Reversibly reactive affinity selection mass spectrometry and uses thereof

Abstract

This disclosure relates to reversible affinity selection methods useful for the isolation and enrichment of covalent peptide inhibitors directly from large synthetic peptide libraries. Also disclosed herein are synthetic peptides for targeting HPV16 E6 and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein.

Description

REVERSIBLY REACTIVE AFFINITY SELECTION MASS SPECTROMETRY AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 601,677, filed on November 21, 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] Peptides have gained traction as alternatives to small molecule drugs due to larger interaction interfaces towards undruggable proteins or protein-protein interactions (PPIs) (Muttenthaler et al., Nat Rev Drug Discov, 2021). Similar to protein drugs, peptides can mimic native PPI interfaces, achieving high binding affinity, target specificity, and biological potency while maintaining adequate cell penetration. The combination of 21 canonical amino acid residues and various non-canonical amino acid residues allows near-infinite possible peptide sequences that are synthetically accessible by solid-phase peptide synthesis. Furthermore, peptide sidechains are also structurally tunable with new chemical moieties to improve peptide stability and cellular permeability. Combining the advantages of small molecules and proteins, peptide drugs are expected to be adopted more frequently in the near future (Craik et al., Chem Biol Drug Des, 2013).

[0004] Covalent peptide inhibitors have become increasingly attractive since covalent crosslinking enhances the potency of peptides and circumvents various pharmacokinetic limitations, facilitating translation in drug discovery. To develop peptide drugs for a protein of interest, parent peptide leads can be generated through rational design based on PPIs that involve the target (Fletcher et al, JR Soc Interface, 2006; Wang et al., Front Chem, 2021) or de novo library selections including phage display and high-throughput screening(Smith et al., Chem Rev, 1997; McGuire et al., Methods Mol Biol, 2009; Blondelle et al., Curr Pharm Des, 2010). Parent peptides discovered by these methods typically have a low binding affinity within the micro- to milli-molar range. Affinity maturation is crucial to improve the binding affinity of the parent peptides (Yu et al., Methods Enzymol, 1996). Affinity selectionfrom synthetic peptide libraries has emerged as a promising technique to improve the affinity of identified peptide ligands (Zuckermann et al., Proc Natl Acd Sci USA, 1992; Annis et al., J Am Chem Soc, 2004). There remains a need for affinity selection and peptide enrichment method from synthetic “focused” peptide libraries, particularly those containing noncanonical amino acid residues.SUMMARY

[0005] The present disclosure relates to methods for isolating and enriching covalent reactive peptides from synthetic libraries. These methods are label-free affinity selection methods that allows for the isolation and enrichment of covalent reactive peptides that have high affinity (e.g., KD ~ 1-100 nM) and / or moderate affinity (e.g., KD ~ 0.1-10 pM) to their protein targets. Furthermore, the affinity selection methods disclosed herein are useful for large synthetic non-canonical peptide libraries (e.g., > 106member diversity).

[0006] Provided herein is a method for enriching one or more peptide sequences, the method comprising:(a) incubating one or more target proteins with a peptide library comprising a covalent peptide in solution under suitable conditions to form a reaction mixture comprising one or more peptide-target protein conjugates, wherein the conjugate peptide comprises a residue conjugated to a warhead, wherein the warhead is a cysteine-reactive covalent warhead, optionally disulfide cysteamine, and wherein the target protein comprises a cysteine residue that is targeted by the warhead, wherein the covalent peptide crosslinks with the cysteine residue forming a disulfide bond with the target protein sequence, thereby forming the peptide-target protein conjugates;(b) fractionating the reaction mixture using high-pressure size exclusion chromatography (HPSEC) into a plurality of fractions;(c) isolating from the plurality of fractions, one or more fractions comprising the peptide-target protein conjugates;(d) reducing the disulfide bond in each of the peptide-target protein conjugates to form one or more isolated target peptides.

[0007] In some embodiments, the cysteine-reactive covalent warhead is dehydroalanine (Dha). In some embodiments, the residue conjugated to the warhead is a cysteine. Variouscysteine conjugated warheads are known to those of ordinary skill in the art. In some embodiments, the warhead comprises the following structure:

[0008] In some embodiments, the wavy bond indicates the attachment point to the residue conjugated to the warhead.

[0009] In some embodiments, the method further comprises (e) sequencing the one or more isolated target peptides, optionally using liquid chromatography-tandem mass spectrometry (LC-MS / MS), wherein each peptide sequence is the sequence of a modulator. In some embodiments, the method further comprises (f) resynthesizing the one or more isolated target peptides to enrich the one or more target peptides in solution.

[0010] In some embodiments, the peptide library has diversity up to 106. In some embodiments, each distinct member of the peptide library has concentration of 0.1 nM or lower. In some embodiments, the one or more isolated target peptide have a binding affinity (KD) of 0.1-10 pM. In some embodiments, the one or more isolated target peptide have a binding affinity (KD) less than 0.1 pM. In some embodiments, the one or more isolated target peptide have a binding affinity (KD) of 1-100 nM.

[0011] In some embodiments, the method further comprises (g) validating binding of the one or more isolated target peptides to the target protein using an assay.

[0012] In some embodiments, wherein the peptide-target protein conjugates are reduced using a dithiothreitol (DTT) treatment. In some embodiments, the peptide-target protein conjugates are reduced using a tris(2-carboxyethyl)phosphine (TCEP) treatment.

[0013] In some embodiments, the peptide library comprises a plurality of peptides comprising non canonical amino acid residues. In some embodiments, wherein the peptide library comprises a peptide having the amino acid sequence of X1X2X3X4X5X6X7X8CEER, wherein Xi comprises a Glu analog, X2 comprises a Leu analog, X3 comprises a Thr analog, X4 comprises a Leu analog, X5 comprises a Gin analog, Xe comprises a Glu analog, X7 comprises a Leu analog, and Xs comprises a Leu analog.. In some embodiments, Xi is independently selected from the group consisting of glutamic acid (Glu, E), Aad, and Cya. In some embodiments, X2 is independently selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf. In some embodiments, X3 is independently selected from the group consisting of threonine (Thr, T), Cpg, Cbg, Ceg, Hyp, and asparagine (Asn, N). In some embodiments, X4 is independently selected from the group consisting ofleucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf. In some embodiments, Xs is independently selected from the group consisting of glutamine (Gin, Q), Gly, DAla, and Aib. In some embodiments, Xe is independently selected from the group consisting of glutamic acid (Glu, E), Aad, and Cya. In some embodiments, X7 is independently selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf. In some embodiments, Xs is independently selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

[0014] In some embodiments, the peptide library comprises the amino acid sequence of ELTLQELLCEER. In some embodiments, the C is the residue conjugated to the warhead. In some embodiments, the warhead comprises the following structure:

[0015] In some embodiments, the one or more target proteins comprises HPV 16E6 or MBP- 16E6 4C4S.

[0016] In some embodiments, wherein the peptide library comprises a peptide comprising the structure of:wherein Yi comprises a Phe analog, Y2 comprises Pip analog, Y3 comprises a Trp analog, and Y4 comprises an Arg analog. In some embodiments, Yi is independently selected from the group consisting of: Phe, Pya, Cnf, Eaf, F2f, Dmf, Amf, Nof, Phg, Hof, Qua, Naf, Phf, Mpf, Trp, and Cl-Trp. In some embodiments, Y2 is independently selected from the group consisting of: Pip, Ach, Tic, Chg, Ceg, Cpg, Ala, Amb, Hyp, 4Fp, Nip, Aib, Gly, Thz, and Nib. In some embodiments, Y3 is independently selected from the group consisting of: Trp, Cl-Trp, Qua, Phf, Mpf, Naf, Nva, Nle, Aoc, Cba, Cpea, and Cha. In some embodiments, YHs independently selected from the group consisting of: Arg, Hor, Hoq, Dmr, Dab, Dap, Orn, Lys, Tml, Cit, Het, Amf, Gnf, Eaf, and His.

[0017] In some embodiments, the target protein is Peptidyl-prolyl cis-trans isomerase NIMA- interacting 1 protein (Pinl).

[0018] Also provided herein is a synthetic peptide comprising the amino acid sequence of X1X2X3X4X5X6X7X8CEER, wherein Xi comprises a Glu analog, X2 comprises a Leu analog,Xs comprises a Thr analog, X4 comprises a Leu analog, X5 comprises a Gin analog, Xe comprises a Glu analog, X7 comprises a Leu analog, and Xs comprises a Leu analog. In some embodiments, Xi is independently selected from the group consisting of glutamic acid (Glu, E), Aad, and Cya. In some embodiments, X2 is independently selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf. In some embodiments, X3 is independently selected from the group consisting of threonine (Thr, T), Cpg, Cbg, Ceg, Hyp, and asparagine (Asn, N). In some embodiments, X4 is independently selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf. In some embodiments, X5 is independently selected from the group consisting of glutamine (Gin, Q), Gly, DAla, and Aib. In some embodiments, Xe is independently selected from the group consisting of glutamic acid (Glu, E), Aad, and Cya. In some embodiments, X7 is independently selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf. In some embodiments, Xs is independently selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

[0019] In some embodiments, the synthetic peptide comprises the amino acid sequence of ELTLQELLCEER. In some embodiments, the synthetic peptide comprises the amino acid sequence of Aad-Naf-Ceg-Leu-Aib-Glu-Cba-Leu-CEER. For example:

[0020] In some embodiments, the C is the residue conjugated to reversible cysteine-targeted warhead. In some embodiments, the reversible cysteine-targeted warhead has the following structure:

[0021] In some embodiments, the C is replaced with dehydroalanine (Dha). In some embodiments, wherein the C is replaced with an irreversible cysteine-reactive covalent warhead. In some embodiments, the C is conjugated to an irreversible cysteine-reactive covalent warhead.

[0022] The present disclosure also provides a synthetic peptide comprising the structure of:

[0023] wherein R is absent or 1-40 amino acid residues residues, wherein Yi comprises a Phe analog, Y2 comprises Pip analog, Y3 comprises a Trp analog, and Y4 comprises an Arg analog. In some embodiments, Yi is independently selected from the group consisting of: Phe, Pya, Cnf, Eaf, F2f, Dmf, Amf, Nof, Phg, Hof, Qua, Naf, Phf, Mpf, Trp, and Cl-Trp. In some embodiments, Y2 is independently selected from the group consisting of: Pip, Ach, Tic, Chg, Ceg, Cpg, Ala, Amb, Hyp, 4Fp, Nip, Aib, Gly, Thz, and Nib. In some embodiments, Y3 is independently selected from the group consisting of: Trp, Cl-Trp, Qua, Phf, Mpf, Naf, Nva, Nle, Aoc, Cba, Cpea, and Cha. In some embodiments, YHs independently selected from the group consisting of: Arg, Hor, Hoq, Dmr, Dab, Dap, Om, Lys, Tml, Cit, Het, Amf, Gnf, Eaf, and His. In some embodiments, Yi is Dmf, Y2 is Pip, Y3 is Trp, and Y4 is Het.

[0024] The present disclosure also provides a synthetic peptide comprising an amino acid sequence having the following structure of:

[0025] The present disclosure also provides a composition comprising any one of the synthetic peptides herein. The present disclosure also provides a pharmaceutical composition comprising any one of the synthetic peptides herein and a pharmaceutically acceptable salt or carrier.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] A better 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:

[0027] FIGS. 1A-1E show a conceptual overview of the ReAct-ASMS platform to identify covalent peptide inhibitors. (FIG. 1 A) Peptides from a synthetic library are first covalently crosslinked to the cysteine of the target protein. During HPSEC, the fraction corresponding to the target protein crosslinked with the reactive peptides can be separated from the fraction containing non-crosslinked library members. (FIG. IB) After separation on column, reduction of disulfide bonds of the protein fraction allows downstream sequencing of the eluted crosslinked peptides by an Orbitrap nLC-MS / MS protocol. (FIG. 1C) Identified hit peptides were selected and resynthesized by automated fast-flow synthesis. (FIG. ID) Resynthesized peptides were validated by crosslinking and binding studies. (FIG. IE) Comparison of ReACT-ASMS platform with traditional HPSEC-ASMS as well as other common methods for discovery of peptide binders with non-covalent enrichment.

[0028] FIGS. 2A-2E show the development of ReAct-ASMS to identify covalent peptide inhibitors. (A) X-ray crystal structure of the ternary protein complex formed by E6, MBP fused to E6AP peptide, and p53. PDB: 4XR8. (B) Interaction interface of E6 and the binding motif of E6AP. The E6AP peptide binds the E6 hydrophobic groove and drives peptide recognition. Cys58 of E6 is highlighted in red and can be targeted by electrophiles. (C) Scheme of crosslinking studies with covalent peptide ELT-G9Dha equipped with a Dha warhead against MBP-16E6. ELT-G9Dha and MBP-16E6 were both at 2 pM. (D) Deconvoluted mass spectra of crosslinking reactions against MBP-16E6 with various disulfide peptides showing different reactivities. ELT-G9-|JME showed the highest crosslink yield of 86% after 6 hours. Cysteamine was chosen as the covalent warhead for further studies. Disulfide peptides and MBP-16E6 were both at 2 pM. (E) Deconvoluted mass spectra of dissociating peptide-protein conjugates. DTT treatment achieved a 95% yield and therefore was used as dissociation reagent to separate MBP-16E6 and crosslinked peptides. In panels D and E, gray arrows indicate the deconvoluted mass of MBP-16E6 in Daltons (62337.1) while black arrows indicate the deconvoluted mass of ELT-G9C-MBP-16E6 conjugate in Daltons (63812.7). All reactions were performed at 37 °C in l x PBS.

[0029] FIGS. 3A-3B show ReAct-ASMS identified peptide E-10 from Library 1. (A) Design of Library 1 with 1.42 million members. Abbreviations: Aad: aminoadipic acid; Cya: cysteic acid; Nva: 2-aminopentanoic acid; Aoc: 2-aminooctanoic acid; Cpa: cyclopropylalanine; Cba: cyclobutylalanine; Cha: cyclohexylalanine; Phg: phenylglycine; Hof: homophenylalanine; Naf: napthylalanine; Cpg: cyclopropylglycine; Cbg: cyclobutylglycine; Ceg: cyclopentylglycine; Hyp: hydroxyproline; Aib: aminoisobutyric acid. (B) Chemical structures of ELT-G9Dha, twelve resynthesized covalent peptide inhibitors for MBP-16E6,and E-10-3L3A. Below the chemical structures: peptide sequences were provided as abbreviations used in panel A; the average local confidence (ALC, an indicator of sequencing confidence) scores were written as percentage in brackets; apparent Kis were determined by competitive FP and BLI assays.

[0030] FIGS. 4A-4H show that Peptide E-10 selectively crosslinks to Cys58 of MBP-16E6.(A) Deconvoluted mass spectra of crosslinking studies against MBP-16E6 with ELT-G9Dha and twelve resynthesized hit peptides (1 pM). Mono-crosslinked E-10-MBP-16E6 conjugate was the only species observed in MS with near-quantitative yield after 2 hours.Representative plots of ELT-G9Dha, E-10 and E-10-3L3A determined in competitive BLI(B) and competitive FP (C) assays. (D) Kinetic deconvoluted mass spectra of crosslinking studies against MBP-16E6 (1 pM) with peptide E-10 (10 pM). (E) Deconvoluted mass spectra of crosslinking reaction against MBP-16E6 (1 pM) with peptide E-10-3L3A (10 pM). No appreciable amount of crosslinking product was observed after 24 hours. In panels D and E, gray arrows indicate the deconvoluted mass of MBP-16E6 in Daltons (62337.1) while colored arrows indicate the deconvoluted mass of the E-10-MBP-16E6 conjugate in Daltons (63869.1). (F) Deconvoluted mass spectra of crosslinking reaction against C58S MBP-16E6 (1 pM) with peptide E-10 (10 pM). No appreciable amount of crosslinking product was observed after 24 hours. Gray arrows indicate deconvoluted mass of C58S MBP-16E6 in Daltons (62321.0). (G) Apparent kinetic constant Tapp was calculated from a kinetic crosslinking study, where 25 nM MBP-16E6 was incubated with different concentration of E-10. (H) A series value of Tfapp was plotted against the corresponding E-10 concentration to estimate Ain act and Ki, resulting in Ain act of 0.018 s-1, Ki value of 792 nM and Ain act / n ratio of 22.7 mM-1 s-1. All reactions were performed at 37 °C in l x PBS.

[0031] FIGS. 5A-5E show a design of a peptide library against Pinl for ReAct-ASMS. (A) Covalent peptide inhibitor BJP-07-017-3 selectively crosslinks Cysl 13 (*) of Pinl45. PDB: 6034. (B) Chemical structures of BJP-07-017-3, P-BJP, and P-BJP-DS. (C) Scheme of crosslinking studies with covalent peptides equipped with a-chloroacetamide or disulfide warhead against Pinl. (D) Deconvoluted mass spectra of crosslinking studies against Pinl. P- BJP mono-crosslinked with Pinl after 1 hour with >99% yield while P-BJP-DS has a crosslinking yield of 48% after 12 hours incubation. Grey peak has the deconvoluted mass of Pinl in Daltons (18110.8). Shown are the deconvoluted mass of P-BJP -Pinl conjugate in Daltons (18768.6). Shown are the deconvoluted mass of P-BJP-DS-Pinl conjugate in Daltons (18814.8). Peptides and Pinl were both at 2.5 pM. All reactions were performed at RT in l x PBS. (E) Design of Library 2 with 43,200 members. Abbreviations: Pya:pyridinylalanine; Cnf: 4-nitrile phenylalanine; Eaf: 4-methylamine phenylalanine; F2f: 3,4- difluorophenylalanine; Dmf: dimethoxyphenylalanine; Amf: 4-aminophenylalanine; Nof: 4- nitrophenylalanine; Phg: phenylglycine; Hof: homophenylalanine; Qua: Quinolyl Alanine; Naf: naphthylalanine; Phf: 4-phenylphenylalanine; Mpf: 2-methoxy-4’ -methyl- 1,1’- biphenylalanine; Cl-Trp: 2-chlorotryptophan; Pip: homoproline; Ach: 2- aminocyclohexanecarboxylic acid; Tic: tetrahydroisoquinoline; Chg: cyclohexylglycine; Ceg: cyclopentylglycine; Cpg: cyclopropylglycine; Amb: 4-aminobenzoic acid; Hyp: hydroxyproline; 4FP: 4-fluoroproline; Nip: imidazolidine-2-carboxylic acid; Aib: aminoisobutyric acid; Thz: thiazolidine; Nib: 3-azetidinecarboxylic acid; Nva: 2- aminopentanoic acid; Nle: 2-aminohexanoic acid; Aoc: 2-aminooctanoic acid; Cba: cyclobutylalanine; Cpea: cyclopentylalanine; Cha: cyclohexylalanine; Hor: homoarginine; Hoq: homoglutamine; Dmr: symmetric dimethylarginine; Dab: diaminobutyric acid; Dap: diaminopropionic acid; Orn: ornithine; Tml: trimethyllysine; Cit: citrulline; Het: homocitrulline; Amf: 4-aminophenylalanine; Gnf: 4-guanidinylphenylalanine.

[0032] FIGS. 6A-6C show peptide P-12 was identified by ReAct-ASMS as the lead covalent inhibitor against Pinl. (A) Chemical structures of Pinl BJP, seventeen resynthesized covalent hit peptides for Pinl, and P-NC. Below the chemical structures: peptide sequences were provided as abbreviations used in FIGS. 4A-4H; the ALC scores were written as percentage in brackets; apparent Kis were determined by the competitive FP assay. (B) Scheme of crosslinking studies with covalent peptides equipped with a- chloroacetamide warhead against Pinl. Peptides and Pinl were both at 2.5 pM. All reactions were performed at RT for 1 hour in 1 * PBS. (C) Deconvoluted mass spectra of crosslinking studies against Pinl with Pinl_BJP and in a mixture with seventeen resynthesized hit peptides. Mono-crosslinked P-12-Pinl conjugate was the only species observed in MS, suggesting it has the highest affinity against Pinl among all eighteen peptides. P-NC displayed no appreciable amount of crosslinking product after 12 hours incubated with Pinl. Grey and red peaks have deconvoluted mass of Pinl in Daltons (18110.8). Shown are the deconvoluted mass of P-BJP-Pinl conjugate in Daltons (18768.6). Shown are the deconvoluted mass of P-12-Pinl conjugate in Daltons (18842.7).

[0033] FIGS. 7-22 show the total ion chromatograms and mass spectra for major compound plots for peptides targeting E6.

[0034] FIGS. 23-59 show the total ion chromatograms and mass spectra for major compound plots for peptides targeting Pinl .

[0035] FIGS. 60A and 60B show rational design of E6AP-mimicking peptides. (FIG 60A) Left: X-ray crystal structure of the ternary protein complex formed by 16E6, MBP fused to the E6AP LXXLL peptide, and p53. PDB: 4XR8. Right: Interaction interface of 16E6 and the LXXLL motif of E6AP. The E6AP LXXLL peptide binds the 16E6 hydrophobic groove and drives peptide recognition. (FIG. 60B) Shows N- and C-terminal modified E6AP peptide mimics. The binding dissociation constant KD was measured by bio-layer interferometry (BLI) competition assay, except for *KD which was measured by direct BLI.

[0036] FIGS. 61A-61C depicts the determination of peptide 1 and 16E6 binding constant by direct binding assay in BLI and competition BLI binding assay. (FIG. 61A) Structure of peptide 1 and peptide 1 -biotin (FIG. 61B) BLI measurement of 1 -biotin and 16E6, parameters are estimated to be: kon= 40200 M’1, kOff = 0.0851 s'1and KD = 2.1± 0.1 pM (N=3). Indicated MBP-16E6 protein concentration was used in each channel on the right. (kon= on rate constant, koff = off rate constant, KD= kinetic apparent dissociation constant). (FIG. 61C) Peptide 1 binding was assessed by competition binding assay. Various concentration of peptide 1 was mixed with 30 nM MBP-16E6 protein. Peptide 1 -biotin was immobilized onto the streptavidin sensor tips to compete for MBP-16E6 protein with various concentrations of unlabeled peptide 1 in the solution (33333 nM, 8333 nM, 2083 nM, 520 nM, 130 nM, 32 nM, 2.0 nM or 0.1 nM). The dissociation binding constant was estimated to be KD = 2.3±0.5 pM. Error is the curve fitting standard error of the mean (SEM) reported by the Prism 8 software (N=3).

[0037] FIG. 62 shows peptides used in alanine scanning of the N-terminal modified E6AP peptide. 17 single alanine mutants of E6AP peptides were synthesized. Binding affinity was measured by competition BLI binding assay (N = 3). (*NB: no binding. *bAla= beta- Alanine)

[0038] FIG. 63 shows the D-alanine scanning of a IPESSELTLQELLGEER peptide. KD was measured by competition BLI binding assay. Biotin-peg4-IPESSELTLQELLGEER was immobilized to the SA tip. Various concentrations of analyte in the solution compete with the immobilized peptide for 40 nM MBP-16E6. Representative competition curves are shown.

[0039] FIG. 64 shows the E6 Library I design with a library size of 1.42 M.

[0040] FIG. 65 shows a sequence table of resynthesized 16E6 binding peptides. Apparent Ki is determined by BLI. Peptides were in competition with immobilized 1 -Biotin following a 30 min incubation with 16E6. incubation with 16E6. ICso was determined by FP. Peptides were in competition with 1-FITC following a 30 min incubation with 16E6.

[0041] FIG. 66 is a WEBLOGO plot showing that most of the monomers were spotted in each randomized position.

[0042] FIG. 67 shows a representative HPSEC separation chromatogram for chromatogram of identified peptides from Library 1 for E6 (ALO80).

[0043] FIG. 68 is a WEBLOGO plot showing that most of the monomers were spotted in each randomized position.

[0044] FIG. 69 is a plot showing a calibration curve of sensor response (nm) vs. MBP-16E6 concentration (nM).

[0045] FIG. 70 shows a characterization of a ELT peptide (ELTLQELLGEER). (A) Direct fluorescence polarization (FP) assay of FITC-ELT (FITC-ELTLQELLGEER). FP was recorded after incubating various concentration of MBP-16E6 with 100 nM FITC-ELT. FP was performed with a BioTek Hl plate reader. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50. Temperature: 27 °C. (B) Direct Biolayer Interferometry (BLI) measurement of Biotin-ELT (Biotin-PEG4-ELTLQELLGEER). Biotin-ELT was at first immobilized onto the streptavidin tips. BLI was recorded after incubating various concentrations of MBP-16E6.

[0046] FIG. 71 shows apparent Ki of resynthesized Dha peptides for MBP-16E6 determined by competitive FP assay. Various concentrations of analyte peptide were mixed with 100 nM FITC-ELT and 500 nM MBP-16E6 solution, incubated overnight. FP was measured using a BioTek Hl plate reader. n=2 biological replicates. Competition curves are shown. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50.

[0047] FIG. 72 shows apparent Ki of resynthesized Dha peptides for MBP-16E6 determined by competitive BLI assay. Biotin-ELT was immobilized onto the streptavidin tips. Various concentrations of analyte peptide in the solution compete with the immobilized Biotin-ELT for 40 nM of MBP-16E6. n=2 biological replicates. Competition curves are shown.

[0048] FIG. 73 shows crosslinking of L57A MBP-16E6 (1 pM) and peptide E6-10 (10 pM). No appreciable amount of crosslinking product was observed after 24 h. Gray arrows indicate deconvoluted mass of L57A MBP-16E6 in Daltons (62295.6).

[0049] FIG. 74 shows apparent Ki of resynthesized reactive peptides for Pinl determined by competitive FP assay. Various concentrations of analyte peptide were mixed with 5 nM of an N-terminal fluorescein-labeled peptide (Bth-d-phos.Thr-Pip-Nal) and 250 nM of Pinl solution, incubated for overnight. FP was measured using a BioTek Hl plate reader. n=2 biological replicates. Competition curves are shown. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50.

[0050] FIG. 75 shows a small molecule library of IPESS peptide. KD is measured by competition BLI binding assay. Biotin-PEG4-IPESSELTLQELLGEER was immobilized to the streptavidin (SA) tips. Various concentrations of analyte in the solution compete with the immobilized peptide for 40 nM of MBP-16E6 (n=2).

[0051] FIG. 76 shows a structures of 18 analogs. Apparent Ki is determined by competition BLI binding assay. Biotin-PEG4-IPESSELTLQELLGEER was immobilized to the streptavidin (SA) tips. Various concentrations of analyte compete with the immobilized peptide for 40 nM or lOOnM MBP-16E6 in the solution (n=2). Competition curves are shown.

[0052] FIG. 77 shows a crosslink mass-spec of analog of 18. Crosslink assay: MBP-16E6 (1 pM) and covalent peptide (4 pM), mono-crosslinked MBP-16E6-18 was observed at 1 h with a yield of 83%(18-Naf2car), 85% (18-car-IPQSA) and 76% (18-car-IPQSA-truncEER). IC50 is measured by competition FP assay. Various concentrations of analyte peptide were mixed with lOOnM FITC-IPESSELTLQELLGEER and 450nM MBP-E6 solution, incubated for 30 min. FP was performed with a BioTek Hl plate reader. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50. Temperature 27 °C.

[0053] FIGS. 78A-78B show a focused Library 1-18 was designed for Re Act- ASMS. (A) Design of Library 1-18 with 80 thousand members. Abbreviations: Aad: aminoadipic acid; Hoc: Homocysteic acid; Naf: napthylalanine; Chg: cyclohexylglycine; Ceg: cyclopentylglycine; Aib: aminoisobutyric acid. (B) Chemical structures of twelve resynthesized covalent peptide inhibitors for MBP-16E6.

[0054] FIG. 79 shows chromatograms from LC-MS and SEC-MALS and an image of a Gel results for E6PAP.

[0055] FIG. 80A shows an LC-MS chromatogram and an image of a Gel from MBP-16E6 4C4S.

[0056] FIG. 80B shows an LC-MS chromatogram and an image of a Gel from MBP-16E6 4C4S C58S.

[0057] FIG. 80C shows an LC-MS chromatogram and an image of a Gel from MBP-16E6 4C4S L57A.DETAILED DESCRIPTION

[0058] The present disclosure is based, in part, upon reversible affinity selection methods useful for the isolation and enrichment of covalent peptide inhibitors directly from large synthetic peptide libraries (e.g., synthetic non-canonical peptide libraries). Also disclosedherein are synthetic peptide sequences that target human papillomavirus (HPV)-encoded early protein 6 (E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pinl), and libraries thereof. The affinity selection platform introduced herein can efficiently assist with the maturation of covalent peptide inhibitors of a wide range of other undruggable protein targets.

[0059] Various components and aspects of the disclosure are described in further detail in the subsections below.I. Definitions

[0060] 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.

[0061] 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 invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0062] 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.

[0063] 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. Inother 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.

[0064] 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’.

[0065] 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’.

[0066] 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.

[0067] 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.

[0068] The term “and / or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.

[0069] 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.

[0070] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should beunderstood 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.

[0071] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0072] 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 specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0073] 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.

[0074] As used herein, “residue” refers to a position in a protein and its associated amino acid identity.Chemical Definitions

[0075] 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.

[0076] 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.

[0077] The term “peptide” refers to a short polymer of amino acid residues linked by peptide bonds. It has the same chemical (peptide) bonds as proteins but is commonly shorter in length. The shortest peptide is a “dipetide” consisting of two amino acid residues 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.

[0078] The term “amino acid” or “residue” refers to a molecule containing both an amino group and a carboxyl group. Amino acid residues include alpha-amino acid residues and betaamino acid residues, the structures of which are depicted below. In certain embodiments, an amino acid is an alpha amino acid.alpha-amino acid beta-amino acid

[0079] Suitable amino acid residues include, without limitation, natural alpha-amino acid residues such as D- and L-isomers of the 20 common naturally occurring alpha-amino acid residues 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 acid residues (as depicted in Table B below), natural beta-amino acid residues (e.g., beta-alanine), and unnnatural betaamino acid residues.

[0080] Amino acid residues used in the construction of peptides of the present invention may be prepared by organic synthesis, or obtained by other mean, including, but not limited to, degradation of or isolation from a natural source and automated peptide synthesis. Additional examples of amino acid residues and methods for synthesis are described in U.S. Patent No. 11,279,734, the relevant disclosures of which are herein incorporated by reference.

[0081] There are many known non-canonical amino acid residues any of which may be included in the peptides of the present invention. See for example, S. Hunt, The Non-Protein Amino acid residues: In Chemistry and Biochemistry of the Amino acid residues, edited by G. C. Barrett, Chapman and Hall, 1985. Some non-limiting examples of non-canonical aminoacid residues are 4-hydroxyproline, desmosine, gamma-aminobutyric acid, betacyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L- leucine, 1 -amino-cyclopropanecarboxylic acid, l-amino-2-phenyl-cyclopropanecarboxylic acid, 1 -amino- 1 -cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3 -aminocyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-l-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2- aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and para-substituted phenylalanines (e.g., substituted with — C(=O)C6Hs; — CF3; — CN; - halo; — NO2; CH3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with — C(=O)C6Hs; — CF3; — CN; -halo, — NO2; CH3), and statine. Additionally, the amino acid residues suitable for use in the present invention may be derivatized to include amino acid residues that are hydroxylated, phosphorylated, sulfonated, acylated, and glycosylated, to name a few.

[0082] As used herein, the term “warhead” refers to a moiety of an inhibitor which participates, either reversibly or irreversibly, in 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.

[0083] As used herein, the term “peptide library” refers to a collection of cloned free peptides, frequently consisting of different combinations of peptide. In some embodiments, a peptide library refers to a collection of peptides that are modified variations of a peptide sequence, e.g. a peptide fragment having one or more of its amino acid residues substituted with an analog, wherein the peptide library comprises multiple, or all, different combinations of that modified peptide.

[0084] 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 racemic mixtures, and mixtures enriched in one or more stereoisomer. Isomers can be isolated frommixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC), and the formation and crystallization of chiral salts. 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.

[0085] 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.

[0086] In the compositions provided herein, an enantiomerically pure compound can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising 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 the compound. In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier.

[0087] 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.

[0088] “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). An aryl group may be described as, e.g., a C6-C 10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Aryl groups include, but are not limited to, phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted C6-C14 aryl. In certain embodiments, the aryl group is substituted C6-C14 aryl.

[0089] “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.

[0090] 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. Reversibly Reactive Affinity Selection Mass Spectrometry

[0091] There are multiple selection techniques for discovery of peptide binders (FIG. IE). Molecular biology-based techniques such as phage display (Clackson et al., Trends Biotechnol, 1994) and mRNA display (Wilson et al., Proc Natl Acad Sci USA, 2001) are used to identify novel peptide binders due to their ability to apply on large libraries (108- 1012), however their choices of non-canonical amino acid residues is limited by the lack oftranslation efficiency. DNA-encoded library (DEL) can include non-canonical amino acid residues but typically limited to the small peptides, i.e., four varied positions on the peptide due to inefficiency in the chemistry used for their assembly (Zhao et al., Expert Opin Drug Discov, 2019). One-bead-one-compound (OBOC) method is another technique to cover non- canonical amino acid residues, but its screening can be a challenge and its library size is restricted (Lam et al., Nature, 1991; Lam et al., Chem Rev 1997).

[0092] The library diversity for non-covalent HPSEC-ASMS is usually below a million due to the following reasons (FIG. IE). The concentration of peptide library is below 100 pM to ensure solubility. When the peptide library diversity is over a million, the concentration of each member is below 0.1 nM. The recovery yield is defined as the amount of peptide detected in the eluted protein fraction after HPSEC divided by the total amount of peptide mixed with the target protein before HPSEC. Considering the recovery yield of non-covalent HPSEC-ASMS is usually below 1% and the solution volume used in the selection is usually below 1 mL, the ideal amount of peptide we can obtain for each member at a concentration of 0.1 nM is only 1 fmol. This low amount of recovered peptide renders it unreliable for de novo sequencing due to the 10 fmol detection limit of the Orbitrap nLC-MS / MS protocol (Touti et al., Nat Chem Biol, 2019). Since the library diversity is the crucial determinant of selection outcome, it is very difficult to generate hit peptides with a hundred-fold improved affinity compared to the starting peptide with such relatively low library diversity.

[0093] Affinity maturation is crucial to improve the binding affinity of parent peptides8. Affinity selection from synthetic peptide libraries has emerged as a promising technique to improve the affinity of identified peptide ligands (Zuckermann et al., Proc Natl Sci Acad USA, 1992; Annis et al., J Am Chem Soc, 2004). Previous methods for affinity-based isolation of peptides from peptide libraries included in-solution affinity selection mass spectrometry (ASMS) platform for affinity maturation of peptide binders from synthetic ‘focused’ peptide libraries containing non-canonical amino acid residues (Touti et al., Nat Chem Biol, 2019)..

[0094] Among all selection techniques, in-solution enrichment by high performance size exclusion chromatography (HPSEC)-ASMS is the only one that allows us to screen an intact protein target without using any labels, tags or beads against synthetic peptide libraries with full chemical control over the non-canonical amino acid residues (Touti et al., Nat Chem Biol, 2019). It can be referred to as traditional non-covalent HPSEC-ASMS. For well-studied target proteins, these features can accurately identify sequences associated with high-affinitybinding. HPSEC-ASMS leverages high performance size exclusion chromatography to separate bound and unbound peptides without attaching affinity or decoding tags on the target proteins. However, the rapid elution on SEC necessitates peptides with mid-to-low nanomolar binding affinity and slow dissociation rates to have sufficient residence time on the target protein, resulting in limited peptide library diversity (< a million members) and omission of peptides with moderate binding affinity (apparent dissociation constant, KD ~ 0.1-10 pM). HPSEC-ASMS only identifies peptide sequences with affinity less than 100 nM since low-affinity binders dissociate from the target protein on column11(FIG. IE). For newly established target proteins, non-covalent HPSEC-ASMS may generate very few hit peptides since the starting peptides for library design might have affinity near or over 10 pM.

[0095] Rapid enzymatic degradation and renal clearance of peptide drugs result in short circulation times, presenting a significant challenge for their development (Diao et al., Clin Pharmacokinet, 2013). Covalent inhibition is an established strategy for peptide inhibitors to overcome these limitations. More importantly, peptides equipped with covalent warheads have enhanced inhibitory potency since covalently crosslinking to the target protein stabilizes their binding conformation and extends their residence time on the protein (Abdeldayem et al., Chem Soc Rev 2010). This “bind-and-react” approach is a proven strategy enabling the study of molecular mechanisms that cannot be addressed using early-stage, noncovalent molecules (Berdan et al., Bioorg Med Chem, 2021; Paulussen et al., J Pept Sci, 2017). There does not appear to be a label-free affinity selection strategy currently available to enrich covalent peptide inhibitors directly from large synthetic non-canonical peptide libraries. The inventors of the present disclosure have developed an affinity selection platform termed reversibly reactive affinity selection mass spectrometry (ReAct-ASMS). Peptides from a synthetic library are first covalently crosslinked to the cysteine of the target protein (FIG.1 A). Thereafter, HPSEC can be used to separate fractions from the peptide library, and the fraction corresponding to the target protein crosslinked with the reactive peptides can be separated from the fraction(s) containing non-crosslinked library members (FIG. 1 A). After separation on column, reduction of disulfide bonds of the protein fraction allows downstream sequencing of the eluted crosslinked peptides, e.g., by using an Orbi-trap nLC-MS / MS protocol (FIG. IB). The eluted crosslinked peptides were selected and resynthesized by automated fast-flow synthesis (FIG. 1C) (Mijalis et al., Nat Chem Biol, 2017; Hartrampf et al., Science, 2020), then validated by crosslinking studies by mass spectrometry (MS) to investigate if resynthesized peptides covalently crosslink to the target proteins and by bindingstudies using biophysical assays (FIG. ID). Since crosslinking offsets the dissociation step in this method, it effectively enriches the peptides on the target protein, allowing detection at reduced concentrations, with a preference for binders with higher on-rate. Therefore, the affinity selection method disclosed herein (also referred to as ReAct-ASMS) is amenable for selections of peptide libraries with more than a million members (FIG. IE). Also, since ReAct-ASMS enriches binders by covalent transformations, it can be used to identify peptides with moderate binding affinity ( D - 0.1-10 pM) because their rapid dissociation is abrogated (FIG. IE). In comparison, the previously described non-covalent HPSEC-ASMS platform is only applicable for identifying high affinity binders (AD - 1-100 nM) (Zuckermann et al., Proc Natl Acd Sci USA, 1992; Ye et al., Commun Chem, 2022).

[0096] In some embodiments, enriching one or more peptide sequences comprises incubating one or more target proteins with a peptide library comprising a covalent peptide in solution under suitable conditions to form a reaction mixture comprising one or more peptide-target protein conjugates, wherein the covalent peptide comprises a residue conjugated to a warhead, wherein the warhead can reversibly crosslink to a cysteine residue in the target protein (e.g., a disulfide cysteamine warhead), and wherein the target protein comprises a cysteine residue that is targeted by the warhead, wherein the covalent peptide crosslinks with the cysteine residue forming a disulfide bond with the target protein sequence, thereby forming the peptide-target protein conjugates. In some embodiments, enriching one or more peptide sequences comprises fractionating the reaction mixture using high performance size exclusion chromatography (HPSEC) into a plurality of fractions. In some embodiments, enriching one or more peptide sequences comprises isolating from the plurality of fractions, one or more fractions comprising the peptide-target protein conjugates. In some embodiments, enriching one or more peptide sequences comprises reducing the disulfide bond in each of the peptide-target protein conjugates to form one or more isolated target peptides. In some embodiments, enriching one or more peptide sequences comprises sequencing the one or more isolated target peptides, wherein each peptide sequence is the sequence of a modulator for the target protein. In some embodiments, the sequencing is conducted using liquid chromatography-tandem mass spectrometry (LC-MS / MS). In some embodiments, enriching one or more peptide sequences comprises resynthesizing the one or more isolated target peptides to enrich the one or more target peptides in solution.

[0097] In some embodiments, enriching one or more peptide sequences comprises:(a) incubating one or more target proteins with a peptide library comprising a covalent peptide in solution under suitable conditions to form a reaction mixture comprising one or more peptide-target protein conjugates, wherein the covalent peptide comprises a residue conjugated to a warhead, wherein the warhead can reversibly crosslink to a cysteine residue in the target protein (e.g., a disulfide cysteamine warhead), and wherein the target protein comprises a cysteine residue that is targeted by the warhead, wherein the covalent peptide crosslinks with the cysteine residue forming a disulfide bond with the target protein sequence, thereby forming the peptide-target protein conjugates;(b) fractionating the reaction mixture using high performance size exclusion chromatography (HPSEC) into a plurality of fractions;(c) isolating from the plurality of fractions, one or more fractions comprising the peptide- target protein conjugates; and(d) reducing the disulfide bond in each of the peptide-target protein conjugates to form one or more isolated target peptides.

[0098] In some embodiments, enriching one or more peptide sequences further comprises (e) sequencing the one or more isolated target peptides, wherein each peptide sequence is the sequence of a modulator for the target protein. In some embodiments, the sequencing is conducted using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0099] In some embodiments, enriching one or more peptide sequences comprises:(a) incubating one or more target proteins with a peptide library comprising a covalent peptide in solution under suitable conditions to form a reaction mixture comprising one or more peptide-target protein conjugates, wherein the covalent peptide comprises a residue conjugated to a warhead, wherein the warhead can reversibly crosslink to a cysteine residue in the target protein (e.g., a disulfide cysteamine warhead), and wherein the target protein comprises a cysteine residue that is targeted by the warhead, wherein the covalent peptide crosslinks with the cysteine residue forming a disulfide bond with the target protein sequence, thereby forming the peptide-target protein conjugates;(b) fractionating the reaction mixture using high performance size exclusion chromatography (HPSEC) into a plurality of fractions;(c) isolating from the plurality of fractions, one or more fractions comprising the peptide- target protein conjugates;(d) reducing the disulfide bond in each of the peptide-target protein conjugates to form one or more isolated target peptides; and(e) sequencing the one or more isolated target peptides, wherein each peptide sequence is the sequence of a modulator for the target protein. In some embodiments, the sequencing is conducted using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0100] In some embodiments, enriching one or more peptide sequences further comprises (f) resynthesizing the one or more isolated target peptides to enrich the one or more target peptides in solution.

[0101] In some embodiments, enriching one or more peptide sequences comprises:(a) incubating one or more target proteins with a peptide library comprising a covalent peptide in solution under suitable conditions to form a reaction mixture comprising one or more peptide-target protein conjugates, wherein the covalent peptide comprises a residue conjugated to a warhead, wherein the warhead can reversibly crosslink to a cysteine residue in the target protein (e.g., a disulfide cysteamine warhead), and wherein the target protein comprises a cysteine residue that is targeted by the warhead, wherein the covalent peptide crosslinks with the cysteine residue forming a disulfide bond with the target protein sequence, thereby forming the peptide-target protein conjugates;(b) fractionating the reaction mixture using high performance size exclusion chromatography (HPSEC) into a plurality of fractions;(c) isolating from the plurality of fractions, one or more fractions comprising the peptide- target protein conjugates;(d) reducing the disulfide bond in each of the peptide-target protein conjugates to form one or more isolated target peptides;(e) sequencing the one or more isolated target peptides, wherein each peptide sequence is the sequence of a modulator for the target protein. In some embodiments, the sequencing is conducted using liquid chromatography-tandem mass spectrometry (LC-MS / MS); and(f) resynthesizing the one or more isolated target peptides to enrich the one or more target peptides in solution.III. Warheads

[0102] The methods of the present disclosure contemplate the use of a warhead as a residue in the covalent peptide and / or conjugated to a residue in the covalent peptide. In some embodiments, the warhead reversibly crosslinks with a residue in the target protein. In some embodiments, the warhead is a reversible cysteine-targeted warhead. Nonlimited examples of a reversible cysteine-targeted warhead include cysteine (e.g. in ELT-G9C-C of FIG. 2D), cysteamine (e.g., in ELT-G9C-|JME of FIG. 2D) and N-acetyl cysteamine (e.g., ELT-G9C- NACPME of FIG. 2D).

[0103] In some embodiments, the warhead is at the N- or C-terminus of the peptide or peptidomimetic. In some embodiments, the warhead is on a side chain of an amino acid in the peptide or peptide. In some embodiments, the warhead is on a cysteine (Cys, C), lysine (Lys, K), tyrosine (Tyr, Y), histidine (His, H), serine (Ser, S), or threonine (Thr, T). In some embodiments, the warhead is on a cysteine (Cys, C). In some embodiments, the warhead is on a lysine (Lys, K). In some embodiments, the warhead is on a tyrosine (Tyr, Y). In some embodiments, the warhead is on a histidine (His, H). In some embodiments, the warhead is on a serine (Ser, S). In some embodiments, the warhead is on a threonine (Thr, T). In some embodiments, the warhead is on a cysteine (Cys, C), lysine (Lys, K), tyrosine (Tyr, Y), histidine (His, H), serine (Ser, S), or threonine (Thr, T) of the peptide or peptidomimetic. In some embodiments, the warhead is on a cysteine (Cys, C) of the peptide or peptidomimetic. In some embodiments, the warhead is on a lysine (Lys, K) of the peptide or peptidomimetic. In some embodiments, the warhead is on a tyrosine (Tyr, Y) of the peptide or peptidomimetic. In some embodiments, the warhead is on a histidine (His, H) of the peptide or peptidomimetic. In some embodiments, the warhead is on a serine (Ser, S) of the peptide or peptidomimetic. In some embodiments, the warhead is on a threonine (Thr, T) of the peptide or peptidomimetic. In some embodiments, the warhead is on a Cys in the covalent peptide. In some embodiments, the warhead is conjugated to the Cys via the Sulfur atom of the Cys.

[0104] In some embodiments, a synthetic peptide of the present disclosure has an irreversible covalent warhead. In some embodiments, a synthetic peptide of the present disclosure has an irreversible covalent warhead, such that it irreversibly crosslinks with a residue in the target protein. Such covalent warheads can be useful for validation studies (e.g., crosslinking studies by mass spectrometry (MS) to investigate if resynthesized peptides covalently crosslink to the target proteins and by binding studies using biophysical assays). Suchcovalent warheads can also be useful in a peptides synthesized for treatment. Nonlimiting examples of irreversible covalent warhead include dehydroalanine (Dha) and a- chloroacetamide.

[0105] In some embodiments, the warhead is selected from the group of phenyl acrylamide (Ph-acr), Dap-acrylamide (dap-acr), Dab-acrylamide (dab-acr), Dap-propiolamide (dap-ppa), and dehydroalanine (Dha).

[0106] Additional nonlimiting examples of warheads include phenyl fluorosulfate (FS), phenyl sulfonyl fluoride (SF), phenyl Carbamate (p-PhC), phenyl Carbamate (m-PhC), and disulfide (DS).

[0107] In some embodiments, the warhead is selected from the group of phenyl acrylamide (Ph-acr), Dap-acrylamide (dap-acr), Dab-acrylamide (dab-acr), Dap-propiolamide (dap-ppa), and dehydroalanine (Dha), phenyl fluorosulfate (FS), phenyl sulfonyl fluoride (SF), phenyl Carbamate (p-PhC), phenyl Carbamate (m-PhC), and disulfide (DS), and a-chloroacetamide.

[0108] In some embodiments, the warhead is dehydroalanine (Dha). In some embodiments, the warhead is a-chloroacetamide. In some embodiments, the warhead is phenylacrylamide (Ph-acr). In some embodiments, the warhead is Dap-acrylamide (dap-acr). In some embodiments, the warhead is Dab-acrylamide (dab-acr). In some embodiments, the warhead is Dap-propiolamide (dap-ppa). In some embodiments, the warhead is phenyl fluorosulfate (FS). In some embodiments, the warhead is phenyl sulfonyl fluoride (SF). In some embodiments, the warhead is phenyl Carbamate (p-PhC). In some embodiments, the warhead is phenyl Carbamate (m-PhC). In some embodiments, the warhead is disulfide (DS).IV. Human Papilloma Virus 16

[0109] The high-risk virus variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. Without wishing to be bound by a particular theory, this degradation is mediated by the HPV early protein 6 (16E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV 16E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53.

[0110] HPV-encoded early protein 6 (E6) and early protein 7 (E7) are primary transforming viral proteins enhancing cancer cell proliferation and contributing to cancer progression. Without wishing to be bound by a particular theory, HPV E7 primarily binds and inactivatesretinoblastoma protein (pRB) and related pocket proteins, pl 07 and pl 30, inducing their proteasome-dependent degradation and promoting cell cycle entry. Multiple host proteins interact with 16E6 via a leucine-rich LXXLL motif, including E6BP, IRF3, paxillin and tuberin; PDZ proteins such as MAGI-1; and other proteins such as p53, E6AP, MAML1, and p300 / CBP. Nevertheless, the interaction of 16E6 with the E3 ubiquitin ligase, E6AP, and p53 is thought to be a central transformative pathway of cell immortalization. E6 and E7 are active in different cell stages. E7 promotes cell entry to the division phase S, in turn, E6 prevents E7-induced apoptosis by degrading the apoptosis-inducing protein p53. HPV+ tumors mostly contain non-mutant p53, as a result, silencing E6 with siRNA can rescue p53 and initiate apoptosis in HPV+ cancer cell lines. Therefore, checkpoint networks are primed in HPV+ cells awaiting E6 disruption, providing support for its suitability as an oncology target.[oni] Without wishing to be bound by a particular theory, HPV16 E6 (16E6) hijacks E6AP to form a complex with p53 that promotes ubiquitination of p53, leading to its subsequent proteasome-mediated degradation, while neither E6 nor E6AP interact with p53 alone. p53 mediates stress response, cell proliferation, and apoptosis, and its downregulation or mutation is a hallmark of carcinogenesis directly affecting efficacy of cancer therapy. Targeting the ubiquitination catalytical domain of ubiquitin ligase E6AP (ELECT domain) represses p53 ubiquitination in vitro. However, E6AP is a regulator of the proteostasis signaling network and has wide distribution patterns in humans. Additionally, E6AP have been associated with Angelman syndrome and Prader-Will syndrome, diseases associated with developmental defects. As such, targeting E6AP could result in on-target toxicity although acute inactivation has not been assessed. Given the potential toxicity associated with targeting E6AP and the lack of 16E6 in healthy human cells, targeting the viral protein 16E6 can provide a wide therapeutic index in HPV+ cancers to limit on-target side-effect.

[0112] Efforts have been made in the past decades to target the 16E6ZE6AP protein-protein interactions (PPIs). Ribozymes and gene-silencing siRNA that reduce or remove the activity of the HPV E6 oncogene and its protein product have been shown to induce apoptosis in cancer cells. Polyhydroxy flavonoid display low micromolar E6 inhibition ICso values and cytotoxicity in HPV+ cancer cells but have not been successful in clinical trials, possibly due to unclear structure-activity relationships (SAR), poor stability, off-target binding, and low specificity. Biomolecules, including E6-binding antibodies and mini-proteins with dissociation constants (KD) of 10 nM - 60 nM, exhibit nanomolar affinity to E6. However,their inhibitory effect is unsurprisingly hindered by poor penetration through the cell membrane.

[0113] In some embodiments, a synthetic peptide that targets HPV 16E6 or MBP-16E6 4C4S comprises the amino acid sequence of:X1X2X3X4X5X6X7X8CEER, wherein the identity of each Xi, X2, X3, X4, X5, Xe, X7, and Xs are as provided for in Table 41 below.Table 41. Amino acid residues at positions Xi to Xs.

[0114] In some embodiments, Xi is selected from the group consisting of glutamic acid (Glu, E), Aad, Cya, and any analog or derivative thereof. In some embodiments, Xi is glutamic acid (Glu, E). In some embodiments, Xi is Aad. In some embodiments, Xi is Cya.

[0115] In some embodiments, X2 is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, Naf, and any analog or derivative thereof. In some embodiments, X2 is leucine (Leu, L). In some embodiments, X2 is Nva. In some embodiments, X2 is Aoc. In some embodiments, X2 is Cpa. In some embodiments, X2 is Cba. In some embodiments, X2 is Cha. In some embodiments, X2 is Phg. In some embodiments, X2 comprises Hof. In some embodiments, X2 is Naf.

[0116] In some embodiments, X3 is selected from the group consisting of threonine (Thr, T), Cpg, Cbg, Ceg, Hyp, asparagine (Asn, N), and any analog or derivative thereof. In some embodiments, X3 is threonine (Thr, T). In some embodiments, X3 is Cpg. In some embodiments, X3 is Cbg. In some embodiments, X3 is Ceg. In some embodiments, X3 is Hyp. In some embodiments, X3 is asparagine (Asn, N).

[0117] In some embodiments, X4 is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, Naf, and any analog or derivative thereof. In some embodiments, X4 is leucine (Leu, L). In some embodiments, X4 is Nva. In some embodiments, X4 is Aoc. In some embodiments, X4 is Cpa. In some embodiments, X4 is Cba. In some embodiments, X4 is Cha. In some embodiments, X4 is Phg. In some embodiments, X4 is Hof. In some embodiments, X4 is Naf.

[0118] In some embodiments, X5 is selected from the group consisting of glutamine (Gin, Q), Gly, DAla, Aib, and any analog or derivative thereof. In some embodiments, X5 is glutamine (Gin, Q). In some embodiments, X5 is glycine (Gly, G). In some embodiments, X5 is Dala. In some embodiments, X5 is Aib.

[0119] In some embodiments, Xe is selected from the group consisting of glutamic acid (Glu, E), Aad, Cya, and any analog or derivative thereof. In some embodiments, Xe is glutamic acid (Glu, E). In some embodiments, Xe is Aad. In some embodiments, Xe is Cya.

[0120] In some embodiments, X7 is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, Naf, and any analog or derivative thereof. In some embodiments, X7 is leucine (Leu, L). In some embodiments, X7 is Nva. In some embodiments, X7 is Aoc. In some embodiments, X7 is Cpa. In some embodiments, X7 is Cba. In some embodiments, X7 is Cha. In some embodiments, X7 is Phg. In some embodiments, X7 is Hof. In some embodiments, X7 is Naf.

[0121] In some embodiments, Xs is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, Naf, and any analog or derivative thereof. In some embodiments, Xs is leucine (Leu, L). In some embodiments, Xs is Nva. In some embodiments, Xs is Aoc. In some embodiments, Xs is Cpa. In some embodiments, Xs is Cba. In some embodiments, Xs is Cha. In some embodiments, Xs is Phg. In some embodiments, Xs is Hof. In some embodiments, Xs is and Naf.

[0122] In some embodiments, Xi is selected from the group consisting of glutamic acid (Glu, E), Aad, and Cya; X2 is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf; X3 is selected from the group consisting of threonine (Thr, T), Cpg, Cbg, Ceg, Hyp, and asparagine (Asn, N); X4 is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf; X5 is selected from the group consisting of glutamine (Gin, Q), Gly, DAla, and Aib; Xe is selected from the group consisting of glutamic acid (Glu, E), Aad, and Cya; X7 is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf; and Xs is selected from the group consisting of leucine (Leu, L), Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf. In someembodiments, Xi comprises a Glu analog, X2 comprises a Leu analog, X3 comprises a Thr analog, X4 comprises a Leu analog, X5 comprises a Gin analog, Xe comprises a Glu analog, X7 comprises a Leu analog, and Xs comprises a Leu analog. The present disclosure further contemplates peptide libraries comprising the combinations resulting from that synthetic peptide.V. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pinl)

[0123] Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pinl) is frequently overexpressed in many types of cancers. It recognizes phosphorylated serine or threonine (pSer / Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. Pinl inhibition was reported to reduce tumorigenic potential (Zhou et al., Nat Rev Cancer, 2016; Wulf et al., EMBO J, 2004).

[0124] In some embodiments, a synthetic peptide that targets a peptide comprises the structure of:wherein Yi comprises a Phe analog, Y2 comprises Pip analog, Y3 comprises a Trp analog, and Y4 comprises an Arg analog. In some embodiments, Yi is a phenylalanine (Phe, F) analog. In some embodiments, Y2 is a Pip analog. In some embodiments, Y3 is a tryptophan (Trp, W) analog. In some embodiments, Y4is an arginine (Arg, R) analog.

[0125] In some embodiments, provided herein are peptide libraries comprising variants of the synthetic peptide according to the above formula.VI. Peptides Residues and Noncanonical Amino acid residues

[0126] In some embodiments, the peptide comprises a modification, such as those provided herein. In some embodiments, the modification is a mutation, such as those provided herein. In some embodiments, the mutation is a substitution, a deletion, or an insertion. In some embodiments, the peptide comprises an amino acid residue substitution. In some embodiments, the substituted amino acid is a canonical amino acid. Canonical amino acidresidues for use in substitutions are listed in TABLE A. In some embodiments, the canonical substituted amino acid residues are an Ala, a Ser, a Gin, or an Arg.TABLE A. Canonical amino acid residues used in the peptides and peptidomimetics.

[0127] In some aspects of the disclosure, the synthetic peptide or peptidomimetic comprises one or more non-canonical amino acid residues. Non-canonical amino acid residues that can be used for substitution are shown in TABLE B.TABLE B. Non-canonical amino acid residues for use in the peptides and peptidomimetics.

[0128] In some embodiments the amino acid residues of the peptide are mixed canonical and non-canonical amino acid residues. In some embodiments, the peptide comprises canonical and non-canonical amino acid residues.

[0129] In some embodiments the library is 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, or 10 million peptides in size. In some embodiments the library comprises about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, or about 10 million peptides. In some embodiments the library comprises at least 1 million, at least 2 million, at least 3 million, at least 4 million, at least 5 million, at least 6 million, at least 7 million, at least 8 million, at least 9 million, or at least 10 million peptides.VII. Modifications

[0130] The central limitation in the development of peptide therapeutics is their short circulation time resulting from rapid enzymatic degradation and renal clearance. Methods to evade renal elimination by increasing the molecular weight have emerged, but extensive modifications can cause undesired steric hindrance during target binding. For small molecules, an alternative 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 acid residues, and backbone modifications.

[0131] In some embodiments, a therapeutic small molecule ligand equipped with an electrophilic warhead binds covalently to nucleophilic groups of the target protein in a proximity-driven reaction. Without wishing to be bound by a particular theory, irreversible covalent inhibition of an interaction can results in increased potency, selectivity, sustained pharmacodynamics, and could alleviate the effects of fast renal elimination. In some embodiments, therapeutic peptides comprising covalent binding mode of action have improved pharmacokinetics.

[0132] In some embodiments, the peptides, such as those provided herein, are chemically modified. In some embodiments, the peptides, such as those provided herein, comprise chemical modifications, such as those provided herein. In some embodiments, the peptide that is modified is selected from TABLES 3A-40. In some embodiments, any one peptide of TABLES 3 A-40 comprises at least one chemical modification, such as those provided herein.

[0133] Modifications may comprise chemical modifications for example such as warheads, protective groups, and pegylation. 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 acetylation, formylation, propanoylation, hexanoylation, or myristoylation. In some embodiments, the modification is an amidated C-terminus. 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 one or more amino acid with a methyl-amino acid. In some embodiments, the modification is a substitution of an a-amino acid with a P-amino acid residues.

[0134] In some embodiments, any one peptide of TABLES 3 A-40 comprises at least one chemical modification, such as those provided herein, for example such as warheads, protective groups, and pegylation. In some embodiments, any one peptide of TABLES 3 A-40comprises at least one chemical modification selected from a warhead, a protective group, pegylation, acetylation, formylation, propanoylation, hexanoylation, myristoylation, an amidated C-terminus, a substitution of one or more L-amino acid with a D-amino acid, a substitution of one or more amino acid with a methyl-amino acid, or a substitution of an a- amino acid with a P-amino acid residues.

[0135] In some embodiments, the N- or C- terminus of the peptide is modified with a chemical moiety.

[0136] In some embodiments, the chemical moiety is attached at the N-terminus and is selected from the group of Fluorescein-5-Isothiocyanate, 9-fluorene acetamido, 1 -fluorene acetamido, 1 -Indane acetamido, 9-fluorenone-2-carboxamido, 9-fluorenone-l-carboxamido, 9-fluorenone-4-carboxamido, anthraquinone-2-carboxamido, xanthene-9-carboxamido, 1- anthracene carboxamido, 2-anthracene carboxamido, 1 -adamantane carboxamido, Triphenyl acetamido, Diphenyl acetamido, 1 -naphthyl carboxamido, 1,6-dihydrophenyl carboxamido, Pentafluorophenyl carboxamido, 6-hydroxy-2-naphthyl carboxamido, 1 -pyrenebutyl carboxamido, 5 -Acenaphthene carboxamide, 7-Methoxycoumarin-4-acetamido, 4-phenyl- phenylalanine, and Cyclohexyl-alanine.

[0137] In some embodiments, any one peptide of TABLES 3A-40 comprises a chemical modification at the N- or C-terminus of the peptide of TABLES 3A-40. In some embodiments, any one peptide of TABLES 3A-40 comprises a chemical modification at the N- or C-terminus of the peptide of TABLES 3A-40, wherein the chemical modification is a chemical moiety selected from Fluorescein-5-Isothiocyanate, 9-fluorene acetamido, 1- fluorene acetamido, 1 -Indane acetamido, 9-fluorenone-2-carboxamido, 9-fluorenone-l- carboxamido, 9-fluorenone-4-carboxamido, anthraquinone-2-carboxamido, xanthene-9- carboxamido, 1 -anthracene carboxamido, 2-anthracene carboxamido, 1 -adamantane carboxamido, Triphenyl acetamido, Diphenyl acetamido, 1 -naphthyl carboxamido, 1,6- dihydrophenyl carboxamido, Pentafluorophenyl carboxamido, 6-hydroxy-2-naphthyl carboxamido, 1 -pyrenebutyl carboxamido, 5 -Acenaphthene carboxamide, 7- Methoxycoumarin-4-acetamido, 4-phenyl-phenylalanine, and Cyclohexyl-alanine.

[0138] In some embodiments, the chemical moiety is attached at the C-terminus and is selected from the group of Fluorescein-5-Isothiocyanate, fluorene-9-acetamido, fluorene- 1- acetamido, 1-Indanecarb oxami do, 9-fluorenone-2-carboxamido, 9-fluorenone-l- carboxamido, 9-fluorenone-4-carboxamido, anthraquinone-2-carboxamido, xanthene-9- carboxamido, 1 -anthracene carboxamido, 2-anthracene carboxamido, 1 -adamantane carboxamido, triphenylacetamido, diphenyl acetamido, 1 -naphthyl carboxamido, and Exo-norbornene-carboxamido. In some embodiments, the chemical moiety is selected from the group of 9-fluorenyl, 1-fluorenyl, 1-Indanyl, 9-oxofluoren-3-yl, 9-oxofluoren-4-yl, 9- oxofluoren-l-yl, 2-anthraquinonyl, 9-xanthyl, 1-anthracenyl, 2-anthracenyl, Adamantyl, Triphenylmethyl, Diphenylmethyl, 1- naphthyl, 2,6-dihydroxy-phenyl, Pentafluoro-phenyl, 6- Hydroxynaphthyl, 1 -pyrenebutyl, 5-acenaphthyl, Coumarin, Biphenyl, Cyclohexyl, Norborenyl, and Fluorescein.

[0139] In some embodiments, any one peptide of TABLES 3A-40 comprises a chemical modification at the C-terminus of the peptide of TABLES 3A-40, wherein the chemical modification is a chemical moiety selected from Fluorescein-5-Isothiocyanate, fluorenesacetamido, fluorene- 1 -acetamido, 1-Indanecarb oxami do, 9-fluorenone-2-carboxamido, 9- fluorenone- 1 -carboxamido, 9-fluorenone-4-carboxamido, anthraquinone-2-carboxamido, xanthene-9-carboxamido, 1 -anthracene carboxamido, 2-anthracene carboxamido, 1- adamantane carboxamido, triphenyl acetamido, diphenylacetamido, 1 -naphthyl carboxamido, and Exo-norbornene-carboxamido. In some embodiments, the chemical moiety is selected from the group of 9-fluorenyl, 1-fluorenyl, 1-Indanyl, 9-oxofluoren-3-yl, 9-oxofluoren-4-yl, 9-oxofluoren-l-yl, 2-anthraquinonyl, 9-xanthyl, 1-anthracenyl, 2-anthracenyl, Adamantyl, Triphenylmethyl, Diphenylmethyl, 1- naphthyl, 2,6-dihydroxy-phenyl, Pentafluoro-phenyl, 6- Hydroxynaphthyl, 1 -pyrenebutyl, 5-acenaphthyl, Coumarin, Biphenyl, Cyclohexyl, Norborenyl, and Fluorescein.

[0140] In some embodiments, the peptide comprises a first chemical moiety and a second chemical moiety. In some embodiments, a first chemical moiety is attached at the N-terminus of the peptide and a second chemical moiety is attached at the C-terminus of the peptide. In some embodiments, a first chemical moiety is selected from the group of Biotin-PEG4, Fluorene, Anthracene, and Fluorene-(Biotin-PEG4-)K, and the second chemical moiety is selected from the group of 9-fluorene, 5 -Acenaphthene, 1-naphthanlene, 2-anthraquinone, and 1 -Anthracene.

[0141] In some embodiments, any one peptide of TABLES 3A-40 comprises a chemical modification at the N-terminus and the C-terminus of the peptide of TABLES 3A-40. In some embodiments, any one peptide of TABLES 3A-40 comprises a first chemical moiety conjugated at the N-terminus of the peptide and a second chemical moiety attached at the C- terminus of the peptide. In some embodiments, any one peptide of TABLES 3A-40 comprises a first chemical moiety conjugated at the N-terminus of the peptide and a second chemical moiety attached at the C-terminus of the peptide, wherein the first chemical moiety is selected from Biotin-PEG4, Fluorene, Anthracene, and Fluorene-(Biotin-PEG4-)K, and thesecond chemical moiety is selected from 9-fluorene, 5 -Acenaphthene, 1-naphthanlene, 2- anthraquinone, and 1 -Anthracene.

[0142] In some embodiments, the N-terminus of the peptide is further modified with a modifier selected from 2-Phenyl-4-quinolinecarboxylic acid, l-(Phenylsulfonyl)-lH-indole- 2-carboxylic acid, 6-Fluoro-2-naphthoic acid, 4-phthalimidobenzoic acid, Xanthene 9- carboxylic acid, 1 -Pyrenebutyric acid, 5-Acenaphthenecarboxylic acid, 1-Phenyl-lH-indole- 2-carboxylic acid, Indole-2-carboxylic acid, quinoline-4-carboxylic acid, and 10- (Carboxymethyl)-9(10H)acridone.

[0143] In some embodiments, any one peptide of TABLES 3A-40 comprises a chemical modification at the N-terminus of the peptide of TABLES 3 A-40, wherein the chemical modification is a modifier selected from 2-Phenyl-4-quinolinecarboxylic acid, 1- (Phenylsulfonyl)-lH-indole-2-carboxylic acid, 6-Fluoro-2-naphthoic acid, 4- phthalimidobenzoic acid, Xanthene 9-carboxylic acid, 1 -Pyrenebutyric acid, 5- Acenaphthenecarboxylic acid, 1 -Phenyl- lH-indole-2-carboxylic acid, Indole-2-carboxylic acid, quinoline-4-carboxylic acid, and 10-(Carboxymethyl)-9(10H)acridone.

[0144] In some embodiments, any one peptide of TABLES 3 A-40 comprises any one, or any combination of modifications provided herein.VIII. Preparation of Peptides

[0145] 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 warhead, a protective group, or pegylation. Alternatively, or additionally, the peptide or peptidomimetic 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 or peptidomimetic can then be purified further. Purification strategies for peptides or peptidomimetics are known in the art, and include FPLC and HPLC based methods.IX. Pharmaceutical Compositions

[0146] 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.

[0147] 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.

[0148] Pharmaceutical compositions containing a synthetic peptide disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method.

[0149] 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.

[0150] 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 amountin an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered 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 as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0151] 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.X. Methods of Use

[0152] 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. In some embodiments, the uses can be based on the binding specificity of the synthetic peptides to HPV 16E6 and on the effect on HPV 16E6 functions of the peptides. In some embodiments, the uses can be based on the binding specificity of the synthetic peptides to Peptidyl-prolylcis-trans isomerase NIMA-interacting 1 protein (Pint) and on the effect on Pint functions of the peptides.

[0153] In some embodiments, the synthetic peptides can be used to modulate the target protein’s function. For example, in some embodiments, the synthetic peptides and peptidomimetics in complex with HPV 16E6 modulate HPV 16E6 engagement with E6AP and its ubiquitination of p53. In some embodiments, the synthetic peptides in complex with HPV 16E6 block or inhibit HPV 16E6 engagement with E6AP in cells.

[0154] Methods for testing for the target protein engagement (e.g., HPV 16E6 - E6AP or Pinl engagement and subsequent cell signaling) are known in the art, for example, by p53 ubiquitination and degradation assays.XI. Kits

[0155] 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.

[0156] 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 such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.EXAMPLES

[0157] 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.

[0158] 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 MOLECULA 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 ENZY OLOGY (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). Additional data and methods are provided in Ye, Xiyun, et al. "Discovery of reactive peptide inhibitors of human papillomavirus oncoprotein E6." Chemical Science 14.44 (2023): 12484-12497, the entire contents of which are incorporated by reference herein for all purposes.

[0159] Unless otherwise stated, all reagents and chemicals were obtained from commercial sources and used without further purification.EXAMPLE 1 - Synthesis of Peptides

[0160] This example describes the synthesis of peptides used in the following examples.Materials

[0161] H-Rink Amide-ChemMatrix resin was purchased from PC AS BioMatrix Inc. Amino acids: Fmoc-Ala-OH, Fmoc-P-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc- Cys(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr( / Bu)-OH, and Fmoc-Val-OH were purchased from Novabiochem (Billerica, MA). Other amino acids: Fmoc-L-citrulline, Fmoc-L-homocitrulline, Fmoc-L-a-aminoadipic acid 5-tert- butyl ester, Fmoc-L-cysteic acid, Fmoc-Om(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc- Dap(Boc)-OH, Fmoc-4-(Boc-amino)-L-phenylalanine, and Fmoc-P-cyclobutyl-L-alanine from ChemPep. Fmoc-HoArg-OH and Fmoc-Phe(4-guanidino-Boc2)-OH from advancedChemTech. Palladium tetrakis (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 (Nashus, NH). Reagents (cleavage): Trifluoroacetic acid (TFA; for HPLC, >99%), triisopropyl silane (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). Fluorescein isothiocyanate isomer I (FITC) from Sigma-Aldrich (St. Louis, MO). Biotin-PEG4- carboxylic acid from ChemPep. 2-Phenyl-4-quinolinecarboxylic acid, 1 -(Phenyl sulfonyl)- 1H- indole-2-carboxylic acid, 6-Fluoro-2-naphthoic acid, 4-phthalimidobenzoic acid, Xanthene 9- carboxylic acid, 1 -Pyrenebutyric acid and 5-Acenaphthenecarboxylic acid are from Sigma- Aldrich (St. Louis, MO). 1 -Phenyl- lH-indole-2-carboxylic acid is from fisher scientific (Hampton, NH). Indole-2-carboxylic acid, quinoline-4-carboxylic acid, lO-(Carboxymethyl)- 9(70J7)acridone are from Enamine (Kyiv, Ukraine).

[0162] Bovine serum albumin (BSA) from VWR (PA). 10x PBS, and 0.25% trypsin-EDTA were obtained from Gibco. Culture flasks, plates, and serological pipettes were obtained from Fisher Scientific. The Pinl recombinant protein was purchased from Novus biology (Centennial, CO) and Abeam (Cambridge, UK).Fast flow synthesis of peptides

[0163] H-Rink Amide-ChemMatrix resin (200 mg, 0.49 mmol / g, 0.10 mmol) was used to prepare peptide-a-carboxamides. Peptides containing noncanonical amino acids were prepared by manual SPPS. Peptides without noncanonical amino acids were prepared by fully automated SPPS. Upon completion, resins were washed with dichloromethane (DCM) three times and dried under reduced pressure.Separation by HPSEC

[0164] High-Performance Size Exclusion Chromatography (HPSEC) was conducted on an Agilent 1260 Infinity II LC System using the Agilent BIOSEC-3 HPLC column (dimensions: 7.8 x 150 mm; particle size: 3 pm; pore size: 100 A). Samples for HPSEC, which included proteins, libraries, or mixtures of protein and library (after 30 min incubation at 4 °C), were prepared in a 100 L buffer. These samples were subsequently eluted using a buffered mobile phase at a flow rate of 1 mL / min for a duration of 15 minutes. During the affinity selectionprocesses, the fraction containing protein-binder complexes was identified using UV detection at wavelengths of 214 and 280 nm, and then collected. Before proceeding to LC- MS / MS for de novo peptide sequencing, this fraction underwent lyophilization. Following the HPSEC affinity selection experiments, the SEC column was cleaned using a mixture of IP A, water, MeCN, and MeOH (1 : 1 : 1 : 1). For MBP-16E6 protein and Pinl protein, an isocratic gradient of IX PBS was used. Collected fractions were desalted by Zip-tip Cl 8 (Agilent, MA) before analyzed by nLC-MS / MS.Peptide library sequencing methods

[0165] Nano liquid chromatography coupled to orbi-trap (nLC-MS / MS) was used for peptide sequencing. Enriched HPSEC-ASMS samples were analyzed on a Thermo Fisher Orbitrap Fusion Eclipse Tribrid Mass Spectrometer with an EASY-Spray source using a Thermo Fisher EASY-nLC 1200 System and Acclaim™ PepMap™ 100 C18 trap columns (20 mm x 75 pm, 3 pm particle size, 100 A pore size, PN164946) and AcclaimTM PepMap TM RSLC C18 HPLC columns (150 mm x 50 pm, 2 pm particle size, 100 A pore size, PN ES901). Liquid chromatography was performed with 0.1% formic acid (FA) in water (solvent A) and 80% MeCN with 0.1% formic acid in water (solvent B) prepared with LiChrosolv® water and MeCN suitable for MS from Millipore Sigma and Optima™LC / MS grade formic acid from Thermo Fisher Scientific. Chromatography was performed at 40 °C, with a flow rate of 300 nL / min using the following gradient: 1% B to 10% B (0-10 min), 10% B to 71% B (10- 70 min), 71% B-90% B (70-80 min) and 91% B-91%(80-90 min) with MS acquisition from 10-70 min in a data-dependent method. Full MS cycle time = 3 s. Detector Type = Orbitrap. Resolution = 120000. Mass Range = Normal. Quadrupole Isolation = True. Scan Range (m / z) = 200-1200. RF Lens (%) = 30. AGC Target = Standard. Maximum Injection Time = Auto. Microscans = 1. Data Type = Profile. Polarity = Positive. The following filters were applied for precursor selection: Monoisotopic Precursor Selection = Peptide. Intensity Threshold = 2.5e4. Charge State = 2-4 or 1-2. Dynamic Exclusion (exclusion after 3n within 3 s for 30 s, mass tolerance = 10 ppm). Fragmentation was induced by higher-energy collisional dissociation (HCD), and electron- transfer dissociation (ETD) with higher-energy collision (EThcD). Specifications HCD: Isolation Mode = Quadrupole. Isolation Window (m / z) = 1.0. Isolation Offset = Off. Collision energy (%) = 30. Detection = Orbitrap. Resolution = 30000. Mass Range = Normal. Scan Range Mode = Define First Mass. First Mass (m / z) = 120. AGC Target = Standard. Maximum Injection Time = Dynamic. 1 Microscan, Data Type = Profile. Specifications EThcD: Isolation Mode = Quadrupole. Isolation Window (m / z) = 1.0 IsolationOffset = Off. Use Calibrated Charge-Dependent ETD Parameters = True. ETD Supplemental Activation = EThcD. SA Collision Energy = 25% or 20%. Detection = Orbitrap. Orbitrap Resolution = 30000. Mass Range = Normal. Scan Range Mode = Define First Mass. First Mass (m / z) = 120. AGC Target = Standard. Maximum Injection Time = Auto. 1 Microscan, Data Type = Profile. ThermoFisher Xcalibur software package and PEAKS Studio 8.5 were used for data analysis.Automated flow peptide synthesis (AFPS) set-up

[0166] 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 (Hartrampf et al., Science, 2020).

[0167] The following settings were used for protein synthesis: flowrate = 40 mL / min, temperature of 90°C (loop) and 85-90°C (reactor). The 50 ml / min pump head pumped 400 pL of liquid per pump stroke; the 5 mL / min pump head pumped 40 pL of liquid per pump stroke. The standard synthetic cycle involved a first step of prewashing the resin at elevated temperatures for 60 s at 40 mL / min. During the coupling step, three HPLC pumps were used: a 50 mL / min pump head pumped the activating agent, a second 50 ml / min pump head pumped the amino acid and a 5 mL / min pump head pumps DIEA. The first two pumps were activated for 8 pumping strokes to prime the coupling agent and amino acid before the DIEA pump was activated. The three pumps were then actuated together for a period of 7 pumping strokes, after which the activating agent pump and amino acid pump were switched using a rotary valve to select DMF. The three pumps were actuated together for a final 8 pumping strokes, after which the DIEA pump was shut off, and the other two pumps continued to wash the resin for another 40 pump strokes. During the deprotection step, two HPLC pumps were used. Using a rotary valve, one HPLC pump selected deprotection stock solution and DMF. The pumps were activated for 13 pump strokes. Both solutions were mixed in a 1 : 1 ratio. The rotary valves selected DMF for both HPLC pumps, and the resin was washed for an additional 40 pump strokes. The coupling-deprotection cycle was repeated for all additional monomers.Method for peptide acetylation

[0168] A 100 mg portion of peptidyl resin from section 1.3 was placed into a 5 mL Torviq fritted syringe and subsequently swelled in DMF. After removing DMF, a solution of AC2O, DIEA, and DMF (2 mL, 85:315: 1600, v / v) were added to the peptidyl resin. The resultingmixture was occasionally agitated for 45 min. After draining the solution, the remaining resin was washed with DMF three times, DCM three times, and dried under reduced pressure.Method for Alloc deprotection

[0169] Peptidyl resin (~10 pmol theoretical loading) was washed with DCM (3 x 5 mL) and then treated with Pd(PPhs)4 (11.0 mg, 10 pmol, 1 equiv) in DCM / piperidine (8:2, 1 mL) for 30 minutes at room temperature under exclusion of light. The resin was then drained and washed with DCM (3 x 5 mL).Method for biotin labeling

[0170] Peptidyl resin (~10 pmol theoretical loading) was loaded into a fritted syringe (6 mL), swollen in DMF (4 mL) for 5 minutes, and then drained. Biotin-PEG4-propionic acid (Biotin- PEG4-OH, 22 mg, 50 pmol, 5 equivalents) and HATU (17 mg, 45 pmol, 4.5 equivalents) were dissolved in DMF (500 pL), activated with DIEA (19 mg, 26 pL, 150 pmol), added to the peptidyl resin and incubated for 30 minutes under exclusion of light. After this time, the resin was drained, washed with DMF (3 x 5 mL), and stored until cleavage.Method for TAMRA labeling

[0171] Peptidyl resin (~ 10 pmol theoretical loading) was loaded into a fritted syringe (6 mL), swollen in DMF (4 mL) for 5 minutes, and then drained. 5- Carboxytetramethylrhodamine (5-TAMRA, 22 mg, 50 pmol, 5 equivalents) and HATU (17 mg, 45 pmol, 4.5 equivalents) were dissolved in DMF (500 pL), activated with DIEA (19 mg, 26 pL, 150 pmol), added to the peptidyl resin and incubated for 30 minutes under exclusion of light. After this time, the resin was drained, washed with DMF (3 x 5 mL), and stored until cleavage.Method for FITC labeling

[0172] Peptidyl resin (~ 10 pmol theoretical loading) was loaded into a fritted syringe (6 mL), swollen in DMF (4 mL) for 5 minutes, and then drained. 5- Carboxytetramethylrhodamine (Fluorescein isomer I, 22 mg, 50 pmol, 5 equivalents) was dissolved in DMF (500 pL), activated with DIEA (19 mg, 26 pL, 150 pmol), added to the peptidyl resin and incubated for 30 minutes under exclusion of light. After this time, the resin was drained, washed with DMF (3 x 5 mL) and stored until cleavage.Cleavage of peptides

[0173] The synthesized peptide was cleaved from the resin and globally deprotected by treating the peptidyl resin with a cleavage cocktail containing 94% TFA, 2.5% water, and 2.5% TIPS (v / 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 was triturated three times with cold Et2O. The resulting material was dissolved in 50% MeCN in water with 0.1% TFA and lyophilized as crude.Dehydroalanine formation

[0174] In a 1.5 mL microcentrifuge tube, cysteine-containing peptide (7 mg) was dissolved in DMF (0.5 mL), and to this, a 10 mg / mL potassium carbonate solution (3.1 mg, 22.3 pmol, 5 equiv) was added. To this mixture, a 10 mg / mL solution of Diethylmeso-2,5-dibromoadipate (151 pL, 1.1 equiv) was added. The mixture was mixed by vortexing for 5 seconds and allowed to react for 4 h. The reaction mixture was diluted with 5% MeCN in H2O + 0.1% TFA and purified by reversed-phase HPLC (Zorbax 300SB-C3, 300A, 5 pm, 9.4 mm x 250 mm) Mobile phase 5% to 55% MeCN in H2O + 0.1% TFA.In solution disulfide dimer formation

[0175] In a 15 mL falcon tube: Dissolve peptide (5.0 mg, 2.75 mmol, 1.0 equiv) in 50% MeCN in H2O + 0.1% TFA (1 mL). Slowly add 50 equiv of 0.3M I2 in MeOH into the peptide solution. Then quench the excessive oxidant with IM of L-Ascorbic acid in water. Mix by vortexing and then purified by reversed-phase flash chromatography using a Sfar Bio C 14 D (300 A 20 pm, 10 g) column (mobile phase 20% MeCN / H2O to 30% MeCN in H2O + 0.1% TFA, 10 column volume).On-resin mixed disulfide formation

[0176] To 50mg resin (0.025 mmol, 1 eq.) add freshly prepared IM Iodine in DMF solution (50 eq., stir for 2 min and drain. Add freshly prepared IM Cysteamine»HCl in DMF (10 eq.) and DIPEA (5 eq.), stir for 10 min. Wash with DMF. Cleavage solution of disulfides: neat TFA, 1 h, RT.General procedure for Cross-linking MBP-16E6 with Covalent Peptides

[0177] A solution of covalent peptides (250 pM solution in 10% DMSO / H2O, 1 equiv, 0.29 pL) was diluted with H2O (29.9 pL) and 10x PBS (3.4 pL, pH = 7.4). A solution of MBP- 16E6 (30 pM in 1 x PBS, 2.4 pL, 1 equiv) and then incubated at room temperature for 2 hours. An 8 pL aliquot of the reaction mixture was removed and quenched with 92 pL of 50:50 MeCN / H2O + 0.1% TFA and analyzed by LC / MS. Yields were obtained by extracting all protein-containing species’ total ion current (TIC) spectra in the chromatogram utilizing Agilent MassHunter Bioconfirm Software 10.0. The extracted chromatograms were deconvoluted utilizing a maximum entropy algorithm, and the abundance of each species was p determined using total ion count. %yield = — — * 100 where Pc is the peak area of the PC + PQ peptide-protein conjugate, and Po is the peak area of the unmodified protein.Purification of the crude peptide.

[0178] Crude peptides were purified by a 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 10 g Biotage SNAP Bio C4 20 pm column. The purification was performed using a gradient as follows: 10% B for 2 column volume (CV), the linear ramp from 30% B to 50% B for 20 CV, 25 mL / min flow rate.EXAMPLE 2 - LC-MS characterization of peptidesLC-MS methods

[0179] 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 methods A-E, as outlined below (solvent A: water with 0.1% formic acid; solvent B: MeCN with 0.1% formic acid).

[0180] Method A. Column used was a Phenom enex Aeris C4 column (1.0 x 150 mm, 5 pm particle size, 300 A pore size). Gradient used was a 1% solvent B (0-2 min), linearly ramped up from 1% solvent B to 91% solvent B (2-10 min). The flow rate was 100 pL / min. MS acquisition was from 2 to 10 min.

[0181] Method B. Column used was a Phenomenex Aeris C4 column (1.0 * 150 mm, 5 pm particle size, 300 A pore size). Gradient used was a 1% solvent B (0-2 min), linearly ramped up from 1% solvent B to 91% solvent B (2-8 min), and 61% solvent B to 95% solvent B (8- 10 min). The flow rate was 100 pL / min. MS acquisition was from 2 to 8 min.

[0182] Method C. Column used was a Agilent Zorbax 300SB C3 column (2.1 * 150 mm, 5 pm particle size, 300 A pore size). Gradient used was a 1% solvent B (0-2 min), linearly ramped up from 1% solvent B to 91% solvent B (2-12 min), and 91% solvent B to 91% solvent B (12-13 min). The flow rate was 500 pL / min. MS acquisition was from 4 to 12 min.

[0183] Method D. Column used was a Phenomenex Jupiter C4 column (1.0 x 150 mm, 5 pm particle size, 300 A pore size). Gradient used was a 1% solent B (0-2 min), linearly ramped up from 1% solvent B to 91% solvent B (2-18 min), and 91% solvent B to 91% solvent B (18-21 min). The flow rate was 100 pL / min. MS acquisition was from 4 to 18 min.

[0184] Method E. Column used was a Agilent Zorbax 300SB C3 column (2.1 x 150 mm, 5 pm particle size, 300 A pore size). Gradient used was a 1% solvent B (0-1 min), linearly ramped up from 1% solvent B to 91% solvent B (1-11 min), and 91% solvent B to 91% solvent B (11-15 min). The flow rate was 500 pL / min. MS acquisition was from 0 to 11 min.

[0185] The total ion chromatograms and mass spectra for major compound plots for peptides targeting E6 are shown in FIGS. 7-22.

[0186] The total ion chromatograms and mass spectra for major compound plots for peptides targeting Pinl are shown in FIGS. 23-59.EXAMPLE 3 - Expression and Purification of ProteinsE6AP

[0187] E6AP (residues 1-875) protein with a C-terminal TEV-6xHis-Avi sequence was subcloned into a pFastBacl vector. Bacmid and viruses of E6AP prepared as described by vendor’s instructions were amplified in Sf9 cells. P2 viruses at 2 uL / mL virus to media were used to infect Sf21 cells for protein expression. Cells were harvested 48 hours post-infection.

[0188] Sf21 cells were lysed by French Press in 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM Imidazole, 5% glycerol, 1 mM PMSF, and cOmplete™ Protease Inhibitor. The supernatant was collected after centrifugation at 39,800 RCF for 30 minutes and loaded onto Ni Resin and washed with 10 CVs of 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol,ImM PMSF, and 20 mM imidazole before eluting with the same buffer supplemented with 500 mM imidazole. The elution was diluted five-fold with 50 mM Tris-HCl pH 7.5 before loading onto a Mono Q 10 / 100 GL column and eluted with a 20 CV linear gradient (Buffer A: 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5% glycerol, 1 mM PMSF; Buffer B: 50 mM Tris-HCl (pH 7.5), 1 M NaCl, 5% glycerol, ImM PMSF). Fractions containing E6AP as determined by SDS-PAGE and Coomassie staining were pooled and concentrated and loaded onto a HiLoad 16 / 600 Superdex 200 pg column equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP (see FIG. 79).

[0189] E6AP Amino Acid Sequence:MEKLHQCYWKSGEPQSDDIEASRMKRAAAKHLIERYYHQLTEGCGNEACTNEFCAS CPTFLRMDNNAAAIKALELYKINAKLCDPHPSKKGASSAYLENSKGAPNNSCSEIKM NKKGARIDFKDVTYLTEEKVYEILELCREREDYSPLIRVIGRVFSSAEALVQSFRKVK QHTKEELKSLQAKDEDKDEDEKEKAACSAAAMEEDSEASSSRIGDSSQGDNNLQKL GPDDVSVDIDAIRRVYTRLLSNEKIETAFLNALVYLSPNVECDLTYHNVYSRDPNYL NLFIIVMENRNLHSPEYLEMALPLFCKAMSKLPLAAQGKLIRLWSKYNADQIRRMM ETFQQLITYKVISNEFNSRNLVNDDDAIVAASKCLKMVYYANVVGGEVDTNHNEED DEEPIPESSELTLQELLGEERRNKKGPRVDPLETELGVKTLDCRKPLIPFEEFINEPLNE VLEMDKDYTFFKVETENKFSFMTCPFILNAVTKNLGLYYDNRIRMYSERRITVLYSL VQGQQLNPYLRLKVRRDHIIDDALVRLEMIAMENPADLKKQLYVEFEGEQGVDEGG VSKEFFQLVVEEIFNPDIGMFTYDESTKLFWFNPSSFETEGQFTLIGIVLGLAIYNNCIL DVHFPMVVYRKLMGKKGTFRDLGDSHPVLYQSLKDLLEYEGNVEDDMMITFQISQT DLFGNPMMYDLKENGDKIPITNENRKEFVNLYSDYILNKSVEKQFKAFRRGFHMVT NESPLKYLFRPEEIELLICGSRNLDFQALEETTEYDGGYTRDSVLIREFWEIVHSFTDE QKRLFLQFTTGTDRAPVGGLGKLKMIIAKNGPDTERLPTSHTCFNVLLLPEYSSKEKL KERLLKAITYAKGFGMLENLYFQGHHHHHHGLNDIFEAQKIEWHE*MBP-16E6

[0190] HPV16E6 (residues 8-158) with a N-terminal HisMBP and four cysteine to serine point mutations to increase solubility was subcloned into a pET45b vector. The plasmid was transformed into BL21(DE3) competent cells. The cells were grown in LB media supplemented with 50 ug / mL carbenicillin shaking at 190 RPM in a 37°C incubator to an optical density of 0.6 at 600 nm before induction with IPTG at a final concentration of 0.5 mM. Protein expression was allowed to proceed for 16 hours shaking at 190 RPM in an 18°C incubator before harvest.

[0191] BL21(DE3) cells were resuspended in 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 2 mM DTT, and cOmplete Protease Inhibitor before lysis by sonification. The supernatant was collected after centrifugation at 30,000 RCF for 45 minutes and loaded onto a MBPTrap HP column equilibrated in the same resuspension buffer and washed for 30 CV. The protein was eluted with 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 2 mM DTT, and 15 mM maltose. The elution was further purified by size exclusion chromatography on a HiLoad 26 / 600 Superdex 200 pg column equilibrated in PBS supplemented with 1 mM DTT (see FIGS. 80A-80C).

[0192] MBP-16E6 amino acid sequence:MAHHHHHHPMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLI AADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAA FNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNK ELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEI MPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNPMSE NLYFQGAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLC IVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEQQYNKPLSDLLIRCINCQKP LSPEEKQRHLDKKQRFHNIRGRWTGRCMSC SRS SRTRRETQL*EXAMPLE 4 - Design of high-affinity peptide binders to E6

[0193] This example describes the design of high affinity peptide binders to HPV16 E6 based on the E6AP protein and subsequent determination of binding affinity of the peptides to HPV16E6 as measured by BLI.

[0194] Briefly, the interaction between 16E6 and E6AP was analyzed in an 16E6-E6AP-p53 ternary complex crystal structure (the crystal structure is represented in PDB: 4XR8) as shown in FIG. 60A. A 17-mer peptide (Peptide 1, SEQ ID NO: 1, sequence: IPESSELTLQELLGEER, as shown in FIG. 60B and FIG. 61 A) that covers residues 401-417 of E6AP and includes the LXXLL motif, was chosen as a starting point for a binder.

[0195] The binding affinity of peptide 1 to HPV16E6 was assessed by bio-layer interferometry (BLI) using recombinant MBP-16E6 4C4S protein (an MBP-16E6 that contains a maltose-binding protein (MBP) tag and four cysteine to serine mutations to stabilize and solubilize HPV16 E6). A competition binding assay was performed as describedbelow using GatorPlus bio-layer interferometry(GatorBio) to estimate the binding affinity of peptides.

[0196] Calibration curve: Streptavidin sensors were soaked in blocking buffer (PBS supplemented with 0.05% Tween-20 and 1 mg / mL bovine serum albumin) for 5 min. After immobilizing the PEG4-Biotinylated 1 peptide (200 nM of Biotin-PEG4- IPESSELTLQELLGEER) (FIG. 61 A) onto streptavidin sensors, 1 : 1 serial dilutions from 1000 nM of E6 in blocking buffer were analyzed for binding (All concentrations: 1000 nM, 500 nM, 250 nM, 125 nM, 62 nM, 31 nM, 15 nM, and 7.8 nM). The response was recorded at equilibrium after 2 min. A curve of sensor response (nm) vs. E6 concentration (nM) was generated to calibrate the free E6 concentration in the solution observed in the competition assay. The curve was generated using Prism 8 software.

[0197] Competition assay: Various concentrations of peptides were incubated in wells with E6 protein in the blocking buffer for 30 min. The PEG4-Biotinylated 1 peptide was immobilized onto streptavidin sensors and dipped into preincubated sample wells. The association events were measured at 30°C, 1,000 rpm. Response at equilibrium after 2 min was recorded. Based on the binding response (nm) values, the concentration of free E6 was interpolated for each sample using the calibration curve. The apparent dissociation constant, KD, can be obtained from the non-linear regression analysis using the equation below as previously described18:[Y]= 0.5x[b-KD-[X]+ (([X]+ KD -b)2+4b* KD ) ] where [Y] is the free [E6] in nM, [X] is the total [peptide] in nM, KD is the binding dissociation constant to be fitted by the equation, and b is the maximal possible E6 concentration to be fitted by the equation. By fitting the free [E6] and [peptide] to the equation, a binding constant with a fitting error was generated by Prism 8 software.

[0198] The BLI binding assay was run in competition mode by immobilizing biotinylated 1 (1-Biotin) onto streptavidin biosensor tips and immersing them across a serial dilution of unlabeled Peptide 1 until equilibrium was reached. The results show that Peptide 1 competed MBP-16E6 binding to 1-Biotin, revealing a competition KD of 2.4 pM (FIGs. 61B and 61C). The competition KD values reported for the peptide variants discussed below were determined by BLI in a similar manner.

[0199] Additionally, peptide 1 was labelled N or C terminally with FITC and binding to 16E6 was measured with BLI. The results show increased binding affinity with N-terminalfluorescein isothiocyanate (FITC)-labeled peptide 1 (N-FITC) compared to the C-terminal FITC-labeled peptide 1 (C-FITC). The N-FITC peptide showed a competition KD of 95 nM, while C-FITC peptide 1 had a competition KD of 640 nM.EXAMPLE 5 - Alanine Scan of Peptide 1

[0200] To determine critical residues in the E6AP peptide designs, an alanine (Ala) scan was performed and 17 single Ala mutants of N-FITC, peptides Al to A17, respectively were synthesized (FIG. 62). Alanine scanning showed the residues that are critical for binding activity. Substituting residues Leu9, Leul 1, and Leul2 to alanine or removing the N-terminal modification significantly decreased or disrupted binding. Four residues, Glu6, Thr8, Glul 1, and Glyl4, showed medium alanine tolerance.

[0201] The result show that hotspot residues Leu9, Leu 12, and Leu 13 to be critical for binding, as the A9, A12, and A13 peptides did not show measurable binding to MBP-16E6. This observation aligns with L12A and L13A single mutated peptides, which showed decrease E6 binding. Peptides simultaneously containing the L9A, L12A, and L13A mutations (or 3L3 A triple mutants) were used as negative control peptides (FIG. 60B).EXAMPLE 6 -Design and Screening of a 1.42-Million-Member Peptide Library

[0202] This example describes the design of a peptide library based on the ELT peptide of E6AP and screen of the library for high affinity binders to 16E6.

[0203] Briefly, a library with 1.42 million members was designed based on the ELT peptide ELTLQELLGEER.

[0204] Results from the Alanine scan showed that alanine mutations in the residues El (El A) and E6 (E6A) of the ELT peptide decreased binding affinity to MBP-16E6 by 9-fold and 16- fold respectively(FIG. 63). These two positions were replaced with Glutamic acid analogs. The D-alanine scanning showed Q5a and Q5A mutations of the ELT peptide did not affect binding, therefore, the Q5 position was mutated to Alanine analogs, including Gly, d-ala and Aib. The four Leu residues were mutated to aliphatic and aromatic sidechains. Thr3 position was replaced by Thr analogs and P-substituted amino acids. The C-term CEER residues were kept constant, with a positively charged Arginine can increase sequencing efficiency. The L- alanine scanning identified hotspot residues Leu4, Leu7 and Leu8 that were critical for binding. L4A, L7A and L8A simultaneous mutated peptides (or 3L3 A) were used as negative control peptides. The library had the consensus sequence of X1X2X3X4QX6X7X8CEER (SEQ ID NO: 6) with Xi, X2, X3, X4, Xe, X7, and Xs chosen from FIG. 64 (Xi is selected from Nva,Leu, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf; X2 is selected from Aad, Glu, and Cya; X3 is selected from Thr, Asn, Hyp, Cpg, Cbg, and Ceg; XHs selected from Aad, Glu, and Cya; Xe is selected from Nva, Leu, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf; X7 is selected from Aad, Glu, and Cya; and Xs is selected from Aad, Glu, and Cya). Residue G9 was replaced with a Cys.

[0205] The ELT peptide library was screened with the ReAct-ASMS method (FIG. 1). Briefly, the affinity selection platform, named Reversibly Active Affinity Selection Mass Spectrometry (ReAct-ASMS), can be used to rapidly identify high-affinity covalent crosslinkers from synthetic libraries of reactive peptides. This approach has several advantages, including label-free screening using High Performance Size Exclusion Chromatography (HPSEC)-ASMS, which eliminates the need to functionalize or label recombinant proteins with affinity tags like biotin, IgA, or GST. The flow-based SEC separation allows for rapid and robust separation of bound and unbound molecules with affinity ranges of 200 pM-1 pM. This platform enables affinity maturation on covalent PPI peptide inhibitors using non-canonical amino acids.

[0206] To mature the E6AP -mimicking peptide, ReAct-ASMS libraries were prepared with fast crosslinker -PME-DS and slow crosslinker NACPME-DS. Library I-PME and library I- NAC0ME were individually screened against MBP-16E6. With the same amount of incubation time, library 1-0ME generate an average of 7 hits with ALC>90, 7-fold more than library I-NAC0ME with an average of 1 hit with ALC>90, with frequent identification of parent peptide, which was supplemented as a competitor in the screening. The finding suggested that fast crosslinker generated more hits and was select for further investigation.

[0207] ReAct-ASMS with MBP-16E6 identified peptides from the X1X2X3X4QX6X7X8CEER library. Three ReAct-ASMS conditions with different ratio of libraries and MBP-16E6 protein were mixed, incubated, and separated by HPSEC. The breakthrough MBP-16E6 protein fraction was collected, dissociated by reductant treatment, and analyzed by an Orbi-trap nLC-MS / MS. De novo peptide sequencing was constructed by PEAKS studio software and filtered by python scripts. Among all library 1-0ME selection conditions, 14.4 hits were identified in the loose condition (>1 ng per member, n=10) and 2.6 hits in the stringent condition (0.1-0.6 ng per member, n=14) on average. Twenty -three hit peptides were select from the most stringent condition with average local confidence (ALC)> 90. Eleven peptides were shortlisted for resynthesis, with Cysteine being replaced by Dha for irreversible inhibition of target protein (FIG. 65).

[0208] Additional peptide libraries screened are listed in TABLES 3A to 40.ASMS of MBP 16E6TABLE 3ANon-canonical amino acid residues: Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenylglycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Homo cysteic acid (Hoc): z, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.Table 3B Resynthesis list from Library 1Non-canonical amino acid residues: Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoicHomophenylalanine (Hof): m, Phenylglycine (Phg): y, Napthylalanine (Nat): o, Aminoadipic acid (Aad): p, Hydroxyproline (Hyp): s, Aminoisobutyric (Aib): w, C-term amide: U.TABLE 4A Quality control of library 1 for MBP-16E6Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclopentylglycine (Ceg): g, Cyclohexylalanine (Cha): j, 2- aminoctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenylglycine (Phg): n, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.

[0209] About 20 beads were cleaved from the library. 1 gram resin contains 2.86 * 106 mono-sized beads. About 12 peptides were identified with ALC>85. Unique sequences that were identified by PEAKS and filtered by python scripts match with the library design.Sequences with ALC>60 were displayed. FIG. 66 shows that most of the monomers were spotted in each randomized position.Table 4B ASMS screening conditions using Library 1 for E6

[0210] FIG. 67 shows a representative HPSEC separation chromatogram of identified peptides from Library 1 for E6 (ALO80).TABLE 5 Condition I-l.l(l)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, 2-aminooctanoicPhenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 6 Condition I-1.2(l)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Homo cysteic acid (Hoc): z, Hydroxyl proline (Hyp): s, C-term amide: U.TABLE 7 Condition I-1.3(l)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Homo cysteic acid (Hoc): z, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 8 Condition I-l.l(2)C-term amide: U.TABLE 9 Condition I-1.3(2)Cyclobutylglycine (Cbg): b, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 10 Condition I-1.3(2)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 11 Condition I-l.l(3)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 12 Condition I-1.2(3)Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 13 Condition I-1.3(3)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 14 Condition 1-2.1Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 15 Condition 1-2.2Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 16 Condition 1-2.3Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 17 Condition 1-2.4Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 18 Condition 1-3.1Cyclopropylglycine (Cpg): d, Cyclobutylalanine (Cba): h, 2-aminooctanoicHomo phenylalanine (Hof): m, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, , C-term amide: U.TABLE 19 Condition 1-3.2Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 20 Condition 1-4.1Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo Phenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Homo cysteic acid (Hoc): z, Hydroxyl proline (Hyp): s, C-term amide: U.TABLE 21 Condition 1-4.2Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, 2-aminooctanoicHomo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, C-term amide: U.TABLE 22 Condition 1-4.3Cyclobutylglycine (Cbg): b, Cyclopropylalanine (Cpa): i, Cyclohexylalanine (Cha): j, 2-aminooctanoicHomo phenylalanine (Hof): m, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, C-term amide: U.TABLE 23 Condition 1-3.3Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 24 Condition 1-3.4Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg):y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 25 Condition 1-4.5] Cyclopropylalanine (Cpa): i, , 2-aminooctanoicHomo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, C-term amide: U.TABLE 26 Condition I- 4.6Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Homo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.Identified binders from peptide E6 library I-NACbME (ALC>80)TABLE 27 Condition 1-5.1Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Aminoadipic acid (Aad): p, C-term amide: UTABLE 28 Condition 1-5.2C-term amide: UTABLE 29 Condition 1-5.3Cyclobutylglycine (Cbg): b, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoicHomo phenylalanine (Hof): m, Phenyl glycine (Phg): y, Napthyl alanine (Naf): o, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.TABLE 30 Condition 1-5.4Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, 2-aminooctanoicPhenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Hydroxyl proline (Hyp): s, C-term amide: U.TABLE 31 Condition 1-5.5Cyclopropylglycine (Cpg): d, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine (Cha): j, 2-aminooctanoic (Aoc): k, Phenyl glycine (Phg): y, Aminoadipic acid (Aad): p, Homo cysteic acid (Hoc): z, Hydroxyl proline (Hyp): s, Amino isobutyric (Aib): w, C-term amide: U.ASMS for PinlTABLE 32 Resynthesis list from Library 2.N-Dimcthyl Lysine (Dmk): m, Dap: t, Dab: a, Om: 1, Homoarginine (Hor): e, N,N-Dimethyl arginine (Dmr): z, Homo citrulline (Het): q, Citrulline (Cit): r, Homo glutamine (Hoe): X, 4-Methylamine phenylalanine (Eaf): f, 4-Guanidinyl phenylalanine( Gnf): u, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylglycine or Cyclopentylalanine (Chg / Cea) : i Cyclohexylalanine (Cha) :j, 2- aminooctanoic (Aoc): h, 2-methoxy-4’ -methyl- 1,1 ’-biphenyl alanine (MpF): s, 4-phenyl phenylalanine (Phf): y, 2-chloro tryptophan (Cl-trp): v, homoproline (Pip): o , Naphthylalanine(Naf): M, p4- aminobenzoic acid (Amb): D, (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid (ABPC): E, thiazolidine (Thz ): J, Hydroxyproline (Hyp): O, 4-Fluoro-Proline (4FP): b, Aib: d, 3,3’,3’-deuterium-LAlanine: Y, 4-nitro phenylalanine (Not): N, 4-nitrile phenylalanine (Cnf): B, 3,4-difluoro phenylalanine(F2f): c, Dimethoxy phenylalanine (Dmf): g, Quinolyl Alanine (Quf): n, tetrahydro isoquinoline (Tic): w, Homo phenylalanine (Hof): k, Phenyl glycine (Phg): Q, 4-amino phenylalanine(Amf): S, Pyridinyl- alanine(Pya): T Azaridine (Aza): Z. Each instance of the one letter code “D” , “E”, “M”, “N”, “T”, and “Y” as used in Tables 32-40 is understood to refer to the non-canonical amino acid as defined in this table legend.TABLE 33 Quality control of Library 2 for PinlOm: 1, Homo citrulline (Het): q, 4-Guanidinyl phenylalanine( Gnf): u, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylglycine or Cyclopentylalanine (Chg / Cea) : i, Cyclohexylalanine(Cha) : j, 2-aminooctanoic (Aoc): h, 4-phenyl phenylalanine (Phf): y, 2-chloro tryptophan (Cl-trp): v, homoproline (Pip): o, 4-nitro phenylalanine (Nof): N, 4-nitrile phenylalanine (Cnf): B, Dimethoxy phenylalanine (Dmf): g, tetrahydro isoquinoline (Tic): w~20 beads were cleaved from the library. 1 gram resin contains 2.86 x 106 mono-sized beads. ~12 peptides were identified with ALC>85. Unique sequences that were identified by PEAKSand filtered by python scripts match the library design. Sequences with ALC>60 were displayed. FIG. 68 shows that most of the monomers were spotted in each randomized positionTABLE 34 ASMS screening conditions using Library 2 for PinlIdentified peptides from Library 2 for Pinl (ALO80)TABLE 35 Hit peptides obtained by Condition 1.1Homoarginine (Hor): e, Citrulline (Cit): r, Homo glutamine (Hoe): X, 4-Guanidinyl phenylalanine ( Gnf): u, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylglycine or Cyclopentylalanine (Chg / Cea) : i Cyclohexylalanine(Cha) :j, 2-aminooctanoic (Aoc): h, 4-phenyl phenylalanine (Phf): y, homoproline (Pip): o, Aib: d, 3,3’,3’-deuterium-LAlanine: Y, 4-nitro phenylalanine (Not): N, 3,4-difluoro phenylalanine (F2f): c, Dimethoxy phenylalanine (Dmf): g, Quinolyl Alanine (Quf): n, Homo phenylalanine (Hof): k, Pyridinyl-alanine(Pya): T. Each instance of the one letter code “N”, T” and “Y” as used in Table 35 is understood to refer to the non-canonical amino acid as defined in this table legend.TABLE 36 Hit peptides obtained by Condition 2.1Homoarginine (Hor): e, 4-Methylamine phenylalanine (Eaf): f, 4-Guanidinyl phenylalanine( Gnf): u, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylglycine or Cyclopentylalanine (Chg / Cea) : i Cyclohexylalanine(Cha) :j, 4-phenyl phenylalanine(Phf): y, homoproline (Pip): o, Naphthyl alanine(Naf): M, Aib: d, 3,3’,3’-deuterium-LAlanine: Y, 4-nitro phenylalanine (Nof): N, Dimethoxy phenylalanine (Dmf): g, Homo phenylalanine(Hof): k, Pyridinyl-alanine(Pya): T. Each instance of the one letter code “M”, “N”, “T”, and ‘ Y” as used in Table 36 is understood to refer to the non-canonical amino acid as defined in this table legend.TABLE 37 Hit peptides obtained by Condition 3.14-Guanidinyl phenylalanine( Gnf): u, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylglycine or Cyclopentylalanine (Chg / Cea) : i, 4-phenyl phenylalanine (Phf): y, 2-chloro tryptophan (Cl -trp): v, homoproline (Pip): o , Naphthyl alanine(Naf): M, Aib: d, 3,3’, 3’-deuterium-LAlanine: Y, 4-nitro phenylalanine (Nof): N, Homo phenylalanine (Hof): k, Pyridinyl-alanine(Pya): T. Each instance of the one letter code “M”, “N”, and ‘ Y” as used in Table 37 is understood to refer to the non-canonical amino acid as defined in this table legend.TABLE 38 Hit peptides obtained by Condition 1.2Om: 1, Homoarginine (Hor): e, 4-Guanidinyl phenylalanine ( Gnf): u, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylalanine(Cha) :j, 4-phenyl phenylalanine(Phf): y, homoproline (Pip): o, Aib: d, 3,3’,3’-deuterium-LAlanine: Y, Dimethoxy phenylalanine (Dmf): g, tetrahydro isoquinoline (Tic): w, 4-amino phenylalanine(Amf): S, Pyridinyl-alanine(Pya): T. Each instance of the one letter code “T” and ‘Y” as used in Table 38 is understood to refer to the non- canonical amino acid as defined in this table legend.TABLE 39 Hit peptides obtained by Condition 2.2Om: 1, Homoarginine (Hor): e, Homo glutamine (Hoe): X, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylglycine or Cyclopentylalanine (Chg / Cea) : i Cyclohexylalanine(Cha) :j, 4- phenyl phenylalanine(Phf): y, homoproline (Pip): o, thiazolidine (Thz ): J, Aib: d, 3,3’,3’-deuterium-LAlanine: Y, phenylalanine (Hof): k, Pyridinyl-alanine(Pya): T. Each instance of the one letter code “T” and “Y” as used in Table 39 is understood to refer to the non-canonical amino acid as defined in this table legend.TABLE 40 Hit peptides obtained by Condition 3.2Om: 1, 4-Guanidinyl, Cyclopentylglycine or Cyclobutylalanine (Ceg / Cba): x, Cyclohexylglycine or Cyclopentylalanine (Chg / Cea) : I, 4-phenyl phenylalanine (Phf): y, homoproline (Pip): o, Aib: d, Homo phenylalanine (Hof): k, Pyridinyl-alanine(Pya): T. Each instance of the one letter code “T” as used in Table 40 is understood to refer to the non-canonical amino acid as defined in this table legend.EXAMPLE 7 - Binding MeasurementsDirect binding measurement by BLI

[0211] Streptavidin sensors were soaked in blocking buffer (1 * PBS supplemented with 0.05% Tween-20 and 1 mg / mL bovine serum albumin) for 5 min. After immobilizing the Biotin-ELT (Biotin-PEG4-ELTLQELLGEER) at 200 nM onto streptavidin sensors, serial dilutions of MBP-16E6 in blocking buffer were analyzed for binding. The response was recorded at equilibrium after 2 min. Association lasted for 100 seconds, and dissociation lasted for another 120 seconds. The curve was reported by GatorPlus software and replotted by Prism 8 software.Competitive binding assay by BLI

[0212] A competition binding assay was performed as described below using GatorPlus biolayer interferometry (GatorBio) to estimate the binding affinity of peptides.

[0213] Calibration curve: Streptavidin sensors were soaked in blocking buffer (PBS supplemented with 0.05% Tween-20 and 1 mg / mL bovine serum albumin) for 5 min. After immobilizing the Biotin-ELT (Biotin-PEG4-ELTLQELLGEER) onto streptavidin sensors, 1 : 1 serial dilutions from 1000 nM of MBP-16E6 in blocking buffer were analyzed for binding (All concentrations: 1000 nM, 500 nM, 250 nM, 125 nM, 62 nM, 31 nM, 15 nM, and 7.8 nM). The response was recorded at equilibrium after 2 min. A curve of sensor response (nm) vs. MBP-16E6 concentration (nM) was generated to calibrate the free MBP-16E6 concentration in the solution observed in the competition assay. The curve, as shown in FIG. 69, was generated using Prism 8 software.

[0214] Competition assay: Various concentrations of peptides were incubated in wells with MBP-16E6 protein in the blocking buffer for 4 h at 37°C. The Biotin-ELT was immobilized onto streptavidin sensors and dipped into preincubated sample wells. The association events were measured at 30°C, 1,000 rpm. Response at equilibrium after 2 min was recorded. Based on the binding response (nm) values, the concentration of ‘free’ MBP-16E6 was interpolated for each sample using the calibration curve. The apparent dissociation constant, KD, can be obtained from the non-linear regression analysis using the equation:[Y] = 0.5 x [b - Kd- [X] + 7([X] + Kd- b)2+ 4b x Kd] , where [Y] is the free [MBP-16E6] in nM, [X] is the total [peptide] in nM, KD is the binding dissociation constant to be fitted by the equation, and b is the maximal possible MBP-16E6 concentration to be fitted by the equation. By fitting the free [MBP-16E6] and [peptide] to the equation, a binding constant with a fitting error was generated by Prism 8 software.Direct fluorescent polarization (FP) binding assay to MBP-16E6

[0215] Direct FP binding assay of FITC-ELT (FITC-ELTLQELLGEER). FP was recorded after incubating various concentration of MBP-16E6 with 100 nM of FITC-ELT. FP was performed with a BioTek Hl plate reader. n=2 biological replicates (Excitation: 485 / 20, Emission: 528 / 20; (Green) Filter set; Read Height: 10.5 mm; Gain sets to 50; Room temperature).Competitive Fluorescent polarization (FP) assay to MBP-16E6

[0216] Binding affinity is measured by competitive FP assay. Various concentration of analyte peptide was mixed with 100 nM FITC-ELT and 500nM MBP-16E6 in the solution, incubated for overnight. Polarization was recorded using Biotek Hl. n=2 biological replicates. FP was performed with a BioTek Hl plate reader. n=2 biological replicates (Excitation: 485 / 20, Emission: 528 / 20; (Green) Filter set; Read Height: 10.5 mm; Gain sets to 50; Room temperature).Competitive FP binding assay against Pinl

[0217] Binding affinity as shown as apparent Ki to Pinl was determined using a competitive FP assay to assess competition with an N-terminal fluorescein-labeled peptide (Bth-d- phos.Thr-Pip-Nal) as previously described45. Briefly, the indicated concentrations of analyte peptides were pre-incubated for 12 h at 4°C with a solution containing 250 nM Pinl recombinant protein, 5 nM N-terminal fluorescein-labeled peptide, 10 pg / mE1BSA, 0.01% (v / v) Tween-20 and 1 mM DTT supplemented with 10 mM HEPES, 10 mM NaCl and 1% glycerol (pH 7.4). Polarization was recorded using Biotek Hl plate reader. n=2 biological replicates (Excitation: 485 / 20, Emission: 528 / 20; (Green) Filter set; Read Height: 10.5 mm; Gain sets to 50; Room temperature).

[0218] Apparent values obtained from the FP binding assay results were derived from the Kenakin equation:Kenakin = (Lb)(EC5o)( D) / (Lo)(Ro)+Lb(Ro-Lo+Lb- o), where KD [M], KD of the probe; ECso [M], concentration of unlabeled peptide that results in 50% inhibition of binding (obtained from competitive FP assay); total tracer Lo [M], fluorescein-labeled peptide concentration in competitive FP assay; bound tracer Lb [M], 85%, fraction of fluorescein-labeled peptide bound to Pinl; and total receptor Ro [M], Pinl concentration in the competitive FP assay.EXAMPLE 8 - ReAct-ASMS to identify covalent peptide inhibitors to target HPV16 E6

[0219] Human papillomavirus (HPV)-encoded early protein 6 (E6) and early protein 7 (E7) are primarily transforming viral proteins contributing to multiple cancers, including more than 99% of cervical, vaginal, oropharyngeal, and potentially a subset of prostate cancers (Roden et al., Nat Rev Cancer, 2018; McBride et al., Nat Rev Microbiol, 2022). HPV16 E6 recruits E6AP (encoded by UBE3A) to form a complex with p53 that promotes ubiquitination of p53, leading to its subsequent proteasome-mediated degradation (Scheffner et al, Cell,1990; Scheffner et al., Cell, 1993). A decrease of p53 accelerates cancer development since p53 plays a crucial role in tumor suppression mainly by inducing growth arrest and apoptosis (Chao et al., Clin Chim Acta, 2015; Hafner et al., Nat Rev Mol Cell Biol, 2019). Therefore, the PPI between HPV16 E6 and E6AP is a promising and novel target for drug development. However, E6AP-mimicking peptides were not able to find success in pre-clinical studies. An E6AP-mimicking peptide ( D = 4 pM) (Liu et al., Biochemistry, 2004), a cysteine- containing peptide (AD = 118 nM) (Dymalla et al., J Mol Med (Berl), 2009; Zanier et al., PLoS One, 2014), and a peptide aptamer (AD not determined) (Butz et al., Proc Natl Acad Sci USA, 2000), were reported to target HPV16 E6, but they all showed insufficient affinity toward disrupting the strong interaction between HPV16 E6 and E6AP (~1 nM), possibly due to a rapid dissociation rate constant ( off). The structure of the complex formed between HPV16 E6, E6AP, and p53 has been reported (PDB: 4XR8, FIG. 2A)(Martinez-Zapien et al., Nature, 2016).

[0220] Truncation studies on the E6-E6AP interaction sites revealed the main recognition domain as a peptide fragment of E6AP (residues 406-417), termed the ELT peptide: ELTLQELLGEER (Ramirez et al., Angew Chem Int Ed Engl, 2015; Brimer et al., PLoS Pathog, 2017). This parent peptide showed micromolar binding affinity (FIG. 70), dissociation constant AD = 6.8 pM by a direct fluorescent polarization (FP) assay using FITC-ELT and AD = 6.1 pM by a direct bio-layer interferometry (BLI) assay with Biotin- ELT to the recombinant MBP-16E6 4C4S protein (hereinafter called MBP-16E6), which was stabilized and solubilized by a maltose-binding protein (MBP) tag and mutation of four cysteine residues to serine (Nomine et al., Biochemistry, 2003). Analysis of the binding interface revealed that nucleophilic Cys58 of HP VI 6 E6is in proximity to the residue Gly9 of the E6AP (FIG. 2B).

[0221] To overcome the fast off rate of ELT and disrupt the E6-E6AP interaction, ELT- G9Dha (Chemical structure in FIG. 3B) was developed, in which Gly9 of ELT was replaced by the cysteine-reactive covalent warhead dehydroalanine (Dha) to achieve covalent crosslinking to Cys58 inside the HP VI 6 E6 peptide binding pocket. Increasing concentrations of unlabeled ELT-G9Dha were pre-mixed with a constant concentration of MBP-16E6 (30 nM) for covalent crosslinking. The FP signal was measured and plotted against the concentrations of ELT-G9Dha to generate the apparent Ai. In a competitive BLI assay, Biotin-ELT was immobilized onto the streptavidin biosensor tips. Increasing concentrations of unlabeled ELT-G9Dha were also pre-mixed with a constant concentration of MBP-16E6(30 nM) for covalent crosslinking. Biotin-ELT-immobilized tips were immersed into each solution to estimate the fraction of non-crosslinked MBP-16E6and interpolate the apparent Ki values. ELT-G9Dha showed an apparent Xi of 5.5 pM by a competitive FP assay and an apparent Ki of 3.4 pM by a competitive BLI assay (FIGS. 3B, 4B and 4C). In the crosslinking studies, the ELT-G9C-MBP-16E6 complex formed 74% in 2 hours at 37°C, effectively occupying the binding pocket of E6 and preventing the association of E6AP protein.

[0222] To develop ReAct-ASMS, the crosslinked peptide needs to be able to dissociate from the target protein (FIGS. 1 A-1D). Inspired by the disulfide tethering technique used for small molecule drug discovery, the irreversible Dha was replaced with a reversible disulfide and prepared three different small molecule-peptide mixed disulfides, cysteine (ELT-G9C-C), cysteamine (ELT-G9C-PME) and N-acetyl cysteamine (ELT-G9C-NACPME, FIG. 2D). The three mixed disulfide-containing peptides were produced on resin with quantitative yield. Among the disulfides, ELT-G9-PME showed the highest crosslinking yield of 86% after 6 h, while ELT-G9C-NACPME yielded 37% and ELT-G9C-C yielded 16% of crosslinked product. Therefore, cysteamine was chosen as the covalent warhead for further studies. For the peptide-protein complex dissociation conditions, dithiothreitol (DTT) treatment achieved a 95% yield over 82% yield of tris(2-carboxyethyl)phosphine (TCEP) treatment (FIG. 2E). DTT treatment was thus used further as dissociation reagent to separate MBP-16E6 from the crosslinked peptidesDesign of a 1.42 million-member peptide library against E6.

[0223] Based on alanine scanning results of ELT (ELTLQELLGEER), El A and E6A mutations decreased binding affinity to MBP-16E6 by 9-fold and 16-fold, respectively, therefore Glul and Glu6 positions were replaced with glutamic acid analogs. The Q5A mutation did not affect binding and was mutated to alanine analogs, including Gly, DAla, and Aib. Leu4, Leu7, and Leu8 are three major hotspots as their mutations decrease binding by more than 50-fold. These three leucine residues along with Leu2 were mutated to aliphatic and aromatic sidechains. Thr3 position was replaced by threonine analogs. The C-term EER residues of ELT were kept constant as these three residues have no contributions to binding and the positively charged arginine at the C-terminus increases sequencing fidelity by MS / MS. Ultimately, a focused Library 1 (X1X2X3X4X5X6X7X8CEER) with 1.42 million members was prepared, in which C represents a cysteamine warhead similar to ELT-G9C- PME (FIG. 3 A). The size of Library 1 would typically be challenging for application to thepreviously described standard in-solution HPSEC-ASMS platform developed for non- covalent libraries (Touti et al., Nat Chem Biol, 2019).Peptide E-10 was identified by ReAct-ASMS as the top covalent peptide inhibitor for E6.

[0224] Three ReAct-ASMS conditions with different ratios of peptide libraries versus MBP- 16E6 protein were mixed, incubated, and separated by HPSEC (FIG. 1 A). The breakthrough MBP-16E6 protein fraction was collected, dissociated by DTT treatment, and analyzed by the Orbitrap nLC-MS / MS protocol (FIGS. 1 A and IB). De novo peptide sequencing was performed with the PEAKS Studio software suite (Ma et al., Rapid Commun Mass Speclrom, 2033) and filtered by Python scripts (Vinogradov et al., ACS Comb Sci, 2017). Among all focused Library 1 selection conditions, 14.4 hits were identified on average per replicate in the “loose” selection conditions (>1 ng per member, n = 10) and 2.6 hits were identified on average in the “stringent” condition (0.1-0.6 ng per member, n = 14). Twenty -three hit peptides were selected from the most stringent condition with average local confidence scores (ALC, an indicator of sequencing confidence) >90. Twelve structurally distinct peptides were shortlisted for resynthesis, with cysteamine warheads being replaced by Dha for irreversible inhibition of target MBP-16E6 protein and biophysical characterization (FIG. 3B).

[0225] To compare crosslinking efficiency towards MBP-16E6, twelve resynthesized Dha peptides E-l to E-12 and the parent ELT-G9Dha were mixed each at 1 pM equally with MBP-16E6 protein at 1 pM for 2 hours at 37°C. Mono-crosslinked E-10- MBP-16E6 conjugate was the only observed conjugate with a yield of 83%, indicating that E-10 outcompeted the other peptides in the reaction with MBP-16E6 (FIG. 4 A).

[0226] Dha peptides were also evaluated by competitive FP assay and competitive BLI assay against FITC-ELT (FIG. 71) and Biotin-ELT (FIG. 72), which were reported in FIG. 3B as apparent Kis. Five out of twelve peptides showed at least 10-fold improved binding relative to the starting peptide ELT-G9Dha (E-l, E-4, E-5, E-10, and E-12, respectively). This >40% positive hit rate demonstrates the efficiency of the ReAct-ASMS platform toward discovery of covalent peptide binders with improved affinities.

[0227] The other peptides showed comparable binding affinity to the parent peptide ELT- G9Dha. Notably, these seven peptides with micromolar binding affinity would be overlooked by the previous in-solution HPSEC-ASMS platform but were reliably recovered with our reactivity -based protocol. With ReAct-ASMS, more putative binders can be identified andanalyzed to understand structure-activity relationships (SAR). For example, higher binding affinity could be achieved by employing branched hydrophobic side chains at position 4 and cyclic hydrophobic side chains at position 7. Opposite situations such as E-2 have drastically decreased binding affinity with cyclic hydrophobic Cpa at position 4 and branched hydrophobic Leu at position 7 (FIG. 3B). In both assays, peptide E-10 (structure in FIG. 3B) demonstrated the lowest value toward MBP-16E6 with 188 nM in competitive FP assay and 177 nM in competitive BLI assay, respectively, corresponding to a 30-fold improvement compared to ELT-G9Dha (FIGS. 3B, 4B and 4C). Three major non-canonical structural changes, Leu2Naf, Thr3Ceg, and Gln5Aib were observed within E-10, which make it distinct from the parent peptide ELT-G9Dha. Since Leu4, Leu7, and Leu8 are three major hotspots, the L4A, L7A, and L8A simultaneously mutated peptide (or E-10-3L3A) was used as a negative control in the binding studies (FIG. 3B). The E-10-3L3A showed no observable binding to the MBP-16E6 protein (FIGS. 4B and 4C).Peptide E-10 selectively crosslinks to Cys58 of E6.

[0228] E-10 uses a two-stage bind-and-react strategy to covalently crosslink to E6 and block the E6AP binding site. When E-10 (10 pM) and MBP-16E6 (1 pM) were incubated in PBS at 37°C, protein deconvolution mass spectra revealed 89% yield of mono-crosslinked E-10- MBP-16E6 at 2 hours and 96% yield of mono-crosslinked E-10-MBP-16E6 at 4 hours. (FIG. 4D). This high conversion was achieved despite the presence of 10 cysteine residues on MBP-16E6 and 10 times more peptide E-10 than MBP-16E6 in the mixture. The negative control E-10-3L3A showed no observable crosslinking to MBP-16E6 protein (FIG. 4E), supporting the target and sequence specificity of E-10. To confirm cysteine 58 targeting sitespecificity, E-10 was incubated with the MBP-16E6 mutant C58S for 24 hours in PBS at 37°C. No crosslinking was observed between E-10 and C58S MBP-16E6 (FIG. 4F), which supports Cys58 as the sole crosslinking site in MBP-16E6 by E-10. Leu57 of E6 is a critical residue for full-length E6APZE6 PPI. Mutation of Leu57 disrupts E6AP binding to MBP- 16E6. Since E-10 was derived from E6AP, no crosslinking was observed between E-10 and L57A MBP-16E6 (FIG. 73), further supporting the selective binding of E-10 to MBP-16E6.

[0229] A kinetic crosslinking study was performed with E-10 to assess the AinactWi of E-10, in which Ki describes the reversible binding and Ain act the maximum rate of inactivation (Singh et al., NatRev Drug Discov, 2011). The crosslinking yield of E-10 to 25 nM ofMBP- 16E6 was monitored over time by LC-MS (FIG. 4G). The apparent kinetic rate constant for crosslinking, .Kapp, at each concentration of E-10 was determined from the slope of a semi-logarithmic plot of crosslinking versus time (FIG. 4G). The ATapp values are re-plotted against the E-10 concentration (FIG. 4H), and fitted to a hyperbolic equation, Ahpp = Ainact[E-10] / (^fi+[E-10]), to obtain a Ainact of 0.018 s-1, a Ki value of 792 nM, and a Ainact / A ratio of 22.7 mM-1 s-1.13 The Ainact / Aa ratio represents the second-order rate constant for the crosslinking of the covalent inhibitor with the target (Singh et al., Nat Rev Drug Discov, 2011). E-10 displayed a ~50-fold improvement over ELT-G9Dha (Ainact of 0.022 s-1, Ki of 50 u M, and Ainact / A"i ratio of 446 M-l s-1). For comparison, the FDA- approved cysteine-targeting small molecular drug Nirmatrelvir showed a Ainact / Aa value of 55 mM-1 s-1 crosslinking to Cysl45 of SARS-CoV-2 3CLpro protease (Eng et al., Drug Metal Dispos, 2022), which is 2.4-fold slower than E-10. E-10 is composed of highly distinct residues apart from the parent peptide ELT-G9Dha. E-lO’s mutations, Leu2Naf, Thr3Ceg and Gln5Aib change sidechain polarity, hydrophobicity, and potentially secondary structures. Aib is a helical inducing building block known for preforming helices (Bellanda et al., J Pept Res, 2001; Banerjee et al., J Pept Res, 2022). Since E6AP adopt a helix in the E6APZE6 binding interface, Aib residue may lower the binding entropy to improve binding as suggested by the crystal structure (PDB: 4XR8) and MD simulations. The SAR work should navigate further medicinal chemistry to optimize the molecular for therapeutical advances.

[0230] In summary, ReAct-ASMS identified E-10 endowed with 30-fold lower K crosslinking activity to E6 and higher selectivity compared with its parent peptide ELT- G9Dha, supporting that this platform can enable rapid maturation of covalent peptide inhibitors.Structural activity relationship studies to further improve binding of E-10 (hereinafter called “18”)

[0231] There is room for further structural optimization and affinity improvement. N- terminal small molecule modifications, that contains tricyclic, planar and aromatic moieties, can improve the binding affinity of E6AP -mimicking peptides (IPESS peptide). Among these moieties, fluorene showed the highest improvement. An analog campaign of fluorene reveals that N-terminal carbazole maintains the binding affinity improvement (FIG. 75). As carbazole derivatives are a common pharmacophore seen in FDA-approved drugs, i.e., Carbazomycin A, Staurosporinone, and Alectini (Singh et al., OrgLett, 2019). Carbazole was thereafter, chosen as the small molecule to modify 18.

[0232] N-term small molecule modifications improved 18’ s binding to MBP-16E6 (FIG. 76). N-term carbazole modification improved 18’s binding to MBP-16E6 by 3-fold (18-Car showed an apparent Ki of 96 ± 33 nM). Substituting the Naf2 position with Lys (Carbazole acetamido) also improved 18’s binding to MBP-16E6 (18-Naf2Cab showed an apparent K of 51 ± 11 nM). Therefore, there is room for binding improvement possible by replacing Naf to other aromatic side chains. N-term elongated peptides were previously shown to have a binding KD of 1 - 10 nM. Next, 18 was extended with the N-terminal Carbazole-IPQSA modification where 18-Car-IPQSA showed an apparent Ki of 25 ± 5.5 nM. This N-term extension improved 18’s binding to MBP-16E6 by 7-fold. The negative control derivative of 18-Car-IPQSA, (18-Car-IPESA-3L3A) showed no detectable binding to MBP-16E6 protein. Further truncation of C-term EER residues from the peptide did not affect binding where 18-Car-IPQSA-truncEER showed an apparent Ki of 44 ± 11 nM.

[0233] Crosslink assay was performed with 18 analogs (FIG. 77). 18-Car, 18-Naf2Cab, 18- Car-IPQSA and 18-Car-IPQSA-truncEER achieved quantitatively mono-crosslink in 2 hours at 37°C, 10 equiv to MBP-16E6. The negative control derivative of 18-Car-IPESA- 3L3A showed no crosslink to MBP-16E6 protein supporting 18-Car-IPESA specificities. Therefore, N-term extension and small molecular modifications can be a viable approach to improve binding affinity and binding specificity. The SAR study expands the molecular diversity of peptide 18.Design of a second-round 80 thousand-member peptide library against E6.

[0234] To further modify peptide 18, an 18 based focused Library 1-18 (Aad-Xl-X2-Leu- Aib-Aad-Cba-Leu-Cys-X3-X3-X3) was designed and composed of 80,000 members (FIGS. 78A-78B). The Naf2 positions were mutated to Naphthalene analogs. The library was designed with a focus on the C-term EER residues, which was randomized into 20 unbiased monomer sets a special ’’null” monomer. The “null” was skipped for coupling to create a set of C-term truncated peptides. Seven rounds of Re Act- ASMS were performed on MBP-16E6 with five conditions. Only conditions with more than 1 ng per member enriched binders, 12 unique hits with ALC >90 were selected using clustering analysis. Selected binders are resynthesized with Cys9Dha modification and subjected for further characterizations.

[0235] Here, ReAct-ASMS was used to overcome these two limitations of traditional non- covalent HPSEC-ASMS (FIG. IE). Since ReAct-ASMS enriches binders by covalent enrichment, it can be used to identify peptides with moderate binding affinity (XD - 0.1-10pM) because their dissociation is abrogated. All hit peptides of E6 protein had affinity over 100 nM and were not identified when a non-covalent HPSEC-ASMS was used to screen against E6. Also, ReAct-ASMS allowed library diversity to over a million since covalent crosslinking prevents peptide to dissociate from target proteins, granting detection at reduced concentrations.EXAMPLE 9 - ReAct-ASMS to identify covalent peptide inhibitors to target peptidyl- prolyl cis-trans isomerase NIMA-interacting 1 protein (Pinl)Design of a peptide library against Pinl

[0236] This novel platform was applied to maturate covalent peptide inhibitors of peptidyl- prolyl cis-trans isomerase NIMA-interacting 1 protein (Pinl), a peptidyl-prolyl isomerase encoded by PIN1 gene. Pinl is frequently overexpressed in various cancers and its inhibition was reported to reduce tumorigenic potential (Zhou et al., Nat Rev Cancer, 2016; Wulf et al., EMBO J, 2004). A selective and potent Pinl inhibitor is hence required to effectively regulate the biological function of Pinl in oncogenesis. Oxidation of the conserved Cysl 13 in the Pinl active site eliminates its isomerase activity and impairs its cellular function (Chen et al., Neurobiol Dis, 2015). This allows the development of covalent inhibitors to irreversibly inhibit Pinl by targeting its Cysl 13 (Hennig et al., Biochemistry, 1998; Campaner et al., Nat Commun, 2017).

[0237] A non-cell-permeable DPEPTIDE (Ac-Phe-Dphos.Thr-Pip-Nal-Gln-NH2) was modified into a potent and cell-permeable peptide inhibitor BJP-07-017-3 that selectively crosslinks Cysl 13 of Pinl with apparent Ki of 48 nM (FIGS. 5 A and 5B) (Pinch et al., Nat Chem Biol, 2010). Since BJP-07-017-3 is a covalent peptide inhibitor, ReAct-ASMS was applied to BJP-07-017-3 for further affinity maturation. The citrulline of BJP-07-017-3 was replaced with arginine to enhance sequencing fidelity by MS / MS (Dikler et al., J Mass Spectrom, 2997), generating P-BJP (FIG. 5B). P-BJP mono-crosslinked with Pinl protein at room temperature (RT) after 1 hour with quantitative yield (FIGS. 5C and 5D) and had an apparent Ki of 41 nM in a competitive FP assay (FIGS. 6A and 6C). P-BJP thus served as the starting peptide for affinity selection. The irreversible a -chloroacetamide warhead was replaced with the reversible disulfide cysteamine and created P-BJP-DS (FIG. 5B) to investigate if P-BJP disulfide can still crosslink with Pinl (FIG. 5C). After 12 hours incubation at RT, P-BJP-DS crosslinked with Pinl with a yield of 48% shown by protein deconvolution mass spectra, which was deemed sufficient to perform ReAct-ASMS (FIG. 5D).

[0238] Based on the reported crystal structure (PDB: 6034), all four residues in P-BJP contributed to binding. Therefore, all residues were mutated to similar analogs. Focused Library 2 (Z-Y1Y2Y3Y4) with 43,200 members was prepared, in which Z represents cysteamine disulfide warhead as P-BJP-DS (FIG. 5D). Yi represents Phe analogs containing aromatic systems with electron donating and withdrawing groups. Y2 represents Pip analogs with residues that favor or disfavor helix formation. Y3 are Trp analogs with aliphatic and aromatic sidechains. Y4 are Arg analogs with various positive charge-containing residues and hydrogen bond donors.Peptide P-12 was identified by ReAct-ASMS as the lead covalent peptide inhibitor for Pinl.

[0239] Three ReAct-ASMS conditions with different ratios of peptide libraries versus Pinl protein were applied in duplicate, wherein 28 hits were identified on average in each trial. Twenty-one hit peptides with ALC >90 were selected for structural analysis. Seventeen structurally distinct peptides were shortlisted for resynthesis, with the cysteamine warhead being replaced by a -chloroacetamide for irreversible inhibition of Pinl (FIG. 6A).

[0240] All seventeen resynthesized covalent peptides P-1 to P-17 and the parent P-BJP were combined as a mixture at 2.5 pM concentration each and mixed with Pinl protein at 2.5 pM for 1 hour at RT. Mono-crosslinked P-12-Pinl conjugate was the only species observed in LC-MS, suggesting it is the variant endowed with highest affinity against Pinl among all eighteen peptides (FIG. 6B). Indeed, P-12 (FIG. 6A) showed an apparent Ki of 2.8 nM in an FP binding assay (FIGS. 6A and 6C), 15-fold lower than the parent peptide P-BJP (apparent Ki = 41 nM). By scrutinizing apparent is of all hits in FIG. 6A generated from the competitive FP assay (FIG. 74), electron-donating groups on the benzene ring at Yi boost binding, as observed for P-5, P-6, P-10, and P-12. These observations supported a cation-pi interaction between the Phe benzene and Arg68 or Arg69 in the Pinl phosphate-binding pocket, in accord with the prior hypothesis that electron-donating groups on the benzene ring strengthen the interaction (Pinch et al., Nat Chem Biol, 2010). t Pip and Trp at Y2 and Y3 positions were also mandatory for binding, as any mutations at Y2 and Y3 significantly reduced binding affinity. Het was to some extent superior to Cit and Arg at Y4 as seen from the the Ki values obtained for P-5 versus P-10 and P6 versus P-12. Accordingly, a P-NC was synthesized as a negative control peptide with Pip mutated to DAla, Trp mutated to Naf, and Het mutated to His (FIG. 6A). The P-NC peptide showed no observable binding orcrosslinking to Pint (FIGS. 6B and 6C). Altogether, the ReAct-ASMS affinity selection platform is a general approach that can be utilized for other protein targets like Pinl for rapid maturation of covalent peptide inhibitors.

[0241] Collectively, ReAct-ASMS is established as a screening platform with tunable crosslinking chemistry, high target selectivity, and potential to access highly diverse non- canonical chemical space. Compared with previously reported non-covalent HPSEC-ASMS protocols, we showed that ReAct-ASMS tolerates larger peptide libraries, discovers peptides with both high and moderate binding affinity, and significantly increases the likelihood for peptide candidates to covalently crosslink to a specific reactive site of the target protein. The platform was successfully applied on two structurally different proteins, HP VI 6 E6 and Pinl, providing covalent peptide inhibitors with 30-fold and 15-fold improved affinity, respectively, relative to parent peptide binders for each target. The resulted peptides composed of highly distinct residues and chemical moi eties apart from the parent peptides, benefiting from large diversity of synthetic peptide library. Future work can be focused on further improve the library diversity by development of multi-stage selections or more specialized and sensitive nLC columns to bring synthetic peptide libraries up to the level of DNA-encoded library (109) or even molecular biology -based combinatorial libraries (IO10).INCORPORATION BY REFERENCE

[0242] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0243] 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 is :

1. A method for enriching one or more peptide sequences, the method comprising:(a) incubating one or more target proteins with a peptide library comprising a covalent peptide in solution under suitable conditions to form a reaction mixture comprising one or more peptide-target protein conjugates, wherein the conjugate peptide comprises a residue conjugated to a warhead, wherein the warhead is a cysteine-reactive covalent warhead, optionally a disulfide cysteamine, and wherein the target protein comprises a cysteine residue that is targeted by the warhead, wherein the covalent peptide crosslinks with the cysteine residue forming a disulfide bond with a target protein sequence, thereby forming the peptide-target protein conjugates;(b) fractionating the reaction mixture using high-pressure size exclusion chromatography (HPSEC) into a plurality of fractions;(c) isolating from the plurality of fractions, one or more fractions comprising the peptide-target protein conjugates;(d) reducing the disulfide bond in each of the peptide-target protein conjugates to form one or more isolated target peptides.

2. The method of claim 1, wherein the residue conjugated to the warhead is a cysteine.

3. The method of claim 1 or 2, wherein the warhead comprises the structure of:

4. The method of any one of the preceding claims, further comprising (e) sequencing the one or more isolated target peptides, optionally using liquid chromatography -tandem mass spectrometry (LC-MS / MS), wherein each peptide sequence is the sequence of a modulator .

5. The method of any one of the preceding claims, further comprising (f) resynthesizing the one or more isolated target peptides to enrich the one or more target peptides in solution.

6. The method of any one of the preceding claims, wherein the peptide library has diversity of up to 106.

7. The method of any one of claims 1-6, wherein each distinct member of the peptide library has a concentration of 0.1 nM or lower.

8. The method of any one of claims 1-6, wherein the one or more isolated target peptide have a binding affinity (KD) of 0.1-10 pM.

9. The method of any one of claims 1-6, wherein the one or more isolated target peptide have a binding affinity (KD) less than 10 pM.

10. The method of any one of claims 1-6, wherein the one or more isolated target peptide have a binding affinity (KD) less than 0.1 pM.

11. The method of any one of claims 1-6, wherein the one or more isolated target peptide have a binding affinity (KD) of 1-100 nM.

12. The method of any one of the preceding claims, further comprising (g) validating binding of the one or more isolated target peptides to the target protein using an assay.

13. The method of any one of the preceding claims, wherein the peptide-target protein conjugates are reduced using a dithiothreitol (DTT) treatment.

14. The method of any one of claims 1-12, wherein the peptide-target protein conjugates are reduced using a tris(2-carboxyethyl)phosphine (TCEP) treatment.

15. The method of any one of the preceding claims, wherein the peptide library comprises a plurality of peptides comprising non canonical amino acid residues.

16. The method of any one of the preceding claims, wherein the peptide library comprises a peptide having the amino acid sequence of X1X2X3X4X5X6X7X8CEER, wherein Xi comprises a Glu analog, X2 comprises a Leu analog, X3 comprises a Thr analog, X4 comprises a Leu analog, X5 comprises a Gin analog, Xe comprises a Glu analog, X7 comprises a Leu analog, and Xs comprises a Leu analog.

17. The method of claim 16, wherein Xi is independently selected from the group consisting of: Glu, Aad, and Cya.

18. The method of claim 16 or 17, wherein X2 is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

19. The method of any one of claims 16-18, wherein X3 is independently selected from the group consisting of: Thr, Cpg, Cbg, Ceg, Hyp, and Asn.

20. The method of any one of claims 16-19, wherein X4 is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

21. The method of any one of claims 16-20, wherein X5 is selected from the group consisting of: Gin, Gly, oAla, and Aib.

22. The method of any one of claims 16-21, wherein Xe is independently selected from the group consisting of: Glu, Aad, and Cya.

23. The method of any one of claims 16-22, wherein X?is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

24. The method of any one of claims 16-23, wherein Xs is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

25. The method of any one of claims 16-24 wherein the peptide comprises the amino acid sequence of ELTLQELLCEER.

26. The method of any one of claims 16-25, wherein the warhead is conjugated to the peptide via the C residue.

27. The method of claim 26, wherein the warhead comprises the structure of:

28. The method of any one of claims 16-27, wherein the one or more target proteins comprises HPV 16E6 or MBP-16E6 4C4S.

29. The method of any one of claims 16-28, wherein the peptide library comprises a peptide comprising the structure of:wherein Yi comprises a Phe analog, Y2 comprises Pip analog, Y3 comprises a Trp analog, and Y4 comprises an Arg analog.

30. The method of claim 29, wherein Yi is selected from the group consisting of: Phe, Pya, Cnf, Eaf, F2f, Dmf, Amf, Nof, Phg, Hof, Qua, Naf, Phf, Mpf, Trp, and Cl-Trp.

31. The method of claim 29 or 30, wherein Y2 is selected from the group consisting of: Pip, Ach, Tic, Chg, Ceg, Cpg, Ala, Amb, Hyp, 4Fp, Nip, Aib, Gly, Thz, and Nib.

32. The method of any one of claims 29-31, wherein Y3 is selected from the group consisting of: Trp, Cl-Trp, Qua, Phf, Mpf, Naf, Nva, Nle, Aoc, Cba, Cpea, and Cha.

33. The method of any one of claims 29-32, wherein YHs selected from the group consisting of: Arg, Hor, Hoq, Dmr, Dab, Dap, Om, Lys, Tml, Cit, Het, Amf, Gnf, Eaf, and His.

34. The method of any one of claims 29-33, wherein the target protein is a Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pint).

35. A synthetic peptide comprising the amino acid sequence of X1X2X3X4X5X6X7X8CEER, wherein Xi comprises a Glu analog, X2 comprises a Leu analog, X3 comprises a Thr analog, X4 comprises a Leu analog, X5 comprises a Gin analog, Xe comprises a Glu analog, X7 comprises a Leu analog, and Xs comprises a Leu analog.

36. The synthetic peptide of claim 35, wherein each Xi is independently selected from the group consisting of: Glu, Aad, and Cya..

37. The synthetic peptide of claim 35 or 36, wherein each X2 is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

38. The synthetic peptide of any one of claims 35-37, wherein X3 is selected from the group consisting of: Thr, Cpg, Cbg, Ceg, Hyp, and Asn.

39. The method of any one of claims 35-38, wherein X4 is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

40. The method of any one of claims 35-39, wherein X5 is selected from the group consisting of: Gin, Gly, oAla, and Aib.

41. The method of any one of claims 35-40, wherein Xe is independently selected from the group consisting of: Glu, Aad, and Cya.

42. The method of any one of claims 35-41, wherein X?is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

43. The method of any one of claims 35-42, wherein Xs is independently selected from the group consisting of: Leu, Nva, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf.

44. The synthetic peptide of any one of claims 35-43, wherein the synthetic peptide comprises the amino acid sequence of ELTLQELLCEER.

45. The synthetic peptide of any one of claims 35-44, wherein the synthetic peptide comprises the amino acid sequence of Aad-Naf-Ceg-Leu-Aib-Glu-Cba-Leu-CEER46. The synthetic peptide of any one of claims 35-45, wherein a reversible cysteine- targeted warhead is conjugated to the synthetic peptide via the C residue.

47. The synthetic peptide of claim 46, wherein the reversible cysteine-targeted warhead has the structure of:

48. The synthetic peptide of any one of claims 35-47, wherein the C residue is replaced with dehydroalanine (Dha).

49. The synthetic peptide of any one of claims 35-45, wherein the C residue is replaced with an irreversible cysteine-reactive covalent warhead.

50. The synthetic peptide of any one of claims 35-45, wherein the C residue is conjugated to an irreversible cysteine-reactive covalent warhead.

51. A synthetic peptide comprising the structure of:wherein R is absent, or comprises 1-40 amino acid residues, wherein Yi comprises a Phe analog, Y2 comprises Pip analog, Y3 comprises a Trp analog, and Y4 comprises an Arg analog.

52. The synthetic peptide of claim 51, wherein Yi is selected from the group consisting of: Phe, Pya, Cnf, Eaf, F2f, Dmf, Amf, Nof, Phg, Hof, Qua, Naf, Phf, Mpf, Trp, and Cl-Trp.

53. The synthetic peptide of claim 51 or 52, wherein Y2 is selected from the group consisting of: Pip, Ach, Tic, Chg, Ceg, Cpg, Ala, Amb, Hyp, 4Fp, Nip, Aib, Gly, Thz, and Nib.

54. The synthetic peptide of any one of claims 51-53, wherein Y3 is selected from the group consisting of: Trp, Cl-Trp, Qua, Phf, Mpf, Naf, Nva, Nle, Aoc, Cba, Cpea, and Cha.

55. The synthetic peptide of any one of claims 51-54, wherein YHs selected from the group consisting of: Arg, Hor, Hoq, Dmr, Dab, Dap, Om, Lys, Tml, Cit, Het, Amf, Gnf, Eaf, and His.

56. The synthetic peptide of any one of claims 51-55, wherein Yi is Dmf, Y2 is Pip, Y3 is Trp, and Y4 is Het.

57. A synthetic peptide comprising an amino acid sequence having the structure of:

58. A composition comprising the synthetic peptide of any one of claims 35-57.

59. A pharmaceutical composition comprising the synthetic peptide of any one of claims 35-57 and a pharmaceutically acceptable salt or carrier.

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