0044] In some embodiments, the present disclosure provides a synthetic peptide, wherein the synthetic peptide is any one of the peptides disclosed in Tables 7-10. In some embodiments, 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.

[0045] In another aspect, the present disclosure provides a pharmaceutical comprising a synthetic peptide as disclosed herein and a pharmaceutically acceptable excipient.

[0046] In another aspect, 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. In some embodiments, the disease or condition is an age-related disease. In some embodiments, the disease or condition is atherosclerosis. In some embodiments, administering the synthetic peptide intravenously, subcutaneously or intraperitoneally.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] A beter understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0048] FIGS. 1A-1E show a schematic for the screening of various zinc-binding groups at the C-terminus of IGFBP5 -anchor peptide.

[0049] FIG. 2 shows a schematic and results from L-Alanine scanning of the peptide 13.

[0050] FIGS. 3A-3B show plots depicting results from the enzymatic degradation of the indicated peptides.

[0051] FIG. 4 shows the potency of IGFBP5-hydroxamate measured for PAPP -A inhibition, determined by the percentage of IGFBP4 and IGFBP5 cleavage.

[0052] FIG. 5 shows a plot depicting the selectivity of IGFBP5-hydroxamate, tested by measuring inhibition of MMPs.

[0053] FIG. 6 shows a plot depicting the selectivity of IGFBP5-hydroxamate, tested by measuring percentage of IGFBP5 cleavage by PAPP-A2.

[0054] 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.

[0055] 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.

[0056] FIG. 9 shows the inhibition for recombinant PAPP-A for the indicated compounds.

[0057] 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.

[0058] 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.

[0059] FIG. 12 shows the PAPP-A / IGFBP5 complex and rationale for picking the modification positions.

[0060] FIG. 13 shows a strategy to incorporate covalent warheads.

[0061] FIG. 14 shows MALDI-MS of PAPP-A using different matrices.

[0062] FIG. 15 shows a schematic representation of reactides.

[0063] FIG. 16 shows the initial screening of the SO2F and OSO2NH2 reactides.

[0064] FIG. 17 shows a control experiment showing importance of covalent binders for PAPP-A staining.

[0065] FIG. 18 shows MALDI-MS of PAPP-A (grey) and mixture obtained after incubation of PAPP-A with reactide 1.

[0066] FIG. 19 shows selective PAPP-A staining in human serum.

[0067] FIG. 20 shows paralog specificity of the screened reactides.

[0068] FIG. 21 shows an experiment demonstrating that PAPP-A staining is sensitive to the catalytic groove availability.

[0069] FIG. 22 shows the inhibitory activity against PAPP-A for the indicated reactides.

[0070] FIG. 23 shows IGFBP5 -based reactides with acrylamide (AM) warheads.

[0071] FIG. 24 shows the inhibitory activity against PAPP-A of 147-cys mutants with AM warheads.

[0072] FIG. 25 shows the binding activity of reactides with AM warheads against PAPP-A 1 in presence of PAPP-A2.

[0073] FIG. 26 shows a potential experiment to target Cys with acrylamides.

[0074] FIG. 27 shows the binding activity of an IGFBP5-based fluorescent probe.

[0075] FIG. 28 shows labeling ability of NHOH-Zn interactions.

[0076] FIG. 29 shows the bioavailability from administering an exemplary peptide inhibitor to mice at various doses and via the indicated routes of administration.

[0077] FIG. 30 shows inhibition of IGFBP5 -anchor peptide featuring amide or carboxylic acid at the C-terminus.

[0078] FIGS. 31A-31C show a schematic and inhibition results from screening various zinc binding groups (ZBGs).

[0079] 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.

[0080] FIG. 33 shows various inhibition results from IGFBP4 cleavage inhibition, IC50 screening, and an N-truncation study of alternative binding moieties — based on IGFBP4 cleavage and PAPP-A autocleavage sites.

[0081] FIG. 34 shows He 124 and Lys 128 hotspot interactions.

[0082] FIG. 35 shows a 2D-map for the identified hotspots in the PAPP-A inhibitory sequence.

[0083] FIG. 36 shows the gel-based IC50 results for the indicated C-terminus hydroxamate peptide variations.

[0084] FIGS. 37A-37E show a schematic and gel-based IC50 results for the IGFBP5-anchor peptide optimization.

[0085] 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.

[0086] FIG. 39 shows the flexible oligoethyleneglycol linkers’ effects on affinity.

[0087] FIG. 40 illustrates the interaction between PAPP-A and the peptide inhibitor.

[0088] FIG. 41 shows the results from a fluorescence polarization study of the FITC-labelled peptides.

[0089] FIG. 42 shows the results from a fluorescence polarization study of the labelled peptides.

[0090] FIG. 43 shows the serum stability results for a mutated variation of the peptide.

[0091] FIG. 44 shows the serum stability results for a mutated variation of the peptide.

[0092] FIGS. 45A-45B show results from optimization conducted on the zinc-binding chemical moiety.

[0093] FIG. 46 shows a 25-30 angstrom distance between two hotspots on the peptide.

[0094] FIG. 47 shows IC50 range results from L-Alanine scanning.

[0095] FIG. 48 shows results from an A549-based assay from measuring the percentage of cleavage for AV-245PK for secreted PAPP -A.

[0096] FIG. 49 shows the A549 assay workflow.

[0097] 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.

[0098] 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.

[0099] FIG. 52 shows lipid-modified peptides inhibit PAPP-A in pregnancy serum.DETAILED DESCRIPTION

[0100] 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. 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.

[0101] Various components and aspects of the disclosure are described in further detail in the subsections below.I. Definitions

[0102] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Mention of techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0103] Throughout the description, where 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.

[0104] In the disclosure, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0105] Further, it should be understood that elements and / or features of a composition or a method provided and described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure and invention(s) herein, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of invention(s) provided, described, and depicted herein.

[0106] As used herein, 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.”

[0107] As used herein, 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.

[0109] The articles “a” and “an” are used in this disclosure to refer to one or more than one (z.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, “an element” means one element or more than one element.

[0110] The term “and / or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.[oni] The term “or” is used in this disclosure to refer to any one of the list elements and any combination thereof, unless the context is inappropriate.

[0112] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0113] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0114] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present disclosure remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0115] At various places in the present specification, 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. For example, 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, and an integer in the range of 1 to 20 is specificallyintended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0116] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention(s) unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of that provided by the present disclosure.

[0117] As used herein, “residue” refers to a position in a peptide and its associated amino acid identity.

[0118] The term “modulation” refers to an increase or decrease in the level of a target molecule or the function of a target molecule. The term “modulator” as used herein refers to modulation of (e.g., an increase or decrease in) the level of a target molecule or the function of a target molecule.Chemical Definitions

[0119] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March ’s Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0120] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0121] The term “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.There can also be tripeptides, tetrapeptides, pentapeptides, etc. A peptide has an amino end and a carboxyl end, unless it is a cyclic peptide.

[0122] The term “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. In some embodiments, the peptides or polypeptides are suspended in a liquid solution. Non-limiting examples of a liquid solution include water, aqueous buffer mixtures, acidic or basic solutions, organic solvents such as alcohol or acetonitrile, or any combination thereof.

[0123] 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.

[0124] 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.11,279,734, the relevant disclosures of which are herein incorporated by reference.

[0125] 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.

[0126] As used herein, the term “biotinylation” refers to the addition of a biotin.

[0127] As used herein, the term “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. The term “glycosyl group” as used herein 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.

[0128] As used herein, the term “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.

[0129] As used herein, the term “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.

[0130] As used herein, the term “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.

[0131] As used herein, the term “hydroxylation” refers to the addition of a hydroxyl group (- OH).

[0132] As used herein, the term “acetylation” refers to the addition of an acetyl group. An acetyl group contains a methyl group single-bonded to a carbonyl.

[0133] As used herein, the term “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. For example, 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. In some embodiments, the warhead is an electrophile and the “donor” is a nucleophile, such as the side chain of a cysteine residue.

[0134] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, 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 racemicmixtures 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. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (E.L.Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0135] As used herein, a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, 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. The term “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. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.

[0136] In the compositions provided herein, an enantiomerically pure compound can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising an enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound. In certain embodiments, 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. For example, a pharmaceutical composition comprising enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound. In certain embodiments, 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 thecompound. In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier.

[0137] As used herein, 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.

[0138] As used herein, the term “ka” (M'1x 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 konvalue.

[0139] As used herein, the term “KD” (M) refers to the dissociation equilibrium constant of a given entity and a target ( particular interaction between an entity and its target (e.g., a particular peptide -target interaction)). KD = kd / ka.

[0140] As used herein, the term “KA” (M'1) refers to the association equilibrium constant of a given entity and a target (e.g., a particular polypeptide-target interaction). KA = ka / kd.

[0141] The affinity of a molecule X for its target Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are as described above. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction. 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®).

[0142] When a range of values is listed, it is intended to encompass each value and subrange within the range. For example, “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.

[0143] As used herein, the term “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”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, 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). Anaryl 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. In certain embodiments, the aryl group is unsubstituted C6-C14 aryl. In certain embodiments, the aryl group is substituted C6-C14 aryl.

[0144] “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. The term “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.

[0145] The term “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. Unless otherwise specified, aliphatic groups can contain 1-6 aliphatic carbon atoms. In some embodiments, 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. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) 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.

[0146] The term “haloaliphatic” refers to an aliphatic group that is substituted with one or more halogen atoms.

[0147] The term “alkyl” refers to a straight or branched alkyl group. Exemplary alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0148] The term “haloalkyl” refers to a straight or branched alkyl group that is substitutedwith one or more halogen atoms.

[0149] The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 n electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “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. The terms “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. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0150] 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. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or 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. Examples of such saturated or partially unsaturated heterocyclicradicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclic group”, “heterocyclic moiety”, and “heterocyclic radical”, are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0151] As used herein, the term “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. In some embodiments, 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. C22 heteroalkyl, C23 heteroalkyl, C24 heteroalkyl, C25 heteroalkyl, C26 heteroalkyl. C27 heteroalkyl, C28 heteroalkyl. C.2.9 heteroalkyl, C30 heteroalkyl, C31 heteroalkyl, C32 heteroalkyl, C33 heteroalkyl).

[0152] As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “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.

[0153] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “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. Unless otherwise indicated, 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 aspecified 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. The term “stable”, as used herein, 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.

[0154] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; — (CFhjo-iR' : — (CH2)o-40R°; — 0(CH2)o-4R°, — O— (CH2)O-4C(0)OR°; — (CH2)O-4CH(OR°)2; — (Cffijo-rSR' : — (CH2)o-4Ph, which may be substituted with R°; — (CH2)o-40(CH2)o-iPh which may be substituted with R°; — CH=CHPh, which may be substituted with R°; — (CH2)o-40(CH2)o-i-pyridyl which may be substituted with R°; — NO2; — CN; — N3; — (CH2)O-4N(R°)2; — (CH2)o-4N(R°)C(0)R°; — N(R°)C(S)R°; — (CH2)O-4N(R°)C(0)NR°2; — N(R°)C(S)NR° 2; — (CH2)o-4N(R°)C(0)OR°; — N(R°)N(R°)C(O)R°; — N(R°)N(R°)C(O)NR° 2; — N(R°)N(R°)C(O)OR°; — (CH2)o-4C(0)R°; — C(S)R°; — (CH2)O-4C(0)OR°; — (CH2)O.4C(0)SR°; — (CH2)0-4C(O)OSiR° 3; — (CH2)o- 4OC(O)R°; — OC(0)(CH2)O-4SR°, SC(S)SR°; — (CH2)O-4SC(0)R°; — (CH2)O-4C(0)NR° 2; — C(S)NR°2; — C(S)SR°; — SC(S)SR°, — (CH2)o-40C(0)NR° 2; — C(O)N(OR°)R°; — C(O)C(O)R°; — C(O)CH2C(O)R°; — C(NOR°)R°; — (CH2)o-4SSR°; — (CH2)o-4S(0)2R°; — (CH2)O-4S(0)2OR°; — (CH2)O-40S(0)2R°; — S(O)2NR° 2; — (CH2)o-4S(0)R°; — N(R°)S(O)2NR°2; — N(R°)S(O)2R°; — N(0R°)R°; — C(NH)NR° 2; — P(O)2R°; — P(O)R° 2; — OP(O)R°2; — OP(O)(OR°)2; SiR° 3; — (C1-4 straight or branched alkylenejO — N(R°)2; or — (C1-4 straight or branched alkylene)C(O)O — N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, — CH2Ph, — 0(CH2)o-iPh, — CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0155] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, — (CH2)O-2R*, -(haloR*), — (CH2)o-2OH, — (CH2)o-2OR*, — (CH2)o-2CH(OR*)2; — O(haloR’), — CN, — N3, — (CH2)O-2C(0)R*, — (CH2)O-2C(0)OH, — (CH2)O-2C(0)OR*, — (CH2)O-2SR*, —(CH2)o-2SH, — (CH2)O-2NH2, — (CH2)O-2NHR*, — (CH2)O-2NR* 2, — N02, — SiR* 3, — OSiR* 3, — C(O)SR*, — (C1-4 straight or branched alkylene)C(O)OR*, or — SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, — CH2Ph, — 0(CH2)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 divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0156] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0, =S, =NNR*2, =NNHC(0)R*, =NNHC(0)0R*, =NNHS(0)2R*, =NR*, =N0R*, — O(C(R*2))2-3O— , or — S(C(R*2))2- 3S — , 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 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.

[0157] Suitable substituents on the aliphatic group of R* include halogen, — R*, -(haloR*), —OH, —OR*, — O(haloR’), — CN, — C(O)OH, — C(O)OR*, — NH2, — 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, — CH2Ph, — 0(CH2)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.

[0158] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include — RT, — NRT2, — C(O)RT, — C(O)ORT, — C(O)C(O)RT, — C(O)CH2C(O)RT, — S(O)2RT, — S(O)2NR:2, — C(S)NR:2, — C(NH)NR:2, or — N(RT)S(O)2RT; 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'. taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partiallyunsaturated, or an aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0159] Suitable substituents on the aliphatic group of R' are independently halogen, — R*, - (haloR*), —OH, —OR*, — O(haloR’), — CN, — C(O)OH, — C(O)OR*, — NH2, — 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(CH2)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.

[0160] The term “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.

[0161] In some embodiments, a presently disclosed synthetic peptide is provided in the form of a pharmaceutical salt.

[0162] As used herein, 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. Other pharmaceutically acceptable 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, persulfate, 3 -phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0163] 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.

[0164] Combinations of substituents and variables envisioned by this disclosure are only those that result in the formation of stable compounds. The term “stable”, as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0165] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0166] As used herein, 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). However, the term “peptide stapling,” as used herein, 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. In some embodiments, hydrocarbon stapling is contemplated. In some embodiments, the staples also comprise nitrogen.

[0167] In some embodiments, one-component stapling is contemplated in the disclosed synthetic peptides. In some embodiments, 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.

[0168] Without being bound by mechanism, in some embodiments, 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). For example, 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.

[0169] As used herein, the term “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.

[0170] The term “biological sample”, as used herein, 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.

[0171] As used herein, 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. In some embodiments, 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. As will be appreciated by those of ordinary skill in this art, 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. For example, 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.

[0172] As used herein, the terms “treatment,” “treat,” and “treating” refer 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. In some embodiments, treatment may be administered after one or more symptoms have developed. In some embodiments, the term “treating” includes preventing or halting the progression of a disease or disorder. In other embodiments, treatment may be administered in the absence of symptoms. For example, 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. Thus, in some embodiments, the term “treating” includes preventing relapse or recurrence of a disease or disorder.

[0173] As used herein, “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. For example, 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. In some aspects, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug or therapeutic.

[0174] The term “subject”, as used herein, refers to an animal, preferably a mammal, and most preferably a human.

[0175] The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to a nontoxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound(s) with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the 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-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0176] 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.

[0177] As used herein, the term “age-related disease” refers to disorders or diseases in which aging is a major risk factor. For example, in some embodiments, age-related disease refers to atherosclerosis. See, e.g., Conover CA, Exp Gerontol. 2013 Jul;48(7):612-3.

[0178] As used herein, 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).

[0179] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention(s) unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of that provided by the present disclosure.II. PAPP-A and IGF Signaling

[0180] Pregnancy-associated plasma protein-A (PAPP-A) is a secreted zinc- metalloproteinase increasing insulin-like growth factor (IGF) availability through cleavage of IGF-binding proteins (IGFBPs). 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.

[0181] 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). Although activation of the IGF pathway is essential for growth and development during the early stages of life, a reduction in IGF signaling has been shown to extend lifespan in diverse species (Barbieri, et al., American Journal of Physiology - Endocrinology and Metabolism, 2003; Fontana, et al., Science, 2010; Kenyon, Cell, 2005; Kenyon, Nature, 2010). PAPP -A knock-out mice demonstrate around 30% extended longevity while being proportional dwarfs, which indicates the role of IGF in early growth modulation (Conover, et al., Aging Cell, 2W)1; Conover, et al., The Journals of Gerontology, 2010). Meanwhile, induced PAPP-A deletion in adulthood results in a similar lifespan extension, without effect on the weight gain (Bale, et al., Aging Cell, 2017). 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). Additional insights into the phenotype of PAPP-A inhibited mice support the idea that PAPP-A targeting provides a safe and effective path towards IGF signalling regulation, without disrupting normal activity (Mohrin, et al., Aging Cell, 2020).

[0182] However, the exact intervention point for downregulating the IGF signaling pathway appears to be critical. Previous attempts in the art to inhibit IGF1R as potential cancer therapy failed in clinical trials due to induced endocrine compensation from other growth factors (Junnila, et al., Nature Reviews Endocrinology, 2013; Beckwith, et al., Molecular Endocrinology, 2015; Gombos, et al., Investigational New Drugs, 2012). PAPP-A deletion is shown to have no effect on circulation levels of the growth hormone and IGF 1 (Conover, et al., Aging Cell, 2007; Conover, et al., Bone, 2004; Laursen, et al., Journal of Biological Chemistry, 2002).

[0183] Natural PAPP-A inhibitors (proMBP and STCs) and developed PAPP-A neutralizing antibodies recognise and bind to PAPP-A’s exosites, sterically hindering protein-proteininteractions (PPI) with its substrates. However, it does not prevent cleavages that are less affected by the steric hindrance e.g., cleavage of IGFBP-5 (in case of mAb) and cleavage of synthetic peptide derived from IGFBP-4 (in case of STC2) (Kobbero, et al., Nature Communications, 2022; Overgaard, et al., Journal of Biological Chemistry, 2003; Boldt, et al., Growth Hormone & IGF Research, 2007: Glerup, et al., Enzyme Catalysis and Regulation, 2007). In this context, development of catalytic site-directed inhibitors of PAPP- A represents a highly promising strategy for anti -aging interventions. 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.

[0184] Rational development of inhibitors is generally based on the pockets adjacent to the active site(S3-S3’). Substrate recognition is more complex and requires more distant recognition (allosteric) sites, access to which could allow more selective inhibition. Peptides are a great modality to gain a facile access to those distant sites. In some case, there are natural example of IGFBP5, where Lys (16 positions away from the cleavage site) is known to be crucial for the PAPP -A mediated peptide probe cleavage. This dual binding mode (e.g., having Lys + ZBG) can facilitate PAPP-A inhibition.

[0185] 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.Additionally, recent structural elucidation of a PAPP-A complex with IGFBP5 -fragment has demonstrated that 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. Within the IGFBP5 anchor peptide, a Lys (16 positions away from the cleavage site) is crucial for the PAPP-A mediated peptide probe cleavage. Without being limited by mechanism of action, 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 forgaining potent inhibitory activity against PAPP-A, whereas incorporation of albumin-binding tags improve pharmacokinetic properties of the peptide.III. Anti-PAPP-A Peptides

[0186] In some embodiments, disclosed herein are synthetic peptides comprising a modified IGFBP5 anchor sequence. The IGFBP5 anchor sequence is PKHTRISELKAEAVKKDRRKKLTQS .

[0187] In some embodiments, in the sequence PKHTRISELKAEAVKKDRRKKLTQS, the lysine residue in position 10 and terminal serine residue (bolded) 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. In some embodiments, 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.

[0188] In some embodiments, a synthetic peptide as disclosed herein comprises the formula L1Z1L2Z2R10or a pharmaceutically acceptable salt thereof, whereinL1is 7 to 10 amino acids in length;L2is an amino acid sequence having a distance of about 20-35 angstroms;Z1is a basic amino acid;Z2is a bond or serine,R10is selected from the group consisting of -R10a, -OH, - NH2, -N(H)0H, -N(H)R10a, -N(R10a)2, -N(R10a)OH, -SR10a, and -CH2R10a; and each R10ais independently selected from the group consisting of optionally substitutedCi-Ce alkyl, optionally substituted 3-6 membered heterocyclyl, optionally substituted -N(H)(Ci-Ce alkyl), andwherein R10ais optionally substituted with 1-3 instances of a group independently selected from oxo, - CH=CH2, -CO2H, -C(O)NH2, -C(O)N(H)OH, -OH, -SH, -OPO3H2, -SO2NH2, -PO3H2, -NHz, -NHCH2CH2CH3, cyclohexyl, optionally substituted 5-6 membered heteroaryl and a peptide comprising 2-10 amino acids; and wherein the synthetic peptide comprises a helical structure when bound to PAPP-A and inhibits pregnancy-associated plasma protein A (PAPP-A).

[0189] In some embodiments, the distance between Z1and Z2is 14 amino acids in length. In some embodiments, Z1is selected from the group consisting of arginine, lysine, and histidine In some embodiments, Z1is lysine. In some embodiments, Z1is arginine or homo-Arginine. In some embodiments, Z1is histidine. In some embodiments, Z2is serine. In some embodiments, the synthetic peptide comprises an amino acid sequence from Insulin Growth Factor Binding Protein 5 (IGFBP5). In some embodiments, L1comprises the amino acid sequence of PKHTRISEL. In some embodiments, L2comprises the amino acid sequence of AEAVKKDRRKKLT, optionally AEAVKKDRRKKLTQ. In some embodiments, R10is X- R10b, wherein X isand R10bis selected from the group consisting of:

[0190] In some embodiments, R10is X-R10b, whereinand

[0191] In some embodiments, R10is X-R10b, X is a bond and R10bis:

[0192] In some embodiments, the synthetic peptide comprises formula II or a pharmaceutically acceptable salt thereof:

[0193] In some embodiments, the synthetic peptide comprises formula II- 1 or a pharmaceutically acceptable salt thereof:

[0194] In some embodiments, the synthetic peptide comprises formula II-2 or a pharmaceutically acceptable salt thereof:

[0195] In some embodiments, the synthetic peptide comprises formula Ila or a pharmaceutically acceptable salt thereof:

[0196] In some embodiments, the synthetic peptide comprises formula lib or a pharmaceutically acceptable salt thereof:

[0197] In some embodiments, 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 R1, R2, R3, R4, R5, R6,R7, R8, or R9modifications relative to formula II.

[0200] In some embodiments, the synthetic peptide comprises formula III or a pharmaceutically acceptable salt thereof:

[0201] In some embodiments, the synthetic peptide comprises formula Illa or a pharmaceutically acceptable salt thereof:

[0202] In some embodiments, the synthetic peptide comprises formula Illb or a pharmaceutically acceptable salt thereof:

[0203] In some embodiments, 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 R1, R2, R3, R4, R5, R6,R7, R8, or R9modifications relative to formula III.

[0206] In some embodiments, the synthetic peptide comprises formula IV or a pharmaceutically acceptable salt thereof:

[0207] In some embodiments, the synthetic peptide comprises formula IVa or a pharmaceutically acceptable salt thereof:

[0208] In some embodiments, the synthetic peptide comprises formula IVb or a pharmaceutically acceptable salt thereof:

[0209] In some embodiments, 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 R1, R2, R3, R4, R5, R6,R7, R8, or R9modifications relative to formula IV.

[0212] In some embodiments, R4is a lipid. In some embodiments, R4is a lipidated tag, optionally an albumin-binding tag. In some embodiments, R1is a lipid. In some embodiments, R1is 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, R4isor

[0214] In some embodiments, R1is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)Rla, and -C(O)ORla;Rlais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, wherein Rlaoptionally comprises a fluorophore.

[0215] In some embodiments, R2is selected the from the group consisting of -NH2, - N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or - N(H)C(=NH)NH2.

[0216] In some embodiments, R3is selected the from the group consisting of -CH2R3a, -NH2, -N(H)C(=NH)NH2, optionally substituted Ci-Ce alkyl, optionally substituted phenyl, and optionally substituted 5-10 membered heteroaryl, wherein R3is optionally substituted with - (CH2)O-3NH2or -(CH2)O-3N(H)C(=NH)NH2.R3ais optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl;

[0217] In some embodiments, R4is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)R4a, -C(O)OR4aand a peptide comprising 2-10 amino acids; wherein R4ais optionally substituted C1-C33 alkyl or optionally substituted Ci- C33 heteroalkyl optionally substituted with a peptide comprising 2-10 amino acids.

[0218] In some embodiments, R5is selected the from the group consisting of -NH2, - N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or - N(H)C(=NH)NH2.

[0219] In some embodiments, R7is selected the from the group consisting of -NH2, - N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or - N(H)C(=NH)NH2.

[0220] In some embodiments, R5and R7are taken together to form a staple comprising 1-10 carbon atoms.

[0221] In some embodiments, R6is selected the from the group consisting of optionally substituted Ci-Ce alkyl substituted with 0-3 instances of optionally substituted 5-6 membered heteroaryl, -NH2 or -N(H)C(=NH)NH2; optionally substituted 5-6 heteroaryl substituted with 1-3 instances of R6a; and -SR6a; whereinR6ais optionally substituted Ci-Ce alkyl substituted or optionally substituted phenyl, wherein R6ais substituted with -SO2F or -OSO2NH2;

[0222] In some embodiments, R8is a warhead;

[0223] In some embodiments, R9is selected the from the group consisting of optionally substituted Ci-Ce alkyl substituted with 0-3 instances of optionally substituted 5-6 membered heteroaryl, -NH2 or -N(H)C(=NH)NH2; optionally substituted 5-6 heteroaryl substituted with 1-3 instances of R9a; and -SR9a; wheirein:R9ais optionally substituted Ci-Ce alkyl or optionally substituted phenyl, wherein R9ais substituted with -SO2F or -OSO2NH2.

[0224] In some embodiments, R1is -C(O)Rlaand R4is -C(O)R4a, Rlais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, optionally comprising a fluorophore, and R4ais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl.

[0225] In some embodiments, Ri is selected from the group consisting of:5

[0226] In some embodiments, as used herein, R1is

[0227] In some embodiments, R2is Ci-Ce alkyl substituted with -NH2. In some embodiments, R3is Ci-Ce alkyl substituted with -NH2. In some embodiments, R2is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2. In some embodiments, R3is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2. In some embodiments, R2is:or

[0228] In some embodiments, R3is -CH2R3awherein R3ais optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl, wherein R3is optionally substituted with - (CH2)O-3NH2 or -(CH2)O-3N(H)C(=NH)NH2. In some embodiments, R3is selected from the group consisting of phenyl, pyridyl, thiophenyl, pyrazolyl, imidazolyl, 1,2,4-triazolyl, 1,2,3- triazolyl, tetrazolyl, and pyrrolyl, wherein R3is optionally substituted with -(CH2)o-3NH2 or - (CH2)O-3N(H)C(=NH)NH2. In some embodiments, R3is optionally substituted Ci-Ce alkyl, substituted with -NH2 or -N(H)C(=NH)NH2. In some embodiments, R3ais optionally substituted indole.

[0229] In some embodiments, R3is selected from the group consisting of:

[0230] In some embodiments, R4is selected from the group consisting of:-Glu-Tyr-Glu-Lys-Glu-Tyr-Glu- , and

[0231] In some embodiments, R5is Ci-Ce alkyl substituted with -NH2, and R7is Ci-Ce alkyl substituted with -NH2. In some embodiments, R5and R7are taken together to form a staple comprising 1-10 carbon atoms. In some embodiments, each of R6and R9is independently selected from the group consisting of:

[0232] In some embodiments, R6is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2. In some embodiments, R6is

[0233] In some embodiments, R8is a warhead, and wherein a warhead is L-R8a, whereinL 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 R8ais optionally substituted phenyl substituted with -N(H)C(O)-R8bor -N(H)S(O)2-R8b, wherein R8bis optionally substituted C2-C6 alkenyl.

[0234] In some embodiments, R9is Ci-Ce alkyl. In some embodiments, R9is . In someembodiments, L is:

[0235] In some embodiments, R8ais selected from the group consisting of:

[0236] In some embodiments, 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 moieties

[0237] 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 (Zn2+).

[0238] In some embodiments, 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. In some embodiments, the zinc-binding chemical moiety is hydroxamate,, or a thiol group.

[0239] In some embodiments, a zinc -binding chemical moiety includes X-R10b, wherein X is ANC| R1ob js seiecte(j fromt|qcgroup consisting of:

[0240] In some embodiments, a zinc -binding chemical moiety includes X-R10b, whereinX is and R10bis;

[0241] In some embodiments, a zinc -binding chemical moiety includes X-R10b, X is a bond and R10bis:IV. Modifications

[0242] 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.

[0243] 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.

[0244] In some embodiments, the N- or C- terminus of the peptide is modified with a chemical moiety.V. Lipidated Tagging

[0245] A central limitation in the development of peptide therapeutics is their short circulation time resulting from rapid enzymatic degradation and renal clearance. Several methods to evade renal elimination by increasing the molecular weight have emerged and areknown to those of ordinary skill in the art, including peptide modifications. However, extensive modifications can cause undesired steric hindrance during target binding.

[0246] In some embodiments, a synthetic peptide of the present disclosure comprises a lipidated tag. Without being bound by mechanism, in some embodiments, a lipidated tag increases the half life of synthetic peptide, e.g. in human serum.

[0247] Non-limiting examples of lipidation include Myristoylation, Palmitoylation, Glycosylphosphatidylinositol (GPI)-anchor addition, and Prenylation.

[0248] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a synthetic peptide as disclosed herein has a half-life of between about 19-20 hours in human serum. Insome 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.

[0249] In some embodiments, 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.VI. Amino Acid Substitutions

[0250] In some embodiments, a synthetic peptide disclosed herein has one or more modifications to its sequence. In some embodiments, the modification is at the N- or C- terminus of the peptide. In some embodiments, the modification is on a side chain of an amino acid in the peptide. In some embodiments, the modification is a substitution of one or more L-amino acid with a D-amino acid. In some embodiments, the modification is a substitution of an a-amino acid with a [3-amino acids.

[0251] In some embodiments, the peptide comprises a substituted amino acid. In some embodiments, 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. In some embodiments, the canonical substituted amino acids are an Ala, a Ser, a Gin, or an Arg.TABLE A: Canonical amino acids used in the peptides and peptidomimetics.

[0252] In some aspects of the disclosure, 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.TABLE B: Non-canonical amino acids for use in the peptides and peptidomimetics.

[0253] In some embodiments the amino acids of the synthetic peptide are mixed canonical and non-canonical amino acids.

[0254] 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.Conservative substitution tables providing similar amino acids are well known in the art. By way of example, the groups of amino acids provided in Tables 1-3 are, in some embodiments, considered conservative substitutions for one another.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.

[0255] Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY. An antibody generated by making one or more conservative substitutions of amino acid residues in a parent antibody is referred to as a “conservatively modified variant.”VII. Preparation of Peptides

[0256] 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. For example, 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. Alternatively or additionally, 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.VIII. Pharmaceutical Compositions

[0257] For therapeutic use, a synthetic peptide or peptidomimetic disclosed herein preferably is combined with a pharmaceutically acceptable carrier and / or an excipient. The term “pharmaceutically acceptable” as used herein 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.

[0258] The term “pharmaceutically acceptable carrier” as used herein 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. For examples of carriers, stabilizers and adjuvants, see, e.g, Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] , 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.

[0259] Pharmaceutical 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.

[0260] 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. Generally, 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. In the case of sterile powders for the preparation of sterile solutions, 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 aslecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0261] The term “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. Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical 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.IX. Kits

[0262] In some embodiments, 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.

[0263] 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, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, 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. As used herein, “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 suchthat 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.EXAMPLES

[0264] Below are examples of specific embodiments for carrying out what is disclosed herein. The examples are offered for illustrative purposes only and are not intended to limit scope.

[0265] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, PROTEINS: STRUCTURES AND MOLECULAR PROPERTIES (W.H. Freeman and Company, 1993); A.L. Lehninger, BIOCHEMISTRY (Worth Publishers, Inc., current addition); Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL (2nd Edition, 1989); METHODS IN ENZYMOLOGY (S. Colowick and N. Kaplan eds., Academic Press, Inc.); REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg ADVANCED ORGANIC CHEMISTRY 3rdEd. (Plenum Press) Vols A and B (1992).

[0266] Unless otherwise stated, all reagents and chemicals were obtained from commercial sources and used without further purification.EXAMPLE 1 - Synthesis of Peptides

[0267] This example describes the synthesis of peptides used in the following examples.Materials

[0268] 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- Cys(Trt)-OH, Fmoc-Glu( / Bii)-OH. Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Scr( / Bii)-OH. Fmoc-Thr( / Bii)-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. Reagents used in solid phase peptide synthesis: Piperidine (ReagentPlus; 99%), formic acid (> 98%) were purchased from Sigma- Aldrich (St. Louis, MO). 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). Reagents (cleavage): 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) from VWR, PA. Recombinant Human Thyroid Hormone Receptor alpha 1 protein (THRA) from Abeam, UK.

[0269] Unless otherwise stated, all other chemicals were purchased from Sigma (St. Louis, MO).Automated flow peptide synthesis (AFPS) set-up

[0270] All peptides were synthesized on automated-flow systems built in the Pentelute lab (“Amidator” and “Peptidator”), which are similar to the published AFPS system. The synthesis conditions were published previously:

[0271] The following settings were used for protein synthesis: flowrate = 40 mL / min, temperature = 90°C (loop) and 85-90°C (reactor). The 50 mL / min pump head pumps 400 pL of liquid per pump stroke; the 5 mL / min pump head pumps 40 pL of liquid per pump stroke. The standard synthetic cycle involves a first step of pre washing the resin at elevated temperatures for 60 s at 40 mL / min. During the coupling step, three HPLC pumps are used: a 50 mL / min pump head pumps the activating agent, a second 50 mL / min pump head pumps the amino acid and a 5 mL / min pump head pumps DIEA. 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 another40 pump strokes. During the deprotection step, 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. Next, 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.

[0272] Preparation ofPd-Aryl Complexes: RuPhos (513 mg, 1.10 mmol, 1.10 equiv) was added to a 20 mL scintillation vial equipped with a magnetic stirbar. The vial was loosely sealed with a screw cap and brought into a nitrogen-filled glovebox. Cyclohexane (3 mL) and bromoaryl compound (1.10 mmol, 1.10 equiv) were added and the reaction mixture stirred for 1 min until a clear solution formed. (cod)Pd(CH2TMS)2 (398 mg, 1.00 mmol, 1.0 equiv) was added. 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.

[0273] Preparation ofReactides: Crude, unprotected peptides bearing a single Cys (approx. 6-8 mg, 5 pmol, 1 equiv.) were dissolved in DMF (0.3 mL) in a 2.0 mL microcentrifuge tube. Pd-OAC (5 mg, 6.5 pmol, 1.3 equiv.) was dissolved in DMF (0.3 mL) and added to the peptides. The solution was mixed thoroughly and allowed to react for 30 min at rt. The reaction was diluted with 5% MeCN + 0.1% TFA in water (10 mL). Reactides were isolated from the reaction mixture by reverse-phase flash chromatography.

[0274] Synthesis methods for covalent warheads are known to those of ordinary skill in the art. Examples of this are provided in Vinogradova, Nature, 2015, and Grob, ACS Chemical Biology, 2023.Manual peptide synthesis

[0275] 200 mg of H-Rink Amide-ChemMatrix resin (0.49 mmol / g, 0.10 mmol) or HMPB- ChemMatrix resin (0.44 mmol / g, 0.088 mmol) was placed in a 6 mL Torviq fritted syringe. After swelling in DMF for 3 minutes, the resin was then washed with DMF (3 x 5 mL). Each Fmoc-protected amino acid (0.70 mmol, 7.0 equivalents) was dissolved in 0.38 M PyAOP solution (DMF as solvent, 1.1 mL, 0.65 mmol). Immediately before the coupling, DIEA (2.10 mmol, 21 equivalents) was added to the mixture. 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.General synthesis method for peptide-zinc ligand conjugates

[0276] 200 mg of H-Rink Amide-ChemMatrix resin (0.49 mmol / g, 0.10 mmol) or HMPB- ChemMatrix resin (0.44 mmol / g, 0.088 mmol) was sequentially modified with Fmoc-Gly- OH, Fmoc-MeDbz-OH and a C-terminal amino acid (Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)- OH, or Fmoc-Gln(Trt)-OH) using the general method for manual coupling. The obtained resin was then used in AFPS to synthesize the peptide sequence. [Optionally, 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). [Optionally, 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]. To activate Dbz-linker, 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). After concentration of the peptides using gentle flow of an inert gas, 10% acetonitrile in water was added (10 mL) and the resulting solution was frozen and lyophilized. The obtained mixture was then deprotected with a cleavage cocktail containing 95% TFA, 2.5% water, and 2.5% TIPS (y / v), for 2 h at room temperature. TFA was removed under a gentle stream of nitrogen gas, and the crude peptide was precipitated by adding cold Et2O (-80°C). After centrifugation at 3220 ref for 3 min, the supernatant was removed, and the precipitated peptide wastriturated three times with cold Et2O. The resulting material was dissolved in 50% MeCN in water with 0.1% TFA and lyophilized as crude.Synthesis in cases whereby protected forms of BoroLeu were used to cleave activated Dawson linker

[0277] In some cases, after nucleophilic cleavage step and prior to purification, the following step was used:

[0278] Crude pellet was dissolved in 5 mL of boronate deprotection solution (prepared by combining 800 mg of MeB(OH)2 in 13.2 mL of acetone and 13.2 mL of 0.1 M HCl(aq)), and allowed to mix at room temperature for 2 hours. Next, the solution was frozen and lyophilized to furnish the crude peptide as a white powder ready for purification. Examples of this synthesis are disclosed in Hinkes, Organic Letters , 2019.Purification of the crude peptide.

[0279] Crude peptides were purified by either flash or high-performance liquid chromatography.

[0280] 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.

[0281] HPLC:Column'. Agilent Zorbax 300 SB C3 (5 mm, 9.4 x 250 mm, 300 A pore size) with an Agilent C3 Zorbax SB 300 guard column.Loading'. < 50 mgFlow Rate'. 4 mL / min.Column Temperature'. 60 °CInstrument'. Agilent mass directed purification system (1260 Infinity LC and 6130 Single Quad MS), affixed with a Timberline Instrument TL105 HPLC column heater. Mobile phases used for LC-MS analysis were Solution A (0. 1% v / v TFA in water) and Solution B (0. 1% v / v TFA in acetonitrile).

[0282] Purification consisted of two stages: gradient generation, and preparative separation.

[0283] Gradient Generation: The purification column was equilibrated to 5% B, and an aliquot of the freshly prepared filtered protein solution accounting for approximately 1 mg of crude protein was injected. 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 65% B over 60 min. The resulting chromatogram was then analyzed to identify the mobile phase composition that began elution of the desired protein peak (% B can be calculated from the gradient and elution time) - this value is termed “C”

[0284] 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.Method for LC-MS characterization

[0285] 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).

[0286] 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.

[0287] 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.

[0288] 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 (2-12min), 91% B to 91% B (12-13 min). The flow rate is 500 pL / min. MS acquisition is from 4 to 12 min.EXAMPLE 2 - Screening of zinc-binding groups at C-terminus of IGFBP5-anchor peptide

[0289] Although both IGFBP4 and IGFBP5 are 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.

[0290] The mechanism behind IGFBP4-PAPP-A recognition appears to be more sophisticated and remains opaque. Thus, 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). Moreover, 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). On this basis, the screening strategy was focused on the ability of the peptides to inhibit PAPP-A mediated cleavage of IFGBP4 in the presence of IGF1.

[0291] IGFBP5-anchor peptide featuring amide or carboxylic acid at the C-terminus (peptides 1 and 2) did not show substantial inhibition below 10 pM (FIG. 30). To improve binding and inhibitory activity 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). For initial assessment, a gel-based assay was used covering several concentrations of the peptide in the ranges of 1 nM -1 pM to identify peptides with submicromolar activity. The screening has revealed that 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-ZBGconjugates did not demonstrate PAPP-A inhibition in the screened range of concentrations (FIGS. 31A-31C).

[0292] Additionally, alternative binding moieties based on IGFBP4 cleavage and PAPP-A autocleavage sites (Laursen, et al., Biochemical Journal, 2002) were also considered. For this purpose, IGFBP4 and PAPP-A derived peptides were prepared containing 20-mer sequence featuring hydroxamate at the C-terminus of the cleavage site. However, the IGFBP4- and PAPP-A-based peptides have shown substantially lower or no inhibitory activity against PAPP-A (FIG. 32). Thus, IGFBP5 -anchor peptide equipped with hydroxamate was further used to investigate how peptide’s composition affects the inhibitory functions.

[0293] 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. To investigate if steric hindrance will have detrimental effect on inhibition the shortest possible extension i.e. 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. Thus, SAR of peptides containing a single free thiol was focused on. First, 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. Several other 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.EXAMPLE 3 - Structure-activity relationship studyN-terminus truncation

[0294] Gained inhibitory function was used as the major indicator of binding efficiency. The gel-based IC50 screening of the N-terminus shortened peptides 3,13-18 has revealed that deletion of 119Pro-120Lys fragment did not result in significant change of the IC50 ranges, while further elimination of N-terminal amino acids resulted in gradual loss of activity with no activity observed in sub-pM ranges after 124Ile deletion (FIG. 33). Peptides 3 and 13 were additionally tested by Simple Western assay demonstrating five-fold better performancein case of the shortened peptide 13 (FIG. 2), which is surprising considering some tight interactions of the 119Pro-120Lys fragment with PAPP -A.L-Alanine scanning and single amino acid substitution

[0295] Based on the truncation study, L-Alanine scanning of the peptide 13 was performed to elucidate the role of each amino acid residue. Ala scanning revealed that mutation of 126Glu and 135 Asp did not affect ranges of the inhibitory activity, while multiple mutations (122Thr, 125Ser, 132Val, 133Lys, 134Lys, 136Arg, 137Arg, 138Lys, 14 IThr) located primarily in the middle of the sequence increased IC50 only marginally (FIG. 2). At the same time modification of several residues (12 IHis, 123Arg, 127Leu, 130Glu, 142Gln and 143Ser) resulted in substantial loss of activity although preserving IC50 in the nanomolar range (FIG. 47). More importantly, evident hotspots were identified, where Ala incorporation dramatically increased IC50 to more than 1 pM (124Ile and 139Lys) or even 10 pM (128Lys). Importance of the latter residue was also pointed out in the previous reports showing its crucial role in PAPP-A mediated IGFBP5 cleavage (Judge, et al., Nature Communications, 2022; Laursen, et al., Biochemical Journal, 2002). While dominating interactions of the 139Lys remain unclear, 124Ile seems to orient 781His of PAPP-A to improve its hydrogen bonding with 128Lys (FIG. 34). Overall, previously available PAPP- A / IGFBP5 -peptide structural data and Ala-scanning of the peptide 13 allow to construct 2D- map of the peptide-protein interactions (FIG. 35).C-terminal amino acid variation

[0296] Since hydroxamate functionality obviously plays vital role in gaining inhibitory activity, the C-terminal residue was also investigated in more details by preparing alternative mutants. Incorporation of more flexible [3-alanine fragment resulted in a similar activity to L- Ala mutant, thus indicating preference of a-amino acid residues. Moreover, His- and Cys- mutants were tested since these two residues possess side chains capable of strong interactions with Zn potentially leading to a dual Zn-binding C-terminus. Cys-mutation was found not to affect the inhibitory potential, whereas His-mutant showed activity only in micromolar range in a gel-based assay (FIG. 36).Modification of middle region.

[0297] 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-cellularmatrix (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.

[0298] An attempt to reduce overall positive charge and remove potential cleavage sites by substituting the middle region and C-terminus with peptidomimetic linkers containing oligoethyleneglycol chains has led to peptide hydroxamates showing no inhibitory activity towards IGFBP4 cleavage below 10 pM in a gel-based assay format, although the designed linkers can cover the necessary distance to deliver hydroxamate to the Zn site (FIGS. 37A- 37E).

[0299] Additional testing of the peptides featuring multiple Ala substitutions in the middle region helped to recover activity below 10 pM, which supposedly indicated importance of the a-helical structure.

[0300] 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. Thus, incorporation of S-(4-pentenyl)alanine at positions 134Lys and 138Lys followed by ring-closing metathesis allowed synthesis of peptides 19 and 20 with alternated ECM-binding motif and showing activity similar to the parent peptides 3 and 13, respectively.Avidity effect

[0301] 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. For this purpose, two peptides were prepared possessing flexible oligoethyleneglycol linkers capable of connecting two IGFBP5 -anchor peptide fragments coordinated to the PAPP-A catalytic grooves of two different subunits(FIGS. 38A and 38B). Gel-based IC50 range screening has shown similar activity of the dimer peptides, indicating lack of affinity improvement using this strategy (FIG. 39).Incorporation of albumin-binding tags

[0302] One of the major drawbacks associated with peptide-based drugs is a short half life time due to enzymatic degradation and renal clearance. However, there are a number of potential strategies to address this issue, e.g. incorporation of peptidomimetics, side chain and backbone modified amino acids, peptide cyclization, lipidation, etc. While combination of these strategies has been shown to drastically improve peptide stability, lipidation is one of the most impactful approaches to achieve extended circulation times via binding to albumin and thus decreasing rates of both enzymatic cleavage and renal elimination (Wang, et al., Signal Transduction and Targeted Therapy, 2022; Werle, et al., Amino Acids, 2006; Zhang, et al., J. Am. Chem. Soc., 2022; Bech, et al., ACS Med. Chem. Lett., 2018). For example, efficacy of the lipidated tags was demonstrated on a series of synthetic analogs of glucagon- like peptide 1, e.g. semaglutide modified with non-dietary fatty acid connected to e-NH2 of 26Lys via an optimized linker composed of y-glutamate and OEG (8-amino-3,6- dioxaoctanoic acid) (Lau, et al., J. Med. Chem., 2015). Although introduction of a bulky substituent can disrupt peptide-protein interactions and lower binding potency in vitro, extended half-life may compensate in vivo efficacy of the peptide (Zhang, et al., J. Am. Chem. Soc., 2022). Semaglutide sets an example showing that rational positioning of the bulky lipidated tags results in prolonged lifetime without decreasing binding potency.

[0303] To minimize sterical hindrance peptides 3 and 13 were modified at N-terminal amino acids with octadecanoic diacid through a spacer consisting of yGlu and two OEG units. While terminal position is less likely to affect binding to the catalytic groove it does not allow equal sterical protection from peptidases to the entire length of the peptide. For this reason, two peptides were also prepared containing the lipidated tag at 133Lys, which was identified as a cold spot during the Ala-scanning, whereas cryo-EM data implies sufficient space to accommodate a large substituent. Gel-based activity screening has identified that exact N- termination is important for preserving binding and inhibition. Thus, 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

[0304] To assess binding affinity of the IGFBP5-anchor peptide and its functionalized versions a series of peptides labelled at N-terminus with a fluorescent tag were prepared. Fluorescence polarization study of the FITC-labelled IGFBP5-amide (peptide 1) has shown KD as 0.83±0.3 pM, whereas its N-terminal fragment (PKHTRISELK) containing two major hotspots did not show any binding in the investigated range (<10 pM) (FIG. 41). Afterwards, the effect of hydroxamate as ZBG and the lipidated tag on the apparent KD was studied. Screening of the peptide 3 functionalized with BODIPY at the N-terminus has revealed that introduction of the ZBG has substantially lowered KD to 2.4±0.3 nM (FIGS. 3A-3B). Meanwhile, modification of 133Lys with the lipidated tag did not seem to disrupt peptide- protein interactions showing FP-estimated KD to be 0.8±0.3 nM , which is three orders of magnitude lower in comparison to the amide -terminated IGFBP5 anchor. Interestingly, mutation of the major hotspot 138Lys to Ala in the peptide 3, resulted only in partial loss of binding with increased KD of 0. 15±0.6 pM, which indicates that the developed peptide has a dual binding mode targeting natural receptor pocket responsible for the IGFBP5 -recognition and Zn-catalytic site. At the same time, substitution of 138Lys with Asp result in complete loss of binding in the tested range and shows similar profile to IGFBP5-scrambled-anchor peptide with hydroxamate functionality at the C-terminus (FIG. 42).EXAMPLE 5A - Potency Studies

[0305] 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.

[0306] The IC50 value of the IGFBP5 -hydroxamate compound for IGFBP4 cleavage was calculated to be approximately 19 nM (FIG. 4).

[0307] The IC50 value of the IGFBP5 -hydroxamate compound for IGFBP5 cleavage was calculated to be approximately 363 nM (FIG. 4).

[0308] The potency of lipidated IGFBP5 -hydroxamate was tested by measuring inhibition for recombinant PAPP-A and a A549-based assay.

[0309] Working Stocks of peptides:20uM in IX PBS

[0310] PCR tubes setup:5 pM -0.0015 pM 1.5-fold series (varies based on the IC50 range)

[0311] Controls:No peptide - PAPP-A + IGFBP4 / IGF1Only IGFBP4 / IGF1WT-PAPP-A - 1 nMIGFBP4 / IGF1- 120 nM / 360 nMTotal reaction volume - 30 HL

[0312] Procedure:1. Peptides were incubated for 1 hour at RT with WT-PAPP-A / Pregnancy serum / A549 cells / A549 Conditioned media, prior to adding the substrate.2. Pre-formed substrate complex of IGFBP4 / IGF1 was added.3. The solution was incubated for 3.5 hours at 37°C and then quenched with 0.1 mM EGTA4. 8 pL of reaction solution was mixed with 2 pL of 5X Fluorescent Master Mix (part of EZ Standard pack, Biotechne product #PS-ST01EZ-8).5. Using the Simple Western protocol, the plate was loaded with 4 pL of boiled / SDS- reduced samples onto the plate and run on the JESS instrument for 3 hours.

[0313] Inhibition for recombinant PAPP-A was tested by measuring the percentage of cleavage of IGFBP4 for AV-21a, AV-233, AV-245PK, and AV-245HT using Simple Western assay. The results are shown in FIG. 9 and revealed that the lipidation of peptides did not result in substantial activity loss.

[0314] 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).

[0315] 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).

[0316] 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).EXAMPLE 5B - In Vivo Pharmacokinetic Study in Mice

[0317] To determine the effect of lipidated tags and various administration modes on the half-life of the synthetic peptides disclosed herein, male C57BL / 6J Mouse, fed mice were divided into nine groups (n = 3) and administered AV-271 at the doses indicated in Table 4. As indicated in Table 4, the groups were administered AV-271 by intravenous (IV), subcutaneous (SC), or intraperitoneal (IP) injection. The results are shown in FIG. 29 and Table 4 and revealed about 9-19 hour half-life time depending on dose and injection type.Table 4: Bioavailability after administration of inhibitor at various dosesEXAMPLE 6 - Selectivity Studies for Matrix Metalloproteinases (MMPs) and PAPP- A2

[0318] The selectivity of IGFBP5-hydroxamate was tested by measuring inhibition of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP13 and inhibition of PAPP-A2.

[0319] The inhibition of MMPs was tested by measuring the relative fluorescence unit (RFU) signal using a concentration of 25 nM of MMP enzyme (Enzo; Cat. # BML-AK013 andBML-AK014) and a peptide concentration of 25 pM , using cleavage of a fluorescent substrate (R&D; Cat. # ES010) as readout (FIG. 5).

[0320] The inhibition of PAPP-A2 (produced recombinantly) was tested by measuring IGFBP5 cleavage over a range of concentrations of peptide using Simple Western assay to measure cleaved IGFBP5 bands against intact IGFBP5 bands (e.g., between 102and 105nM) (FIG. 6)EXAMPLE 7 - Enzymatic Stability and Circulation Times

[0321] 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).

[0322] The stability of 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.Table 5: IGFBP5-hydroxamates for Stability TestingSerum stability.

[0323] To establish effect of the incorporated modifications i.e. stapled bridges and lipidated tags on enzymatic stability, human serum stability of the corresponding peptides was measured.

[0324] Methods: Human serum was thawed and centrifuged (14,000 rpm for 3 min) to remove lipids before incubating at 37 °C for 15 min to activate the proteins. Peptide solutions were prepared at 300 pM in PBS and added to human serum in a 1:5 (v / v) ratio, and the mixed solutions were incubated at 37 °C for 0, 1, 2, 4, 7, and 24 h. At designated times, 10 pL of each sample was removed from the incubator, mixed with 20 pL of acetonitrile: TFA (97,3, v / v), and left for 10 min at 4 °C. Next, 30 pL of 6 M urea was added into the mixture and left for 10 min at 4 °C. Finally, 30 pL of 20% w / v trichloroacetic acid was added and leftfor 10 min at 4 °C. The quenched solutions were centrifuged at 14,000 rpm for 10 min at 4 °C, and 45 pL of the supernatant of each sample was transferred to LC-MS vial. The samples were analyzed with Agilent 6550 quadrupole time-of-flight LC-MS using Agilent Zorbax 300SB C3 column (2.1 x 150 mm, 5 pm particle size, 300 A pore size). Gradient: 10% B (0-5 min), linearly ramp from 10% B to 45% B (5-15 min), 45% B to 91% B (15-16 min), 91% B to 91% B (16-20 min). The flow rate is 500 pL / min. MS acquisition is from 5 to 15 min. Percentage of the remaining peptide was estimated by dividing the area under extract ion chromatogram (EIC) to the area of time zero (N = 2-4).

[0325] Results: The peptides 3 and 13 having hydroxamate functionality as the only modification to the IGFBP5 -anchor, were found to undergo rapid enzymatic degradation with rl / 2 ~2 hours and nearly full degradation after 24 hours (FIGS. 3A, 3B, and 43). In this case, no major fragments could be identified as the peptide is likely shredded into smaller hydrophobic peptides that could not be identified by LC-MS method. Interestingly, that stapled peptides were showing only marginally improved serum stability profile, however stapling clearly resulted in stabilization of the C-terminal half of the peptide and allowed us to identify major cleavage sites closer to the N-terminal side. Moreover, 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. In the meantime, lipidated peptide (AV-245PK) have shown superior stability without significant degradation after 24 hour incubation. Thus, 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. Noteworthy, 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.EXAMPLE 8 - Gel-based IGFBPs Cleavage InhibitionPAPP-A mediated IGFBP4 cleavage inhibition assay

[0326] 2 pL of 20 nM solution of PAPP-A (produced recombinantly) in PBS was added to the mixture of 2 pL of IGF 1 (Abeam; Cat. # ab270062) (7 pM), 2.33 pL of IGFBP4 (produced recombinantly, 3.85 pM) and the corresponding peptide (ranges of concentrations) in PBS to generate 20 pL of a solution with the following final concentrations: PAPP-A: 2 nM ; IGF1: 700 nM ; IGFBP4: 450 nM ; Peptide: 0.1, 1, 10, 50, 100, 500, 1000 or 10000 nM. 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.PAPP-A mediated IGFBP5 cleavage inhibition assay

[0327] 2 pL of 0.2 nM solution of PAPP-A (produced recombinantly) in PBS was added to the mixture of 3 pL of IGFBP5 (produced recombinantly, 5 pM), and the corresponding peptide (ranges of concentrations) in PBS to generate 20 pL of a solution with the following final concentrations: PAPP-A: 2 nM ; IGFBP5: 500 nM ; Peptide: 0.1, 1, 10, 50, 100, 500, 1000 or 10000 nM 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%, BisTris, 1.0-1.5 mm). IC50 ranges were estimated based on disappearances of the band corresponding to intact IGFBP5 or appearance of the corresponding fragments.EXAMPLE 9 - Bioassays

[0328] Simple Western Assay To determine the IC50 of different peptides for PAPP-A as a target by MIT. These peptides are targeted towards cleavage inhibition of IGFBP4.

[0329] MaterialsHiBit-IGFBP4-8X His fusion protein (produced recombinantly) IGF1 Abeam IGF1 (ab270062)PAPP-A (produced recombinantly, 3 nM stock)IX PBS; pH 7.4Multi-channel pipettePCR tubesJESS automated Western blot system (Biotechne Cat. # 004-650)Simple Western Assay Reagents• Anti-His antibody (Primary to capture HiBit-IGFBP4-8X His fusion protein)- 0.5mg / ml (Genscript; Cat.#A00186-THE_His_Tag_Antibody_mAb_Mouse)• Anti-mouse detection module (Biotechne; DM002)• EZ Standard pack 1 (Ladder, reducing agent and fluorescent Master mix) (Cat. #PS- ST01EZ-8)• ProteinSimple 12-230 kDa Separation Module, 200 Capillaries (NCI 193547)

[0330] Procedure

[0331] Inhibition Assay1. Serial dilution of 2X peptide in a total volume of 15 pL For example, 10 pM of peptide in 15 pL would result in a final concentration of 5 pM in 30 pL (total reaction volume).2. IGFBP4 / IGF1 - Prior incubation for complex formation to a final stock concentration of 400 / 600 nM3. PAPP-A stock prepared to a final concentration of 3 nM4. The components were added in the order outlined in Table C below; in 8-strip PCR tubes.5. Prior to addition of substrate IGFBP4 / IGF1, the peptides are incubated with PAPP-A for 1.5 hours at 37°C.Table 66. After 1.5 hours incubation with the peptides, IGFBP4 / IGF1 is added to the reaction and incubated for 3.5 hours at 37°C7. Once the reactions are incubated for 3.5 hours, they are quenched with 0.1 mM EGTA.Simple Western Assay Set-Up1. 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).a. Prepare Standard Pack Reagents i. Open and remove 3 tubes from EZ Standard Pack ii. DTT (Clear Tube)1. Pierce foil with pipette tip2. Gently mix by pipette3. Add 40 uL of deionized water to make a 400 mM solution iii. Fluorescent 5X Master Mic (Pink Tube)1. Pierce foil with pipette tip2. Gently mix by pipette3. Add 20 uL 10X Sample Buffer4. Add 20 uL of prepared 400 mM DTT solution iv. Biotinylated Ladder (Green Tube with Pink Pellet)1. Pierce foil with pipette tip2. Gently mix by pipette3. Add 20 uL of deionized water b. Prepare Samples i. Optimal protein concentration depends on expression level of your protein. Dilute lysate as necessary with 0.1X Sample Buffer ii. Combine 1 part 5X Fluorescent Master Mix with 4 parts diluted lysate in a microcentrifuge tube1. Final concentration of 0.4 mg / mL for chemiluminescence or 1.0 mg / mL for fluorescence iii. Produce enough diluted sample volume required for assay iv. Gently mix by pipette and close tube. c. Denature Samples i. Vortex to mix ii. Incubate at 95 °C for 5 minutes iii. Vortex and spin iv. Store on ice d. Mix Luminol-S and Peroxide (If Applicable) i. Combine 200 uL Luminol-S and 200 uL Peroxide in a microcentrifuge tube ii. Gently pipette to mixiii. Store on ice2. 8 pL of the total reaction are taken in separate tubes and 2 pL of 5X Fluorescent Master mix is added to each of these reactions and boiled at 95°C for 5 min.3 pL of the boiled reactions are added to the plate set-up by reverse-pipetting to ensure no bubbles in each of the wells. Note that the evaporation sensitive cover was not removed until ready to put the plate in the JESS instrument (Biotechne JESS; Cat. # 004-650)3. For the Simple Western assay Read-Out, the run report was saved as a PDF and the readout image was saved separately as JPEG file.EXAMPLE 10 - Cryo-EM structure-based peptide design

[0332] Development of active PAPP-A fluorescent probes is important to enable quantification and localization of active PAPP-A and elucidate its role in aging-related diseases. IGFBP5 -anchor peptide is a great binding modality to exploit for proximity-driven covalent modification of PAPP-A in the area of the catalytic groove. The cryo-EM structure of PAPPA-BP5 allows structure based rational design and incorporation of the corresponding covalent warheads.

[0333] Using the cryo-EM structure (Protein Data Bank ID 7ufg) as design rationale, we have identified several of PAPP-A’s residues that could serve as potential targets for covalent binding. Thus, initial focus was on Arg 136 (or Arg 156) and Leu 140 (or Leu 160) providing access to Lys338 and Lys528 / Tyr558, respectively (FIG. 12). While the mentioned targets represent typical nucleophiles that can be targeted via electrophilic warheads (e.g. SuFEX chemistry electrophiles, sulfonyl fluorides). Although initial focus was on nucleophiles residing in the catalytic groove, we have also considered possibility to target PAPP-A with nucleophilic warheads (sulfamates) and acrylamides preferring free thiols of cysteine residues.

[0334] As a general strategy we have prepared Cys-mutants in the corresponding positions that were equipped with a covalent warhead via S-arylation with Pd-complexes (FIG. 13).

[0335] Firstly, we aimed to set up an assay to test whether a peptide binds to PAPP-A covalently. PAPP-A does not produce any detectable signal in ESI and is only poorly ionized by MALDI (FIG. 14).

[0336] Therefore, we have focused on a gel-based assay with read out from two channels, corresponding to fluorescein and Coomassie blue excitation / emission wavelengths. For this, we have incorporated fluorescent tags at N-terminus of the IGFBP5-anchor peptides and overall design strategy can be illustrated as follows (FIG. 15).

[0337] At first, several reactides with differently substituted sulfonyl fluorides and sulfamates at position 136 and 140 were screened. Here, the signal of Coomassie blue stained PAPP-A (produced recombinantly) bands appears similar across different incubations, whereas intensity of the fluorescence detected in the fluorescein channel clearly varies, indicating different degree of binding to PAPP-A. Reactides based on a scrambled IGFBP5- anchor sequence did not show substantial binding to PAPP-A, which confirms proximity- driven nature of the interactions (FIG. 16).

[0338] To exclude the possibility that non-covalent interactions may contribute to the intensity of the fluorescein channel, we performed control experiment, where PAPP-A was incubated with functionalized reactide and FITC-labelled IGFBP5 -anchor peptide bearing no covalent warhead. The latter has not demonstrated any signal in the fluorescein channel, thus indicating negligible role of non-covalent interactions in PAPP-A staining (FIG. 17).

[0339] Additionally, a study was conducted to identify the stoichiometry of the interactions. MALDI-MS analysis has revealed ~3 kDa shift of the PAPP-A related peaks, which corresponds to the molar weight of a single reactide unit and therefore indicates 1 : 1 stoichiometric ratio (FIG. 18).

[0340] To identify whether covalent binding is indeed proximity-driven and PAPP-A specific, we have incubated the same series of reactides with PAPP-A in 10% human serum. Comparison of Coomassie blue and fluorescein channel suggest the proximity-driven nature of the covalent binding, however albumin appears to be the major off-target band, which is likely caused by high concentration of this protein. Semi quantification of the PAPP- A / albumin fluorescent bands identifies reactides 1 and 7 as the most promising ones (FIG. 19).

[0341] In an additional round of screening we aimed to investigate paralog specificity of the reactides, by incubation with 1: 1 mixture of PAPP-A 1 and PAPP-A2. Here, again peptides 1 and 7 were showing remarkable selectivity towards PAPPA1 over PAPPA2, thus allowing to assume paralog specificity of the developed reactides (FIG. 20).

[0342] Although the exact residue responsible for the covalent binding with the IGFBP5- reactides was not identified, the reaction appears to be sensitive to the availability of the catalytic groove. Thus, reactides do not bind to PAPP-A after pre -incubation with PAPP-A natural covalent inhibitor proMBP (produced recombinantly). proMBP binds covalently via disulfide bridge near to the catalytic groove and thus preventing substrate recognition and cleavage (FIG. 21).

[0343] Current attempts to target cysteine residues of PAPP-A did not result in efficient binding, which is most likely related to the fact that targeted cysteines form intramolecular disulfide bridge. Moreover, these reactides showed poor selectivity for PAPP-A over PAPP- A2. Noteworthy, reactides 1 and 7 showed very low inhibitory activity against PAPP-A (FIG. 22)

[0344] To investigate suitability of the acrylamide warheads, we plan to retarget IGFBP5- reactides to free-thiol functionality. PAPP-A Cys residue that is bound by the natural covalent inhibitor proMBP is also considered a potential target (FIG. 26).

[0345] To study whether incorporation of both zinc-binding group (-NH0H) and covalent warhead (sulfonyl fluoride) will result in improved PAPP-A staining and inhibition, we have prepared a reactide equipped with hydroxamate at C-terminus. While PAPP-A staining gelbased assay has indicated covalent binding, the reactide did not show PAPP-A inhibition in sub-pM ranges (FIG. 27).

[0346] Noteworthy, removal of the covalent warhead resulted in improvement of the inhibitory activity, while PAPP-A staining can still be observed, which is likely caused by strong Zn-hydroxamate interactions (FIG. 28).

[0347] 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.

[0348] As used herein, “Cl 8” or “C18tag” is. In some embodiments, Cl 8 is also referred to as “SG,” e.g., “KSG”.INCORPORATION BY REFERENCE

[0349] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0350] An invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on any invention disclosed herein. Scope of an invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed:

1. A synthetic peptide comprising the formula L1Z1L2Z2R10or a pharmaceutically acceptable salt thereof, whereinL1is 7 to 10 amino acids in length;L2is an amino acid sequence having a distance of about 20-35 angstroms;Z1is a basic amino acid;Z2is a bond or serine,R10is selected from the group consisting of -R10a, -OH, - NH2, -N(H)OH, -N(H)R10a, -N(R10a)2, -N(R10a)OH, -SR10a, and -CH2R10a; and each R10ais independently selected from the group consisting of optionally substitutedCi-Ce alkyl, optionally substituted 3-6 membered heterocyclyl, optionally substituted -N(H)(Ci-Ce alkyl), andwherein R10ais optionally substituted with 1-3 instances of a group independently selected from oxo, - CH=CH2, -CO2H, -C(O)NH2, -C(O)N(H)OH, -OH, -SH, -OPO3H2, -SO2NH2, - PO3H2. -NH2, -NHCH2CH2CH3, cyclohexyl, optionally substituted 5-6 membered heteroaryl and a peptide comprising 2-10 amino acids; and wherein the synthetic peptide comprises a helical structure when bound to PAPP-A and inhibits pregnancy-associated plasma protein A (PAPP-A).

2. The synthetic peptide of claim 1, wherein the distance between Z1and Z2is 14 amino acids in length.

3. The synthetic peptide of claim 1 or 2, wherein Z1is selected from the group consisting of arginine, lysine, and histidine.

4. The synthetic peptide of any one of claims 1-3, wherein Z1is lysine.

5. The synthetic peptide of any one of claims 1-3, wherein Z1is arginine or homo-Arginine.

6. The synthetic peptide of any one of claims 1-3, wherein Z1is histidine.

7. The synthetic peptide of any one of claims 1-6, wherein Z2is serine.

8. The synthetic peptide of any one of claims 1-7, wherein the synthetic peptide comprises an amino acid sequence from Insulin Growth Factor Binding Protein 5 (IGFBP5).

9. The synthetic peptide of any one of claims 1-8, wherein L1comprises the amino acid sequence of PKHTRISEL.

10. The synthetic peptide of any one of claims 1-9, wherein L2comprises the amino acid sequence of AEAVKKDRRKKLT, optionally AEAVKKDRRKKLTQ.

11. The synthetic peptide of any one of claims 1-10, wherein R10is X-R10b, wherein X isand R10bis selected from the group consisting of:

12. The synthetic peptide of any one of claims 1-10, wherein R10is X-R10b, wherein13. The synthetic peptide of any one of claims 1-10, wherein R10is X-R10b, X is a bond andR10bis:

14. The synthetic peptide of any one of claims 1-13, wherein the N-terminus of the peptide is modified with a lipidated tag, optionally an albumin-binding tag.

15. A synthetic peptide of formula I or a pharmaceutically acceptable salt thereof:whereinR1is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)Rla, and -C(O)ORla;Rlais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, wherein Rlaoptionally comprises a fluorophore;R2is selected the from the group consisting of -NH2, -N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or -N(H)C(=NH)NH2;R3is selected the from the group consisting of -CH2R3a, -NH2, -N(H)C(=NH)NH2, optionally substituted Ci-Ce alkyl, optionally substituted phenyl, and optionally substituted 5-10 membered heteroaryl, wherein R3is optionally substituted with - (CH2)O-3NH2or -(CH2)O-3N(H)C(=NH)NH2;R3ais optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl;R4is selected from the group consisting of hydrogen, optionally substituted C1-C30 alkyl, -C(O)R4a, -C(O)OR4aand a peptide comprising 2-10 amino acids;R4ais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl optionally substituted with a peptide comprising 2-10 amino acids;R5is selected the from the group consisting of -NH2, -N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or -N(H)C(=NH)NH2;R7is selected the from the group consisting of -NH2, -N(H)C(=NH)NH2 and optionally substituted Ci-Ce alkyl substituted with -NH2 or -N(H)C(=NH)NH2; alternatively, wherein R5and R7are taken together to form a staple comprising 1- 10 carbon atoms;R6is selected the from the group consisting of optionally substituted Ci-Ce alkyl substituted with 0-3 instances of optionally substituted 5-6 membered heteroaryl, - NH2 or -N(H)C(=NH)NH2; optionally substituted 5-6 heteroaryl substituted with 1-3 instances of R6a; and -SR6a;R6ais optionally substituted Ci-Ce alkyl substituted or optionally substituted phenyl, wherein R6ais substituted with -SO2F or -OSO2NH2;R8is a warhead;R9is selected the from the group consisting of optionally substituted Ci-Ce alkyl substituted with 0-3 instances of optionally substituted 5-6 membered heteroaryl, - NH2 or -N(H)C(=NH)NH2; optionally substituted 5-6 heteroaryl substituted with 1-3 instances of R9a; and -SR9a;R9ais optionally substituted Ci-Ce alkyl or optionally substituted phenyl, wherein R9ais substituted with -SO2F or -OSO2NH2;X is a bond, -C(O)-,or•R10is selected from the group consisting of -R10a, -CO2H, -C(O)OH, -C(0)NH2, - C(O)N(H)OH, -C(O)N(H)R10a, -C(O)N(R10a)2, -C(O)N(R10a)OH, -C(O)SR10aand - C(O)CH2R10a; and each R10ais independently selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted 3-6 membered heterocyclyl, optionally substituted -N(H)(Ci-Ce alkyl), and, wherein R10ais optionally substituted with 1-3 instances of a group independently selected from oxo, -CH=CH2, -CO2H, -C(O)NH2, -C(O)N(H)OH, -OH, -SH, - OPO3H2, -SO2NH2, -PO3H2, -NH2, -NHCH2CH2CH3, cyclohexyl, optionally substituted 5-6 membered heteroaryl and a peptide comprising 2-10 amino acids.

16. The synthetic peptide of claim 15, wherein R1is -C(O)Rlaand R4is -C(O)R4a, Rlais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl, optionally comprising a fluorophore, and R4ais optionally substituted C1-C33 alkyl or optionally substituted C1-C33 heteroalkyl.

17. The synthetic peptide of claim 15 or 16, wherein R1is selected from the group consisting of:

18. The synthetic peptide of any one of claims 15-17, wherein R2is Ci-Ce alkyl substituted with -NH2.

19. The synthetic peptide of any one of claims 15-18, wherein R3is Ci-Ce alkyl substituted with -NH2.

20. The synthetic peptide of any one of claims 15-17 or 19, wherein R2is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2.

21. The synthetic peptide of any one of claims 15-18 or 20, wherein R3is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2.

22. The synthetic peptide of any one of claims 15-17, wherein R2is:

23. The synthetic peptide of any one of claims 15-18, 20, or 22, wherein R3is -CH2R3awherein R3ais optionally substituted phenyl or optionally substituted 5-10 membered heteroaryl, wherein R3is optionally substituted with -(CH2)o-3NH2 or -(CH2)o- 3N(H)C(=NH)NH2.

24. The synthetic peptide of claim 23, wherein R3is optionally substituted Ci-Ce alkyl, substituted with -NH2 or -N(H)C(=NH)NH2.

25. The synthetic peptide of claim 23, wherein R3ais optionally substituted indole.

26. The synthetic peptide of any one of claims 15-18, 20, or 22, wherein R3is selected from the group consisting of:

27. The synthetic peptide of any one of claims 15-26, wherein R4is selected from the group consisting of:

28. The synthetic peptide of any one of claims 15-27, whereinR5is Ci-Ce alkyl substituted with -NH2; and R7is Ci-Ce alkyl substituted with -NH2.

29. The synthetic peptide of any one of claims 15-27, wherein R5and R7are taken together to form a staple comprising 1-10 carbon atoms.

30. The synthetic peptide of any one of claims 15-29, wherein each of R6and R9is independently selected from the group consisting of:

31. The synthetic peptide of any one of claims 15-30, wherein R6is Ci-Ce alkyl substituted with -N(H)C(=NH)NH2.

32. The synthetic peptide of claim 31, wherein R6is33. The synthetic peptide of any of claims 15-32, wherein R8is a warhead, and wherein a warhead is L-R8a, whereinL 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 theoptionally substituted C1-C10 alkyl and optionally substituted C2-C10 alkenyl chain is each independently replaced with -C(O)NH-, -O- or -S-; andR8ais optionally substituted phenyl substituted with -N(H)C(O)-R8bor -N(H)S(O)2- R8b, wherein R8bis optionally substituted C2-C6 alkenyl.

34. The synthetic peptide of any one of claims 15-33, wherein R9is Ci-Ce alkyl.

35. The synthetic peptide of any one of claims 15-33, wherein R9is36. The synthetic peptide of any one of claims 33-35, wherein L is:

37. The synthetic peptide of any one of claims 33-36, wherein R8ais selected from the group consisting of:

38. The synthetic peptide of any one of claims 15-37, whereinX isR10is selected from the group consisting of:

39. The synthetic peptide of claim 38, wherein R10is:

40. The synthetic peptide of any one of claims 15-37, whereinX isand R10is selected from the group consisting of:and , wherein Z is Pro, Thr, Leu, Gly,Lys, or Phe.

41. The synthetic peptide of any one of claims 15-37, wherein:

42. The synthetic peptide of any one of claims 15-37, wherein X is a bond and R10is:

43. The synthetic peptide of claim 15, wherein the synthetic peptide is formula II or a pharmaceutically acceptable salt thereof:

44. The synthetic peptide of claim 15, wherein the synthetic peptide is formula III or a pharmaceutically acceptable salt thereof:

45. The synthetic peptide of claim 15, wherein the synthetic peptide is formula IV or a pharmaceutically acceptable salt thereof:

46. A synthetic peptide comprising the amino acid sequence ofPKHTRISELKAEAVKKDRRKKLTQS, PKHTRISELKAEAVKKDRRKKLTQ, PKHTRISELKAEAVKKDRRKKLT, or a pharmaceutically acceptable salt thereof, wherein the C- terminus of the peptide is modified with a zinc-binding chemical moiety, wherein 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.

47. The synthetic peptide of claim 46, wherein 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.

48. The synthetic peptide of claim 46, wherein the zinc-binding chemical moiety is hydroxamate,, or a thiol group.

49. The synthetic peptide of any one of claims 46-48, wherein the N-terminus of the peptide is modified with a lipidated tag, optionally an albumin-binding tag.

50. The synthetic peptide of any one of claims 46-48, wherein the Pro in position 1 or the Lys in position 15 of the peptide is modified with an albumin-binding tag.

51. The synthetic peptide of any one of the preceding claims, wherein the synthetic peptide shows no or a negligible amount of inhibition of pregnancy-associated plasma protein- A2 and metalloproteinases other than PAPP -A.

52. The synthetic peptide of any one of the preceding claims, wherein the synthetic peptide shows an increased half-life relative to comparators.

53. The synthetic peptide of any one of the preceding claims, wherein 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.

54. The synthetic peptide of any one of the preceding claims, wherein the synthetic peptide has a half-life of up to about 20 hours in human serum.

55. The synthetic peptide of any one of the preceding claims, wherein the synthetic peptide inhibits PAPP-A with an IC50 of 500 nM or less.

56. The synthetic peptide of any one of the preceding claims, wherein the synthetic peptide inhibits PAPP-A with an IC50 of 100 nM or less.

57. A pharmaceutical comprising the synthetic peptide of any one of the preceding claimsand a pharmaceutically acceptable excipient.

58. 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 the synthetic peptide of any one of claims 1-56.

59. The method of claim 58, wherein the disease or condition is an age-related disease.

60. The method of claim 59, wherein the disease or condition is atherosclerosis.

61. The method of any one of claims 58-60, comprising administering the synthetic peptide intravenously, subcutaneously or intraperitoneally.

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