Compositions and methods for peptide-based modulators of human papillomavirus oncoprotein e6

Abstract

This disclosure relates to synthetic peptides of E6AP and complexes of synthetic peptides with HPV E16, methods of making such peptides and complexes, and methods of using such peptides and complexes for blocking, inhibiting, or preventing the interaction of E6AP with HPV E16. The synthetic peptides and complexes of synthetic peptides with HPV E16 can further comprise warheads to introduce covalent linkages between the synthetic peptides with HPV E16.

Description

Attorney Docket No.: CLS-035WO PATENT COMPOSITIONS AND METHODS FOR PEPTIDE-BASED MODULATORS OF HUMAN PAPILLOMAVIRUS ONCOPROTEIN E6 CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 502,313, filed on May 15, 2023, the entire contents of which are incorporated by reference herein for all purposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file copy, created on XXXX, is named XXX and is XXX bytes in size. BACKGROUND

[0003] Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, the fourth most common cancer in women worldwide. High-risk forms of HPV are causative in multiple cancers, including cervical, vaginal, oropharyngeal, and potentially a subset of prostate cancers. Despite the introduction of HPV-directed vaccines over ten years ago, the lack of their widespread distribution, uptake by the general population, and availability continues to make HPV positive (HPV+) cancers prevalent. Among ~200 identified HPV strains, high-risk HPV16 and HPV18 are responsible for ~75% of HPV-associated cervical cancers. Current treatment strategies for HPV cancers include radiation therapy, surgery, chemotherapy, monoclonal antibody(mAb), and checkpoint blockade. There are no currently approved targeted therapeutics against HPV. SUMMARY

[0004] In one aspect, the disclosure provides for a synthetic peptide comprising an amino acid sequence IPESSELTLQELLGEER (SEQ ID NO: 1) or ELTLQELLGEER (SEQ ID NO: 5). In some embodiments, the N- or C- terminus of the peptide is modified with a chemical moiety. 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-1-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-pyrenebutylAttorney Docket No.: CLS-035WO PATENT carboxamido, 5-Acenaphthene carboxamide, 7-Methoxycoumarin-4-acetamido, 4-phenyl- phenylalanine, and Cyclohexyl-alanine. 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-Indanecarboxamido, 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, triphenylacetamido, 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-1-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. 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. In some embodiments, the peptide comprises Fluorene-IPESSELTLQELLGEERRAA-K(1-Anthracene (SEQ ID NO:2) or Fluorene-IPESAELTLQELLGEERRAA-K(1-Anthracene (SEQ ID NO:3).

[0005] In another aspect, the disclosure provides for a synthetic peptide modified with a warhead, and the peptide is selected from TABLE 2C, TABLE 2D, or TABLE 2E. In some embodiments, the warhead is at position 9 with respect to SEQ ID NO: 3. In some embodiments, the warhead is selected from the group of phenylacrylamide (Ph-acr), Dap-acrylamide (dap-acr), Dab-acrylamide (dab-acr), Dap-propiolamide (dap-ppa), and dehydroalanine Dha. In some embodiments, the peptide comprises Fluorene- IPQSAELTLQELL(DHA)RRKKK(Anthracene) (SEQ ID NO: 4). In some embodiments, the warhead is selected from the group of phenyl fluorosulfate (FS), phenyl sulfonyl fluoride(SF), phenyl Carbamate (p-PhC), phenyl Carbamate (m-PhC), and disulfide (DS).

[0006] In another aspect, the disclosure provides for a synthetic peptide comprising an amino acid sequence X1X2X3X2QX1X2X2CEER (SEQ ID NO: 6), wherein X1 is Nva, Leu, Aoc, Cpa, Cba, Cha, Phg, Hof, or Naf; X2 is Aad, Glu, or Cya; X3 is Thr, Asn, Hyp, Cpg, Cbg, or Ceg; and X4 is d-Ala, Aib, Gly, or Gln. In some embodiments, the peptide is selected fromAttorney Docket No.: CLS-035WO PATENT TABLES 3-31. In some embodiments, the peptide is selected from SEQ ID NO: 7-19. In some embodiments, the peptide comprises SEQ ID NO: 17.

[0007] In another aspect, the disclosure provides for a synthetic peptide comprising an amino acid sequence Aad-X1-X2-Leu-Aib-Aad-Cba-Leu-Cys-X3-X3-X3 (SEQ ID NO: 20) wherein X1 is PhF, Naf, Qua, Clw, or Trp; X2 is Chg or Ceg; and X3 is d-Ala, Aib, Gly, Ser, Aad, Glu, Gln, Hoc, Val, Leu, Met, Pro, Dap, Lys, Arg, His, Trp, Phe, Tyr, or Null. In some embodiments, the peptide is 11 or 12 amino acids long. In some embodiments, the N-terminus of the peptide is further modified with a modifier selected from 2-Phenyl-4-quinolinecarboxylic acid, 1- (Phenylsulfonyl)-1H-indole-2-carboxylic acid, 6-Fluoro-2-naphthoic acid, 4-phthalimidobenzoic acid, Xanthene 9-carboxylic acid, 1-Pyrenebutyric acid, 5-Acenaphthenecarboxylic acid, 1- Phenyl-1H-indole-2-carboxylic acid, Indole-2-carboxlic acid, quinoline-4-carboxylic acid, and 10-(Carboxymethyl)-9(10H)acridone. In some embodiments, the peptide is selected from TABLES 32-39.

[0008] In another aspect, the disclosure provides for a synthetic peptide comprising the formulaIn some embodiments, the peptide further comprises a warhead. In some embodiments, the warhead is selected from the group of phenylacrylamide (Ph-acr), Dap-acrylamide (dap-acr), Dab-acrylamide (dab-acr), Dap-propiolamide (dap-ppa), and dehydroalanine Dha.

[0009] In some embodiments, the synthetic peptide is an HPV16E6 / E6AP specific inhibitor.

[0010] In one aspect, the disclosure provides for a synthetic peptide disclosed herein, comprising one or more additional modifications selected from: acetylated, formylated, propanoylated, hexanoylated, or myristoylated N-terminus; amidated C-terminus; substitution of one or more L- amino acid with a D-amino acid; substitution of one or more amino acid with a methyl-amino acid; and substitution of an Į-amino acid with a ȕ-amino acids.

[0011] In one aspect, the disclosure provides for a synthetic peptide / HPV16E6 complex, wherein the peptide is selected from any one of the synthetic peptides disclosed herein. In some embodiments, the synthetic peptide and the HPV16E6 in the complex are covalently linked. InAttorney Docket No.: CLS-035WO PATENT some embodiments, the synthetic peptide is covalently linked to amino acid residue Cys-58, Arg-84, or Arg-13 of HPV16E6. In some embodiments, the peptide / HPV16E6 complex disclosed herein the complex is inhibited in binding of human E6AP.

[0012] In some embodiments, a pharmaceutical composition comprising the synthetic peptide described herein and a pharmaceutically acceptable salt or carrier is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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:

[0014] FIGS.1A-1F depict rational design of E6AP-mimicking peptides. (FIG.1A) Left: X-ray crystal structure of the ternary protein complex formed by 16E6 (“E6”), MBP fused to the E6AP LXXLL peptide (dark grey), 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.1B) Rational design of high-affinity 16E6 binding peptide. The N-terminal and C-terminal lysine side chain primary amines were modified with various small molecules listed in FIG.1C. (FIG.1C) Chemical library of small molecules used for modifying E6AP-based peptides.9-Fluorenyl, 1-fluorenyl, 9-oxofluoren-3-yl, 9-oxofluoren-4-yl, 9- oxofluoren-1-yl, 2-anthraquinonyl, 9-xanthyl, 1-anthracenyl, 2-anthracenyl, 1- naphthyl, 6- Hydroxynaphthyl, 1-pyrenebutyl, 5-acenaphthyl, and Fluorescein are the planar aromatic residues; Adamantyl, Biphenyl, Cyclohexyl, and Norborenyl are the hydrophobic residues; and the remaining indicated are other aromatics. (FIG.1D) Table of N- and C-terminal modified E6AP peptide mimics. The binding dissociation constant KDis measured by bio-layer interferometry (BLI) competition assay, except for *KDmeasured by direct BLI. (FIG.1E) BLI competition assay was used to measure the binding of 6’ and 6’-3L3A. Streptavidin tips were immobilized with 1-biotin and dipped into a solution of various concentration of analyte and 16E6. (FIG.1F) Direct BLI measurement of 6’-biotin, parameters are estimated to be: kon = 1.5E+05 Mí1sí1, koff = 4.3E-03sí1, and KD = 3.0 ± 1.8 nM.

[0015] FIGS.2A-2C depict the determination of peptide 1 and 16E6 binding constant by direct binding assay in BLI and competition BLI binding assay. (FIG.2A) Structure of peptide 1 and peptide 1-biotin (FIG.2B) BLI measurement of 1-biotin and 16E6, parameters are estimated to be: kon = 40200 M-1s-1, koff = 0.0851 s-1and KD = 2.1± 0.1 μM (N=3). Indicated MBP-16E6Attorney Docket No.: CLS-035WO PATENT protein concentration was used in each channel on the right. kon = on rate constant, koff = off rate constant, KD= kinetic apparent dissociation constant. (FIG.2C) Peptide 1 binding was assessed by competition binding assay. Various concentrations of peptide 1 were mixed with 30nM 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 0.1nM). The dissociation binding constant was estimated to be KD= 2.3±0.5 μM. Error is the curve fitting standard error of the mean (SEM) reported by the Prism 8 software (N=3).

[0016] FIG.3 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

[0017] FIGS.4A and 4B show structure-activity-relationship(SAR) study of small molecule modifications. (FIG.4A) N-terminal modifications SAR. Į: modification is not sensitive to ring substitution position. ȕ: tricyclic, planar, and aromatic molecules improve the affinity to the low nano-molar range. *: Removing a phenyl ring from the structure or breaking the strain between two phenyl moieties will decrease the binding by 30-50 folds. (FIG.4B) C-terminal modifications SAR. Į: binding improvement of anthracene modification is not sensitive to ring substitution position. Į: two phenyl rings are required for a mid-micromolar binding. *: Removing a phenyl ring from the structure, breaking the strain between two phenyl moieties, will decrease the binding by 30-50 folds. KDvalues are measured by BLI binding assay in a competition mode.

[0018] FIG.5A shows the structure of peptide 6’-biotin (6’-biotin).

[0019] FIG.5B shows a direct BLI binding assay of 6’-biotin against SUMO-MDM2.

[0020] FIG.5C shows a direct BLI binding assay of peptide 6’-biotin against THRA.

[0021] FIG.6A shows the analysis of the main binding interface of 16E6 and E6AP. Cys58 of 16E6. Residues of E6AP were colored based on their measured reactivity towards Cys58 of 16E6: Gly9, Glu10, Glu11, and Arg12.

[0022] FIG.6B shows structures of electrophiles.

[0023] FIG.6C shows a scheme of the bind-and-react strategy. Structure of peptide E3 containing a Dha highlighted in grey.Attorney Docket No.: CLS-035WO PATENT

[0024] FIG.6D shows the apparent kinetic constant kapp calculated from the time-course of protein depletion assay, where 50nM MBP-16E6 was incubated with different concentrations of E3.

[0025] FIG.6E shows a series value of Kapp that was plotted against the corresponding E3 concentration to estimate kinact and Ki.

[0026] FIG.7A shows cross-linking experiments between MBP-E6 and peptides E0–E11. All reactions were performed using 1X PBS, pH = 7.4, as the buffer. Conditions of crosslinking experiments were listed. Cross-link yield was interpolated using LC-MS protein deconvolution mass spectrum. The percentage was obtained by dividing the peak area of cross-linked protein by the sum of uncross-linked and cross-linked protein peak area (N = 2).

[0027] FIG.7B shows the structure of electrophiles.

[0028] FIG.8 shows as sequence table and results of a binding experiments of E6AP-mimicking peptides (peptides 7-13). Apparent Ki is determined by BLI. Peptides were in competition with immobilized 1-Biotin following a 30 min incubation with 16E6. Dha: dehydroalanine.

[0029] FIGS.9A-9E show examples of affinity matured peptide for improved reactivity. FIG. 9A shows the main binding interface of 16E6 and E6AP. Cys58 of 16E6 is targeted by an electrophile substituted at Gly14. FIG.9B shows sequence table of E6AP-mimicking peptide 13 and peptide 13-3L3A (negative control peptide). Apparent Ki is determined by BLI. n.b. (non- binding). Peptides were in competition with immobilized 1-Biotin following a 30 min. incubation with 16E6. Dha: dehydroalanine. Structure of peptide 13, Dha, tri-leucine. FIG.9C shows the results of a BLI competition assay measurement of 13 and 13-3L3A estimated apparent Ki in unit of nM. FIG.9D shows the apparent kinetic constant kapp calculated from the time-course of protein depletion assay, where 10nM MBP-16E6 was incubated with different concentration of 13. FIG.9E shows a series value of Kapp was plotted against the corresponding peptide 13 concentration to estimate kinactand Ki.

[0030] FIGS.10A-10E show crosslinking of reactive peptides to 16E6. FIG.10A shows a crosslinking reaction of MBP-16E6 (1 μM) and reactide 13 (3 μM). Quantitative mono- crosslinked MBP-16E6-13 was observed at 2 h. (FIG.10B) Crosslinking of MBP-16E6 C58S (1 μM) and peptide 13 (3 μM), no appreciable amount of reaction product was observed after 12 h. FIG.10C shows crosslinking of MBP-16E6 (1 μM) and peptide 13-3L3A (3 μM), no appreciable amount of reaction product was observed after 12 h. FIG.10D shows protein- selective intermolecular crosslinking of MBP-16E6 in a protein mixture. Only 16E6 wasAttorney Docket No.: CLS-035WO PATENT modified, as indicated by the LC-MS analysis. FIG.10E shows a BLI assay determined 16E6 / E6AP complex formation.1 μM of Biotin-E6AP protein was immobilized onto streptavidin tip and dipped into 1 μM of MBP-16E6 only, MBP-16E6 mixed with 1 μM of 13 or 1 μM of 13-3L3A.

[0031] FIG.11 shows the control peptide 13-3L3A crosslink to THRA. Crosslink reaction of 1 μM THRA (PDB: 1nav) and 10 μM 13-3L3A, at 37°C for 12h. PDB codes of structures used: 1nav.

[0032] FIG.12A shows the structure of 13-biotin

[0033] FIG.12B shows a protein deconvolution mass spec of MBP-16E6 conjugates with peptide 13-biotin (Molecular Weight: 3231.90).

[0034] FIG.12C shows a 13-biotin direct KD is determined by BLI assay. koff= 6.79E-05 s-1, kon= 5.31E+04 M-1s-1, apparent direct KD= 1.3 ± 0.6 nM (N = 3).

[0035] FIG.12D shows a 13-biotin association and dissociation to THRA as determined by direct BLI assay (N=1).

[0036] FIG.12E shows a 13-biotin association and dissociation to MDM2 as determined by direct BLI assay (N = 1).

[0037] FIG.12F shows the structure of 13-3L3A-biotin

[0038] FIG.12G shows no observable crosslink between MBP-16E6 and 13-3L3A-biotin. Protein deconvolution mass spec of 1 μM MBP-16E6 that were treated with 20 μM 13-3L3A- biotin (Molecular Weight: 3103.69 ) for 12 h in 1x PBS at 37°C. Mass-spec was recorded using a 6550 Agilent Q-TOF coupled to Infinite II 1290 LC system, with an Aeries C4 column (Phenomenex) using 1 to 61 B% linear ramping gradients.

[0039] FIG.12H shows 13-3L3A-biotin showing no observable association to 1 μM and 0.5 μM MBP-16E6 as determined by direct BLI assay, where 13-3L3A-biotin was immobilized onto the streptavidin tips and dipped into MBP-16E6 solutions.

[0040] FIGS.13A-13F show molecular modeling of the16E6-13 conjugate. (FIG.13A) Structural representation of 16E6 (white) bound to peptide 13 (dark grey) featuring the Dha warhead (black), obtained through ~120 ns MD simulation. Structure represents the averaged configurations of 13 within calculated clusters (see methods). (FIG.13B) Trajectory analysis of root mean squared differences (r.m.s.d) during MD simulatioms, showing stability and modest conformational changes of the complex. FIG.13C shows a comparison of molecular modelingAttorney Docket No.: CLS-035WO PATENT of the 13:16E6 complex with the X-ray structure of 16E6 (coral)-E6AP LXXLL peptide (yellow), p53 (plum) complex (PDB ID: 4XR8). All r.m.s.d. were calculated for the Cα atoms. All structures were compared to the initial struture. FIG.13D shows intermolecular interactions between 16E6 (white) and 13 (blue), identified and analyzed from the MD simulation. Interactions shown in FIG.13D are persistent during the ~120 ns MD simulation. Dotted lines represent hydrogen bonding. (FIG.13E) Covered surfaces by the N term [within 5Å]: Patch1 in black [V38,Y39,C40,K41, R62,E63], Patch2 in yellow [C73,F76,Y77,I80, Y83,R84,H85,R136]. Left: E6 and 13 N-term detailed interactions. FIG.13F shows covered surfaces by the Cterm [within 5Å]: Patch1 [M8,F9,Q10, D11, P12, Q13, E14, R15, P16, R17, K18,L19, P20,Q21,D25]. Patch2 in yellow [A53,D56,R55,L57, C58*]. Patch3 in purple [Y99, K101, D105,L107, I108, R109, C110, C113,Q114,K115, P116,L117, R136,R138,W139,T140]. Left: E6 and C-term 13 detailed interactions.

[0041] FIGS.14A-14B show an illustration of the native E6AP peptide. (FIG.14A) E6AP peptide bound to 16E6 protein, surrounded by ~6K water molecules (lines) and ions (spheres). (FIG.14B) Comparison of X-ray structure of 16E6 (light grey, top) bound to E6AP (light grey, bottom) (PDB ID: 4XR8) with relaxed 16E6 (black / dark grey, top)-E6AP peptide (dark grey, bottom) complex after 1.1 μs MD simulation. P53 and MBP were omitted for clarity. The r.m.s.d. was calculated for the Cα atoms.

[0042] FIGS.15A-15D show an illustration of structural modeling of 16E6-bound Peptide-13. (FIG.15A) Structure of Peptide-13. (FIG.15B) E6AP native core adopts an alpha helical conformation in Peptide-13 structural model obtained by computational conformational sampling. (FIG.15C) molecular docking of Peptide-13 to 16E6 protein. (FIG.15D) Molecular modeling of 16E6-Peptide-13 complex, featuring the covalent link between Peptide-13 warhead (Dha) and the 16E6 hotspot (Cys58).

[0043] FIG.16 shows an illustration of molecular docking of the native E6AP peptide in the 16E6 (left). The MOE molecular docking procedure accurately describes the interactions between 16E6 and E6AP native peptide (right).

[0044] FIG.17 shows an illustration of molecular modeling of 16E6-Peptide-13 complexes. The structures are ordered from left to right based on: the largest binding affinity between the peptide- 13 core sequence and 16E6 and the lowest r.m.s.d of the peptide-13 core sequence compared to the crystallographic native E6AP peptide.

[0045] FIG.18 shows Reactide 13 stability in media stability assay. Ten μM peptide was incubated in RPMI with 10% FBS at 37°C for the indicated time. Proteins were removed fromAttorney Docket No.: CLS-035WO PATENT the mixture and analyzed by LC-MS. Percentage of the remaining peptide was estimated by dividing the area under extract ion chromatogram (EIC) to the area of time zero (N = 3).

[0046] FIGS.19A-B show protein sequence alignment of high-risk type HPV E6 using CLUSTAL O (1.2.4) sequence alignment tool. The alignment includes for the following high- risk subtypes: 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. For example, FIG.19B shows the protein sequence alignment of HPV16E6 and HPV18 E6. In the conserved regions, HPV16E6 (48-64) and HPV18E6 (43-59), the Phe53 of HPV18 E6 aligns with the Cys58 of HPV16 E6. This Phe52 residue made HPV18E6 insensitive to cysteine reactive bind-and-react inhibition strategy.

[0047] FIG.20 shows the structures of various peptides as indicated.

[0048] FIG.21 left panel shows direct BLI measurement of Biotin-PEG4-ELTLQELLGEER. Biotin ligand was immobilized onto the sensor tip. BLI was recorded after incubating various concentrations of MBP 16E6. Right panel shows FP binding assay of FITC-ELTLQELLGEER. FP was recorded after incubating various concentrations of MBP 16E6 with 100nM FITC- ELTLQELLGEER. FP was performed with a BioTek H1 plate reader. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50. Temperature 27ÛC.

[0049] FIG.22 shows the structure of the E3 peptide (ELT-G9DHA) and BLI competition assay, and FP competition assay. The 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. N=2, competition curves are shown. IC50 is measured by competition FP assay. Various concentrations of analyte peptide were mixed with 100nM FITC-IPESSELTLQELLGEER and 450nM MBP-16E6 solution, incubated for 30 min. Polarization was recorded using BioTek H1. N=2 biological replicates, competition curves are shown. FP was performed with a BioTek H1 plate reader. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50. Temperature 27ÛC

[0050] FIG.23 shows the D-alanine scanning of 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.

[0051] FIG.24 shows the E6 Library I design with a library size of 1.42 M.Attorney Docket No.: CLS-035WO PATENT

[0052] FIG.25A shows an illustration of ReAct-ASMS platform. (FIG.25B) Mix disulfide peptides with different R group showcased different crosslink rate. Right: time-course of crosslink reaction between reactides and MBP 16E6 protein, monitored by LC-MS. At 6 h, cysteamine (βME) showed a crosslink yield of 76%. (FIG.25C) A scheme of the bind-and-react reaction and dissociating peptide-protein conjugates. Ki denotes the binding constant. Kinact denotes first-order rate. Peptides were dissociated from protein by DTT and subsequently sequenced by an nLC-MSMS. Right: Mass-spec analysis of dissociating peptide-protein conjugates.

[0053] FIG.26A 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. IC50 was determined by FP. Peptides were in competition with 1-FITC following a 30 min incubation with 16E6.

[0054] FIG.26B shows competitive crosslink assay. MBP-16E6 (1 μM) and Dha peptide mixture (12 μM), mono-crosslinked MBP-16E6-18 was observed at 2 h with a yield of 83%.

[0055] FIG.27A illustrates the structure of peptides 18 and 23, dehydroalanine and unnatural amino acids (highlighted). Apparent Kiis determined by BLI. (FIGS.27A and 27C) Peptides were in competition with immobilized 1-Biotin following a 30 min incubation with 16E6. IC50was determined by FP. (FIGS.27B and 27D) Peptides were in competition with 1-FITC following a 30 min incubation with 16E6.

[0056] FIG.27B shows the structure of peptides 14, 18 and 23, dehydroalanine and unnatural amino acids (highlighted).14: apparent Kiof 1.3± 0.4 μM. IC50of 766 ± 180 nM.18: apparent Kiof 177 ± 29 nM, IC50of 330 ± 55 nM.23: apparent Kiof 179± 33 nM, IC50of 389 ± 109 nM. Apparent Kiis 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 or 100nM MBP-16E6. N=2, competition curves are shown. IC50is measured by competition FP assay. Various concentration of analyte was mixed with 100nM FITC-IPESSELTLQELLGEER and 450nM MBP-16E6 solution, incubated for 30min. FP was performed with a BioTek H1 plate reader. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50. Temperature 27ÛC.

[0057] FIG.27C shows graphs depicting BLI competition assays of peptide ELTLQELL(Dha)EER, peptide 14, peptide 23, peptide 18, and peptide 18-3L3A.Attorney Docket No.: CLS-035WO PATENT

[0058] FIG.27D shows graphs depicting FP competition assays of peptide ELTLQELL(Dha)EER, peptide 14, peptide 23, peptide 18, and peptide 18-3L3A (order from top to bottom).

[0059] FIG.28A shows a crosslinking reaction of MBP-16E6 (1 μM) and reactide 18 (10 μM). Quantitative mono-crosslinked MBP-16E6-18 was observed at 4 h.

[0060] FIG.28B shows crosslinking of MBP-16E6 (1 μM) and peptide 18-3L3A (10 μM).

[0061] FIG.28C shows crosslinking of MBP-16E6 C58S (1 μM) and peptide 18 (10 μM).

[0062] FIG.28D shows crosslinking of MBP-16E6 L57A (1 μM) and peptide 18 (10 μM).

[0063] FIG.28E shows apparent kinetic constant kapp was calculated from the time-course of protein depletion assay, where 25 nM MBP-16E6 was incubated with different concentration of 18.

[0064] FIG.28F shows a series value of Kapp was plotted against the corresponding reactide 18 concentration to estimate kinact and Ki.of 0.018 s-1, Ki value of 792 nM and kinact / KI ratio of 22.7 mM-1s-1.

[0065] FIGS.29A-29B show a small molecule library of IPESS peptides. KDis 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 MBP-16E6. (N=2).

[0066] FIG.30 shows structures of exemplary peptide 18 analogs. Apparent Kiis 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 100 nM MBP-16E6 in the solution (n=2). Competition curves are shown.

[0067] FIG.31A shows the structure of reactide 18’s analogs.

[0068] FIG.31B shows crosslink assay: MBP-16E6 (1 μM) and Dha peptide (4 μM), 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).

[0069] FIG.31C shows the IC50is measured by competition FP assay. Various concentration of analyte peptide was mixed with 100nM FITC-IPESSELTLQELLGEER and 450nM MBP-16E6 solution, incubated for 30 min. FP was performed with a BioTek H1 plate reader. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50. Temperature 27ÛC.Attorney Docket No.: CLS-035WO PATENT

[0070] FIG.32 shows E6 Library II design. Above: library II structure. Below: monomer structures in randomized position 2, 3, 10, 11, and 12.

[0071] FIGS.33A-33D show a design of targeting R84 on HPV16 E6 from the position of A6 in the IPQSA reactide and targeting R138 from the position of R16.

[0072] FIGS.33E-33F show a competition experiment for Pep1 and Pep4.

[0073] FIG.33G shows the structures of Pep1 and Pep4.

[0074] FIGS.34A and 34B show cell toxicity assays towards HPV16 E6 positive cell lines of reactide 13 in SiHa and CaSki cells and control cell lines lacking 16E6 expression: HeLa and c- 33a. At 96 hr, cell viability was estimated using MTT assay. Concentration of the compounds was 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.4μM, and 0.08μM.

[0075] FIG.35 shows p53 protein levels in CaSki cells treated with reactide 13.

[0076] FIGS.36A and 36B shows the results from LC-MS and MALS and gel staining for E E6AP, MBP-16E6 (4C4S), and MBP-16E6 (4C4S C58S).

[0077] FIGS.37A-37D show crosslinking of 13-TAMRA to MBP-16E6 (4C4S) and MBP-16E6 C58S (4C4S) in HPV-negative HT1080 lysate. HT1080 lysates (50 μg) were incubated overnight at 4°C with 2 μM each of MBP-16E6 WT or C58S (each 4C4S), and 13-TAMRA (10 μM). The mixtures were then separated by SDS-PAGE and transferred onto nitrocellulose. Fluorescence scans of the protein gel provided a visualization of both the free 13-TAMRA and 13-TAMRA crosslinked to WT MBP-16E6 with (FIG.37A) short and (FIG.37B) long exposure times. Additional minor fluorescent signals from 13-TAMRA crosslinked to unidentified proteins in the lysate at FIG.37B. (FIG.37C) Ponceau S staining of the nitrocellulose membrane after transfer displayed the combination of MBP-16E6 (either WT or C58S), 13-TAMRA, and HT1080 lysate. (FIG.37D) The Western blot for MBP-16E6 and GAPDH, alongside a 488 nm fluorescence scan for 13-TAMRA, showed selective binding of 13-TAMRA to MBP-16E6 WT but not C58S.

[0078] FIGS.38A-38D show crosslinking of 13-biotin to MBP-16E6 (4C4S) and MBP-16E6 C58S (4C4S) in HPV-negative HT1080 lysate. HT1080 lysates (50 μg) were incubated overnight at 4°C with 2 μM each of MBP-16E6 WT or C58S (each 4C4S), and 13-biotin (10 μM) or 13-3L3A-biotin (10 μM). The mixtures were then separated by SDS-PAGE and transferred onto nitrocellulose. (FIG.38A) Ponceau staining of the nitrocellulose membrane provides an overview of the distribution of MBP-16E6 (WT or C58S), 13-biotin or 13-3L3A- biotin, and HT1080 lysate. (FIG.38B) Western blot analysis of MBP-16E6 and GAPDH, detailing antibody interactions. The same blot was subsequently incubated with streptavidin-680,Attorney Docket No.: CLS-035WO PATENT enabling the visualization of 13-biotin and 13-3L3A-biotin under (FIG.38C) short and (FIG. 38D) long exposures. The interaction between MBP-16E6 and 13-biotin remains observable. Additional bands indicate at 10 μM, binding of 13-biotin and 13-3L3A-biotin to unidentified proteins present in the HT1080 lysate.

[0079] FIGS.39A-39G show streptavidin pull-down analysis of 13-biotin, 13-3L3A-biotin, or control (beads alone with DMSO) using quantitative multiplexed mass spectrometry. HPV- HT1080 or HPV16+ CaSki cell lysates (2 mg) were treated with 13-biotin (5 μM), 13-3L3A- biotin (5 μM), or DMSO (0.5%) overnight at 4°C and subjected to pull-down using streptavidin beads. The samples were then TMT labeled, digested with trypsin, and analyzed by mass spectrometry. (FIG.39A) Heat map showing the relative enrichment in CaSki cells treated with 13-biotin relative to those treated with 13- 3L3A-biotin. Volcano plot comparisons for HT1080 cells are shown for (FIG.39B) 13-3L3A-biotin vs.13-biotin, (FIG.39C) DMSO vs.13-biotin, and (FIG.39D) DMSO vs.13-3L3A-biotin. Volcano plot comparisons for CaSki cells are presented for (FIG.39E) 13-3L3A-biotin vs.13-biotin, (FIG.39F) DMSO vs.13-biotin, and (FIG.39G) DMSO vs.13-3L3A-biotin. The data represent results from two independent experiments. The top five proteins with the highest -Log10(q-value) are highlighted. DETAILED DESCRIPTION

[0080] The present disclosure is based, in part, upon the development of synthetic peptides that bind Human Papilloma Virus 16 E early protein 6 (HPV16E6 or 16E6) in a covalent or non covalent manner to form peptide-16E6 complexes. Additionally, the peptide-16E6 complexes can modulate the function of p53 that promotes ubiquitination. The synthetic peptides and peptidomimetics can be used to modulate or abrogate HPV16E6 / p53 binding and signaling in HPV 16 positive cancer cells.

[0081] Various components and aspects of the disclosure are described in further detail in the subsections below. I. Definitions

[0082] 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 areAttorney Docket No.: CLS-035WO PATENT 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.

[0083] 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.

[0084] 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.

[0085] Further, it should be understood that elements and / or features of a composition or a method provided and described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure and invention(s) herein, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of invention(s) provided, described, and depicted herein.

[0086] 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.

[0087] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.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.Attorney Docket No.: CLS-035WO PATENT

[0088] The term “and / or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.

[0089] 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.

[0090] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0091] 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.”

[0092] 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”.

[0093] 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.

[0094] 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.

[0095] 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 specificationAttorney Docket No.: CLS-035WO PATENT should be construed as indicating any non-claimed element as essential to the practice of that provided by the present disclosure.

[0096] As used herein, “residue” refers to a position in a protein and its associated amino acid identity.

[0097] The term “modulation" refers to an increase or decrease in the level of a target molecule or the function of a target molecule. The term “modulator” as used herein refers to modulation of (e.g., an increase or decrease in) the level of a target molecule or the function of a target molecule. Chemical Definitions

[0098] 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.

[0099] 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.

[0100] 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 from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw–Hill, NY, 1962); andAttorney Docket No.: CLS-035WO PATENT Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0101] 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.

[0102] 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.

[0103] When a range of values is listed, it is intended to encompass each value and sub–range within the range. For example, “C1-C6 alkyl” is intended to encompass, C1, 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.

[0104] “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 6–14Attorney Docket No.: CLS-035WO PATENT 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 (“C10 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-C10-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.

[0105] “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.

[0106] 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. Human Papilloma Virus 16

[0107] The high-risk virus variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. 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. 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. HPV E7 primarily binds and inactivates retinoblastoma protein (pRB) and related pocket proteins, p107 and p130, 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 toAttorney Docket No.: CLS-035WO PATENT 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.

[0108] 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. It was reported that targeting the ubiquitination catalytical domain of ubiquitin ligase E6AP (HECT 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.

[0109] Efforts have been made in the past decades to target the 16E6 / E6AP 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 IC50values 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. III. E6AP Peptides

[0110] Disclosed herein are synthetic peptides based on E6AP and libraries thereof.

[0111] In one aspect, the disclosure provides for a synthetic peptide comprising an amino acid sequence IPESSELTLQELLGEER (SEQ ID NO: 1).Attorney Docket No.: CLS-035WO PATENT

[0112] In one aspect, the disclosure provides for a synthetic peptide comprising an amino acid sequence ELTLQELLGEER (SEQ ID NO: 5).

[0113] In some embodiments, the peptide comprises Fluorene-IPESSELTLQELLGEERRAA- K(1-Anthracene (SEQ ID NO:2) or Fluorene-IPESAELTLQELLGEERRAA-K(1-Anthracene (SEQ ID NO:3).

[0114] In another aspect, the disclosure provides for a synthetic peptide selected from TABLE 2C, TABLE 2D, or TABLE 2E modified with a warhead. In some embodiments, the peptide comprises Fluorene- IPQSAELTLQELL(DHA)RRKKK(Anthracene) (SEQ ID NO: 4).

[0115] In another aspect, the disclosure provides for a synthetic peptide comprising an amino acid sequence X1X2X3X2QX1X2X2CEER (SEQ ID NO: 6), wherein X1 is Nva, Leu, Aoc, Cpa, Cba, Cha, Phg, Hof, or Naf; X2 is Aad, Glu, or Cya; X3 is Thr, Asn, Hyp, Cpg, Cbg, or Ceg; and X4 is d-Ala, Aib, Gly, or Gln.

[0116] In some embodiments, the peptide is selected from TABLES 3-31. In some embodiments, the peptide is selected from SEQ ID NO: 7-19. In some embodiments, the peptide comprises SEQ ID NO: 17. In some embodiments, the peptide is selected from TABLES 32-39.

[0117] In another aspect, the disclosure provides for a synthetic peptide comprising an amino acid sequence Aad-X1-X2-Leu-Aib-Aad-Cba-Leu-Cys-X3-X3-X3 (SEQ ID NO: 20) wherein X1 is PhF, Naf, Qua, Clw, or Trp; X2is Chg or Ceg; and X3is d-Ala, Aib, Gly, Ser, Aad, Glu, Gln, Hoc, Val, Leu, Met, Pro, Dap, Lys, Arg, His, Trp, Phe, Tyr, or Null.

[0118] In some embodiments, the peptide is 11 or 12 amino acids long.

[0119] In another aspect, the disclosure provides for a synthetic peptide comprising the formula.

[0120] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: e6apep Sequence: IPESSELTLQELLGEERR.

[0121] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 1-biotin Sequence: Biotin-(PEG)4-IPESSELTLQELLGEERRAttorney Docket No.: CLS-035WO PATENT.

[0122] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: FITC-ELT.

[0123] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: FITC-IPE.

[0124] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Peptide: ELT-G11DS Sequence: AcELTLQELLC(DS)EER-CONH2.

[0125] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Peptide E4 Sequence: AcELTLQELLGC(DS)ER-CONH2Attorney Docket No.: CLS-035WO PATENT.

[0126] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Peptide E5.

[0127] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Peptide E6 Sequence: AcELTLQELLG(dha)ER -CONH2.

[0128] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Peptide E7

[0129] In another aspect, the disclosure provides for a synthetic peptide comprising the formulaAttorney Docket No.: CLS-035WO PATENT Name: 294-201-001-DHA Sequence: 9-Fluorene carboxyl-IPESAELTLQELL(DHA)-NH2.

[0130] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-201-002-DHA Sequence: 9-Fluorene carboxyl-IPESAELTAQEAA(DHA)-NH2.

[0131] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-201-03-DHA Sequence: 9-Fluorene carboxyl-IPESAELTLQELL(DHA)EER-NH2.

[0132] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-03-DHA Sequence: Aad-Nva-Hyp-Nva-Gly-Glu-Cba-Nva-Dha-Glu-Glu-Arg..

[0133] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-04-DHA Aad-Nva-Asn- Gln-Glu-Leu-Nva-Dha-Glu-Glu-.

[0134] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-05-DHA Sequence: Aad-Leu-Hyp-Nva-Gln-Glu-Nva-Nva-Dha-Glu-Glu-ArgAttorney Docket No.: CLS-035WO PATENT

[0135] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-06-DHA Sequence: Aad-Nva-Asn-Nva-Gln-Glu-Leu-Nva-Dha-Glu-Glu-Arg.

[0136] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-08-DHA Sequence: Aad-Nva-Asn-Nva-Gln-Glu-Leu-Nva-Dha-Glu-Glu-Arg ..

[0137] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-10-DHA Sequence: Aad-Cpa-Cpg-Cpa-ala-Glu-Nva-Nva-Dha-Glu-Glu-ArgAttorney Docket No.: CLS-035WO PATENT.

[0138] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-12-DHA Aad-Hof-Thr-Naf-ala-Glu-Leu-Cba-Dha-Glu-Glu-Arg.

[0139] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-14DHA Sequence: Aad-Naf-Cbg-Cpa-Aib-Glu-Hof-Cpa-Dha-Glu-Glu-Arg.

[0140] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-18-DHA Sequence: Aad-Naf-Ceg-Leu-Aib-Glu-Cba-Leu-Dha-Glu-Glu-Arg.

[0141] In another aspect, the disclosure provides for a synthetic peptide comprising the formula

[0142] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-18-DHA-3L3A Sequence: Aad-Naf-Ceg-Ala-Aib-Glu-Ala-Ala-Dha-Glu-Glu-ArgAttorney Docket No.: CLS-035WO PATENT.

[0143] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-19 DHA Sequence: Aad-Cha-Hyp-Naf-ala-Glu-Cha-Phg-Dha-Glu-Glu-Arg

[0144] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 330-066-23DHA Sequence: Aad-Naf-Ceg-Cpa-Aib-Aad-Leu-Leu-Dha-Glu-Glu-Arg

[0145] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 18-car Structure: (330-066-18-01-Car-Dha).

[0146] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 18-Naf2CarAttorney Docket No.: CLS-035WO PATENT Sequence: (330-066-18-07).

[0147] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 18-Car-IPQSA Sequence: (330-066-18-05).

[0148] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 18-Car-IPQSA-3L3A Sequence: (330-066-18-05-3L3A).

[0149] In another aspect, the disclosure provides for a synthetic peptide comprising the formula 18-Car-IPQSA-truncEER 066-18-. N-terminal modified peptide

[0150] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-20 Structure:Attorney Docket No.: CLS-035WO PATENT.

[0151] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-21 Structure:.

[0152] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-23 Structure:.

[0153] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-24 Structure:.

[0154] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-25 Structure:Attorney Docket No.: CLS-035WO PATENT.

[0155] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-26 Structure:.

[0156] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-30 Structure:.

[0157] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-31 Structure:.

[0158] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-32 Structure:Attorney Docket No.: CLS-035WO PATENT.

[0159] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-33 Structure:.

[0160] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-35 Structure:.

[0161] In another aspect, the disclosure provides for a synthetic peptide comprising the formula Name: 294-190-36.

[0162] In some embodiments, the peptide comprises a substituted amino acid. In some embodiments, the substituted amino acid is a canonical amino acid. Canonical amino acids for use in substitutions are listed in TABLE A. In some embodiments, the canonical substitutedAttorney Docket No.: CLS-035WO PATENT amino acids are an Ala, a Ser, a Gln, or an Arg. TABLE A Canonical amino acids used in the peptides and peptidomimetics. TABLE A

[0163] In some aspects of the disclosure, the synthetic peptide or peptidomimetic comprises one or more non-canonical amino acids. Non-canonical amino acids that can be used for substitution are shown in TABLE B.

[0164] TABLE B Non-canonical amino acids for use in the peptides and peptidomimetics. TABLE BAttorney Docket No.: CLS-035WO PATENT

[0165] In some embodiments the amino acids of the synthetic peptide are mixed canonical and non-canonical amino acids.

[0166] In some embodiments, hydroxyproline is substituted with a hydroxyproline derivative or protected analog (e.g., L-hydroxyproline(tBu)-OH).

[0167] In another aspect of the disclosure, the synthetic peptides and peptidomimetics are designed to bind HPV16E6. In some embodiments, the synthetic peptide binds HPV16E6 with low, medium, or high affinity. In some embodiments, the synthetic peptides and peptidomimetics bind HPV16E6 with higher affinity than SEQ ID NO: 1 or SEQ ID NO: 5. InAttorney Docket No.: CLS-035WO PATENT some embodiments, the synthetic peptides are covalently bound to HPV16E6. In some embodiments, the covalent bond is between the synthetic peptide and HPV16E6 residues Cys- 58, Arg-84, or Arg-13.

[0168] In some aspects, the disclosure is directed to libraries of synthetic disclosed herein. In some embodiments, the peptide library has the design X1X2X3X2QX1X2X2CEER (SEQ ID NO: 6), wherein X1 is Nva, Leu, Aoc, Cpa, Cba, Cha, Phg, Hof, or Naf; X2 is Aad, Glu, or Cya; X3 is Thr, Asn, Hyp, Cpg, Cbg, or Ceg; and X4 is d-Ala, Aib, Gly, or Gln.

[0169] In some embodiments, the peptide library has the design Aad-X1-X2-Leu-Aib-Aad-Cba- Leu-Cys-X3-X3-X3 (SEQ ID NO: 20) wherein X1 is PhF, Naf, Qua, Clw, or Trp; X2 is Chg or Ceg; and X3 is d-Ala, Aib, Gly, Ser, Aad, Glu, Gln, Hoc, Val, Leu, Met, Pro, Dap, Lys, Arg, His, Trp, Phe, Tyr, or Null.

[0170] 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. III. Modifications

[0171] 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 acids, and backbone modifications. A therapeutic small molecule ligand equipped with an electrophilic warhead binds covalently to nucleophilic groups of the target protein in a proximity-driven reaction. Irreversible covalent inhibition of an interaction can result in increased potency, selectivity, sustained pharmacodynamics, and could alleviate the effects of fast renal elimination. Therapeutic peptides may benefit from a covalent binding mode of action and alleviate pharmacokinetic limitations of this class of therapeutics.

[0172] In some aspects, the disclosure is directed to synthetic peptides that are chemically modified. In some embodiments, the peptide that is modified is selected from TABLE 1- TABLE 40. In some embodiments, the peptide that is modified is selected from SEQ ID NOS: 1-20. 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 aminoAttorney Docket No.: CLS-035WO PATENT 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 Į-amino acid with a ȕ-amino acids.

[0173] In some embodiments, the N- or C- terminus of the peptide is modified with a chemical moiety.

[0174] 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-1-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.

[0175] 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- Indanecarboxamido, 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, triphenylacetamido, 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-1-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.

[0176] 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.Attorney Docket No.: CLS-035WO PATENT

[0177] In some embodiments, the N-terminus of the peptide is further modified with a modifier selected from 2-Phenyl-4-quinolinecarboxylic acid, 1-(Phenylsulfonyl)-1H-indole-2-carboxylic acid, 6-Fluoro-2-naphthoic acid, 4-phthalimidobenzoic acid, Xanthene 9-carboxylic acid, 1- Pyrenebutyric acid, 5-Acenaphthenecarboxylic acid, 1-Phenyl-1H-indole-2-carboxylic acid, Indole-2-carboxlic acid, quinoline-4-carboxylic acid, and 10-(Carboxymethyl)-9(10H)acridone. Warheads

[0178] In some aspects, the disclosure is directed to a synthetic peptide comprising a warhead. In some embodiments, the peptide is modified with a warhead that is selected from TABLE 2C, TABLE 2D, or TABLE 2E. In some embodiments, the peptide that is modified is selected from SEQ ID NOS: 1-20. In some embodiments, the warhead facilitates a covalent bond to a cognate protein after a chemical reaction. 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 peptidomimetic In some embodiments, the warhead is on a Cys, Lys, Tyr, His, Ser, or Thr. In some embodiments, the warhead is on a Cys, and the Cys is a position 9 of the peptide with respect to SEQ ID NO: 3. In some embodiments, the warhead is conjugated to the Cys via the Sulfur atom of the Cys.

[0179] In some embodiments, the warhead is selected from the group of phenylacrylamide (Ph- acr), Dap-acrylamide (dap-acr), Dab-acrylamide (dab-acr), Dap-propiolamide (dap-ppa), and dehydroalanine Dha.

[0180] In some embodiments, the warhead is selected from the group of phenyl fluorosulfate (FS), phenyl sulfonyl fluoride(SF), phenyl Carbamate (p-PhC), phenyl Carbamate (m-PhC), and disulfide (DS).

[0181] In some embodiments, the peptide with a warhead has the following formula Peptide E1 Sequence: AcELTLQELLC(Phacr)EER -CONH2. .Attorney Docket No.: CLS-035WO PATENT

[0182] In some embodiments, the peptide with a warhead has the following formula Peptide E2 ER -CONH2. .

[0183] In some embodiments, the peptide with a warhead has the following formula Peptide E3 Sequence: AcELTLQELL(dha)EER-CONH2.

[0184] In some embodiments, the peptide with a warhead has the following formula Peptide E4 Sequence: AcELTLQELLG(dha)ER -CONH2.

[0185] In some embodiments, the peptide with a warhead has the following formula Peptide E5Attorney Docket No.: CLS-035WO PATENT

[0186] In some embodiments, the peptide with a warhead has the following formula Peptide E6.

[0187] In some embodiments, the peptide with a warhead has the following formula Peptide E7 Sequence: AcELTLQELLG(dap-acr)ER-CONH2.

[0188] In some embodiments, the peptide with a warhead has the following formula Peptide E8 Sequence: AcELTLQELLGE(dap-acr)R -CONH2.

[0189] In some embodiments, the peptide with a warhead has the following formula Peptide E9 Sequence: AcELTLQELLG(dab-acr)ER-CONH2

[0190] In some embodiments, the peptide with a warhead has the following formula Peptide E10Attorney Docket No.: CLS-035WO PATENT Sequence: AcELTLQELLGE(dab-acr)R -CONH2.

[0191] In some embodiments, the peptide with a warhead has the following formula Peptide E11. IV. Complexes

[0192] In some aspects, the disclosure is directed to synthetic peptides that are bound in a complex with HPV16E6 to form peptide / HPV16E6. In some embodiments, the peptide that is complexed with HPV16E6 is selected from TABLE 1 to TABLE 40. In some embodiments, the peptide that is complexed with HPV16E6 is selected from SEQ ID NOS: 1-20.

[0193] In some embodiments, the synthetic peptides are designed to bind HPV16E6. In some embodiments, the synthetic peptides bind HPV16E6 with low, medium, or high affinity. In some embodiments, the synthetic peptides bind HPV16E6 with higher affinity than SEQ ID NO: 1 or SEQ ID NO: 5. In some embodiments, the peptides are covalently bound to HPV16E6. In some embodiments, the covalent bond is between the synthetic peptides and HPV16E6 residues Cys- 58, Arg-84, or Arg-13.

[0194] In some embodiments, the peptide / HPV16E6 is modulated in binding of E3 ubiquitin ligase E6AP. In some embodiments, the peptide / HPV16E6 is inhibited in binding or engaging of E3 ubiquitin ligase E6AP. In some embodiments, the peptide / HPV16E6is soluble in a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the HPV16E6 is located in a cancer cell.Attorney Docket No.: CLS-035WO PATENT V. Preparation of Peptides

[0195] 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. VI. Pharmaceutical Compositions

[0196] 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.

[0197] 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.Attorney Docket No.: CLS-035WO PATENT

[0198] Pharmaceutical compositions containing a synthetic peptide disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration, e.g., oral administration. The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form will depend upon the intended mode of administration and therapeutic application.

[0199] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by 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.

[0200] 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-basedAttorney Docket No.: CLS-035WO PATENT substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. VII. Methods of Use

[0201] 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, based on the binding specificity of the synthetic peptides to HPV 16E6 and on the effect on HPV 16E6 functions of the peptides.

[0202] The synthetic peptides and peptidomimetics disclosed herein are designed to be bound covalently or non-covalently by HPV 16E6. In some embodiments, the synthetic peptides can be used to modulate HPV 16E6 function. 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.

[0203] Methods for testing for HPV 16E6 - E6AP engagement and subsequent cell signaling are known in the art, for example by p53 ubiquitination and degradation assays. VIII. Kits

[0204] 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.

[0205] 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 ofAttorney Docket No.: CLS-035WO PATENT pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration. EXAMPLES

[0206] 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.

[0207] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, PROTEINS: STRUCTURES AND MOLECULAR PROPERTIES (W.H. Freeman and Company, 1993); A.L. Lehninger, BIOCHEMISTRY (Worth Publishers, Inc., current addition); Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL (2nd Edition, 1989); METHODS IN ENZYMOLOGY (S. Colowick and N. Kaplan eds., Academic Press, Inc.); REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg ADVANCED ORGANIC CHEMISTRY 3rdEd. (Plenum Press) Vols A and B (l992).

[0208] 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.

[0209] Unless otherwise stated, all reagents and chemicals were obtained from commercial sources and used without further purification. EXAMPLE 1 - Synthesis of Peptides

[0210] This example describes the synthesis of peptides used in the following examples. Fast flow synthesis of peptides

[0211] H-Rink Amide-ChemMatrix resin (200 mg, 0.49 mmol / g, 0.10 mmol) was used to prepare peptide-Į-carboxamides. Peptides containing noncanonical amino acids were prepared by manual SPPS. Peptides without noncanonical amino acids were prepared by fully automated SPPS[1]. Upon completion, resins were washed with dichloromethane (DCM) three times and dried under reduced pressure.Attorney Docket No.: CLS-035WO PATENT Automated flow peptide synthesis (AFPS) set-up

[0212] 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[2]:

[0213] The following settings were used for protein synthesis: flowrate = 40 mL / min, temperature = 90°C (loop) and 85–90°C (reactor). The 50 ml / min pump head pumps 400 ^L of liquid per pump stroke; the 5 mL / min pump head pumps 40 ^L of liquid per pump stroke. The standard synthetic cycle involves a first step of prewashing the resin at elevated temperatures for 60 s at 40 mL / min. During the coupling step, three HPLC pumps are used: a 50 mL / min pump head pumps the activating agent, a second 50 ml / min pump head pumps the amino acid and a 5 mL / min pump head pumps DIEA. The first two pumps are activated for 8 pumping strokes to prime the coupling agent and amino acid before the DIEA pump is activated. The three pumps are then actuated together for a period of 7 pumping strokes, after which the activating agent pump and amino acid pump are switched using a rotary valve to select DMF. The three pumps are actuated together for a final 8 pumping strokes, after which the DIEA pump is shut off, and the other two pumps continue to wash the resin for another 40 pump strokes. During the deprotection step, two HPLC pumps are used. Using a rotary valve, one HPLC pump selects deprotection stock solution and DMF. The pumps are activated for 13 pump strokes. Both solutions are mixed in a 1:1 ratio. Next, the rotary valves select DMF for both HPLC pumps, and the resin is washed for an additional 40 pump strokes. The coupling–deprotection cycle is repeated for all additional monomers. Method for peptide acetylation

[0214] 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 resulting mixture 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

[0215] Peptidyl resin (~10 ^mol theoretical loading) was washed with DCM (3 × 5 mL) and then treated with Pd(PPh3)4 (11.0 mg, 10 ^mol, 1 equiv) in DCM / piperidine (8:2, 1 mL) for 30Attorney Docket No.: CLS-035WO PATENT minutes at room temperature under exclusion of light. The resin was then drained and washed with DCM (3 × 5 mL). Method for biotin labeling

[0216] Peptidyl resin (~10 ^mol 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 ^mol, 5 equivalents) and HATU (17 mg, 45 ^mol, 4.5 equivalents) were dissolved in DMF (500 ^L), activated with DIEA (19 mg, 26 ^L, 150 ^mol), 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 × 5 mL), and stored until cleavage. Method for TAMRA labeling

[0217] Peptidyl resin (~ 10 ^mol 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 ^mol, 5 equivalents) and HATU (17 mg, 45 ^mol, 4.5 equivalents) were dissolved in DMF (500 ^L), activated with DIEA (19 mg, 26 ^L, 150 ^mol), 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 × 5 mL), and stored until cleavage. Method for FITC labeling

[0218] Peptidyl resin (~ 10 ^mol 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 ^mol, 5 equivalents) was dissolved in DMF (500 ^L), activated with DIEA (19 mg, 26 ^L, 150 ^mol), 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 × 5 mL) and stored until cleavage. Cleavage of peptides

[0219] 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 rcf 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.Attorney Docket No.: CLS-035WO PATENT Pd Mediated Conjugation

[0220] In a 50 mL falcon tube: Dissolve peptide (5.0 mg, 2.75 mmol, 1.0 equiv) in water (8.5 mL) and 500 mM HEPES (1.5 mL, pH = 8.0). In a 20 mL glass vial: Dissolve Pd OAC (5.0 mg, 6.19 mmol, 2.25 equiv) in MeCN and add to peptide solution over 30 seconds (final volume = 15 mL, final peptide concentration = 184 μM). Mix by vortexing and let stand for 20 minutes. Add 10 mL AcOH and 30 mL H2O and mix and then purify by reversed phase flash chromatography using a Sfär Bio C18 D (300 Å 20 μm, 6 g) column (mobile phase 5% MeCN / H2O to 55% MeCN in H2O + 0.1% TFA). Dehydroalanine formation

[0221] 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 μmol, 5 equiv) was added. To this mixture, a 10 mg / mL solution of Diethylmeso-2,5-dibromoadipate (151 μL, 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, 300Å, 5 μm, 9.4 mm x 250 mm) Mobile phase 5% to 55% MeCN in H2O + 0.1% TFA. Purification of the crude peptide.

[0222] 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 C420 ^m 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. In solution disulfide dimer formation

[0223] 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 I2in MeOH into the peptide solution. Then quench the excessive oxidant with 1M of L-Ascorbic acid in water. Mix by vortexing and then purified by reversed-phase flash chromatography using a Sfär Bio C14 D (300 Å 20 μm, 10 g) column (mobile phase 20% MeCN / H2O to 30% MeCN in H2O + 0.1% TFA, 10 column volume).Attorney Docket No.: CLS-035WO PATENT On-resin mixed disulfide formation

[0224] To 50mg resin (0.025mmol, 1 eq.) add freshly prepared 1M Iodine in DMF solution (50 eq., stir for 2 min and drain. Add freshly prepared 1M 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. EXAMPLE 2 - LC-MS characterization of peptides LC-MS methods

[0225] LC-MS characterizations were carried out using an Agilent 6550 quadrupole time-of- flight LC-MS. Total ion current (TIC) chromatograms were plotted. Mass spectra were integrated over the principal TIC peaks. High-performance liquid chromatography was done by the following methods: (solvent A: water with 0.1% formic acid; solvent B: MeCN with 0.1% formic acid).

[0226] Method A: Column: Phenomenex Aeris C4 column (1.0 × 150 mm, 5 ^m particle size, 300 Å pore size) Gradient: 1% B (0-2 min), linearly ramp from 1% B to 91% B (2-10 min). The flow rate is 100 ^L / min. MS acquisition is from 2 to 10 min.

[0227] Method B: Column: Phenomenex Aeris C4 column (1.0 × 150 mm, 5 ^m particle size, 300 Å pore size) Gradient: 1% B (0-2 min), linearly ramp from 1% B to 91% B (2-8 min), 61% B to 95% B (8-10 min). The flow rate is 100 ^L / min. MS acquisition is from 2 to 8 min.

[0228] Method C: Column: Agilent Zorbax 300SB C3 column (2.1 × 150 mm, 5 ^m particle size, 300 Å pore size) Gradient: 1% B (0-2 min), linearly ramp from 1% B to 91% B (2-12 min), 91% B to 91% B (12-13 min). The flow rate is 500 ^L / min. MS acquisition is from 4 to 12 min.

[0229] Method D: Column: Phenomenex Jupiter C4 column (1.0 × 150 mm, 5 ^m particle size, 300 Å pore size) Gradient: 1% B (0-2 min), linearly ramp from 1% B to 91% B (2-18 min), 91% B to 91% B (18-21 min). The flow rate is 100 ^L / min. MS acquisition is from 4 to 18 min.

[0230] Method E: Column: Agilent Zorbax 300SB C3 column (2.1 × 150 mm, 5 ^m particle size, 300 Å pore size) Gradient: 1% B (0-1 min), linearly ramp from 1% B to 91% B (1-11 min), 91% B to 91% B (11-15 min). The flow rate is 500 ^L / min. MS acquisition is from 0 to 11 min.Attorney Docket No.: CLS-035WO PATENT EXAMPLE 3 - Expression and Purification of Proteins E6AP

[0231] E6AP (residues 1-875) protein with a C-terminal TEV-6xHis-Avi sequence was subcloned into a pFastBac1 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.

[0232] 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, 1mM PMSF, and 20 mM imidazole before eluting with the same buffer supplemented with 500mM 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, 1mM PMSF). Fractions containing E6AP as determined by SDS-PAGE and Coomassie staining were pooled and concentrated and loaded onto a HiLoad 16 / 600 Superdex 200pg column equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP. (FIG.36A).

[0233] E6AP Amino Acid Sequence: MEKLHQCYWKSGEPQSDDIEASRMKRAAAKHLIERYYHQLTEGCGNEACTNEFCASCP TFLRMDNNAAAIKALELYKINAKLCDPHPSKKGASSAYLENSKGAPNNSCSEIKMNKK GARIDFKDVTYLTEEKVYEILELCREREDYSPLIRVIGRVFSSAEALVQSFRKVKQHTKEE LKSLQAKDEDKDEDEKEKAACSAAAMEEDSEASSSRIGDSSQGDNNLQKLGPDDVSVD IDAIRRVYTRLLSNEKIETAFLNALVYLSPNVECDLTYHNVYSRDPNYLNLFIIVMENRN LHSPEYLEMALPLFCKAMSKLPLAAQGKLIRLWSKYNADQIRRMMETFQQLITYKVISN EFNSRNLVNDDDAIVAASKCLKMVYYANVVGGEVDTNHNEEDDEEPIPESSELTLQELL GEERRNKKGPRVDPLETELGVKTLDCRKPLIPFEEFINEPLNEVLEMDKDYTFFKVETEN KFSFMTCPFILNAVTKNLGLYYDNRIRMYSERRITVLYSLVQGQQLNPYLRLKVRRDHII DDALVRLEMIAMENPADLKKQLYVEFEGEQGVDEGGVSKEFFQLVVEEIFNPDIGMFTY DESTKLFWFNPSSFETEGQFTLIGIVLGLAIYNNCILDVHFPMVVYRKLMGKKGTFRDLG DSHPVLYQSLKDLLEYEGNVEDDMMITFQISQTDLFGNPMMYDLKENGDKIPITNENRK EFVNLYSDYILNKSVEKQFKAFRRGFHMVTNESPLKYLFRPEEIELLICGSRNLDFQALEE TTEYDGGYTRDSVLIREFWEIVHSFTDEQKRLFLQFTTGTDRAPVGGLGKLKMIIAKNGPAttorney Docket No.: CLS-035WO PATENT DTERLPTSHTCFNVLLLPEYSSKEKLKERLLKAITYAKGFGMLENLYFQGHHHHHHGLN DIFEAQKIEWHE* MBP-16E6

[0234] 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.

[0235] 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 200pg column equilibrated in PBS supplemented with 1 mM DTT. (FIG.36B).

[0236] MBP-16E6 amino acid sequence: MAHHHHHHPMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFP QVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAY PIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGG YAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETA MTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLEN YLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNPMSENLYFQGAMFQDPQ ERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKC LKFYSKISEYRHYSYSLYGTTLEQQYNKPLSDLLIRCINCQKPLSPEEKQRHLDKKQRFH NIRGRWTGRCMSCSRSSRTRRETQL* SUMO-MDM2

[0237] Recombinant SUMO-MDM2 was expressed as previously reported. In brief, the 25- 109MDM2 gene was synthesized and cloned into a n expression vector. A SUMO tag was incorporated using the Champion™ pET SUMO Expression System. SUMO-25-109MDM2 was expressed in Rosetta (DE3) pLysS cells. The bacteria were inoculated to reach OD600 = 0.5 atAttorney Docket No.: CLS-035WO PATENT 37°C, induced with 0.4 mM IPTG for 4 hours, and pelleted. Approximately 1 L broth produced 10 g cell pellet, which was resuspended in 30 mL of 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 buffer containing 40 mg lysozyme, 1 mg Roche DNAase I, and one tablet of protease inhibitor cocktail, and then sonicated for three times for 20 s. The suspension was then centrifuged at 30,000 rcf for 30 min to clarify the lysate. The supernatant was loaded onto a 5 mL HisTrap FF crude Ni-NTA columns , and washed sequentially with 30 mL of 20 mM Tris-HCl, 150 mM NaCl, pH 8.5 and then 30 mL 20 mM Tris-HCl 150 mM NaCl, 80 mM imidazole pH 8.5. The protein was eluted with 10 mL 20 mM Tris-HCl, 500 mM imidazole, 500 mM NaCl, pH 8.5. The eluted protein was buffer exchanged into 20 mM Tris-HCl, 50 mM NaCl, pH 8.5 using a HiPrep 26 / 10 Desalting column. The resulting protein solution was purified the same day using a 5 mL HiTrap Q HP anion exchange columns with a linear NaCl gradient (B% graded from 5% to 25%, B = 20 mM Tris-HCl, 500 mM NaCl, pH 8.5). Fractions containing pure SUMO-25- 109MDM2, as determined by SDS-PAGE, were collected, and concentrated to 0.5 mg / mL using a 10,000 Da Centrifuge Filter, and immediately flash frozen and stored at í80 °C. EXAMPLE 4 - Design of high-affinity peptide binders to E6

[0238] 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.

[0239] 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) FIG.1A. A 17-mer peptide (Peptide 1, SEQ ID NO: 1, sequence: IPESSELTLQELLGEER, FIG.1D and FIG.2A) that covers residues 401-417 of E6AP and includes the LXXLL motif, was chosen as a starting point for a binder.

[0240] The binding affinity of peptide 1 to HPV16E6 was assessed by bio-layer interferometry (BLI) using recombinant MBP-16E64C4S protein (MBP-16E6, which contains a maltose- binding protein (MBP) tag and four cysteine to serine mutations to stabilize and solubilize HPV16 E6. Direct binding measurement by BLI

[0241] 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 1-biotin peptide (200 nM) onto streptavidin sensors, serial dilutions of MBP-16E6 in the blocking buffer were analyzed for binding. The response was recorded at equilibrium after 2 min. AssociationAttorney Docket No.: CLS-035WO PATENT lasted for 100 seconds, and dissociation lasted for another 120 seconds. The curve was reported by GatorPlus software (version 2.7.3.1013) and replotted by Prism 8 software. Competition binding assay by BLI

[0242] A competition binding assay was performed as described below using GatorPlus bio- layer interferometry(GatorBio) to estimate the binding affinity of peptides.

[0243] 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.2A) onto streptavidin sensors, 1:1 serial dilutions from 1000 nM of E6 in blocking buffer were analyzed for binding (All concentrations: 1000nM, 500 nM, 250 nM, 125 nM, 62 nM, 31nM, 15nM, and 7.8nM). 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.

[0244] 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: [Y]= 0.5×[b-K_d-[X]+¥(([X]+ K_d -b)^2+4b× K_d ) ] 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.

[0245] 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 μM (FIGs.2B and 2C). The competition KD values reported for the peptide variants discussed below were determined by BLI in a similar manner.Attorney Docket No.: CLS-035WO PATENT

[0246] 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-terminal fluorescein 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 KDof 95 nM, while C-FITC peptide 1 had a competition KDof 640 nM. EXAMPLE 5 - Alanine Scan of Peptide 1

[0247] To determine critical residues in the E6AP peptide designs, an alanine (Ala) scan was performed and 17 single Ala mutants of N-FITC, peptides A1 to A17, respectively were synthesized (FIG.3). Alanine scanning showed the residues that are critical for binding activity. Substituting residues Leu9, Leu11, and Leu12 to alanine or removing the N-terminal modification significantly decreased or disrupted binding. Four residues, Glu6, Thr8, Glu11, and Gly14, showed medium alanine tolerance.

[0248] The result show that hotspot residues Leu9, Leu12, and Leu13 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 3L3A triple mutants) were used as negative control peptides (FIG.1D). EXAMPLE 6 - Design of N- or C- terminal modified high-affinity peptide binders to E6

[0249] Additional peptides with N- and / or C-terminal modifications were designed for analysis (Peptides N1-N22, SEQ ID NO: 2-23, Peptides C1-C15, SEQ ID NO: 24-). A chemical library of small molecules used for modifying E6AP-based peptides is shown in FIG.1C.

[0250] To determine the structure-affinity relationships (SAR) contributing to the binding enhancement of N or C terminal FITC modifications, FITC was replaced with a panel of small molecules at the C- or N-terminus of parent peptide 1 (FIGs.1B, C and TABLE 1 and TABLE 2A). Peptide binding to E6 was measured with BLI as described in Example 4. TABLE 1 N-terminal modified E6AP peptides. Binding affinity was measured by BLI competition assay.Attorney Docket No.: CLS-035WO PATENTError distribution is reported as the standard error of the mean (SEM) of curve fitting reported by Prism 8 software. Wild-type (WT) peptide was used as a reference. KD values are measured by BLI binding assay in a competition mode (N = 2 or 3). The ratio is calculated by dividing competition KDin this row to the competition KDof WT showed in the first row. TABLE 2A C-terminal modified E6AP peptides. Binding affinity was measured by BLI competition assay.Attorney Docket No.: CLS-035WO PATENTError distribution is reported as the standard error of the mean (SEM) of curve fitting reported by Prism 8 software. Wild-type (WT) peptide was used as a reference. KD values are measured by BLI binding assay in a competition mode (N = 2 or 3). The ratio is calculated by dividing competition KDin this row to the competition KDof WT showed in the first row.

[0251] The results show that from the 36 synthesized peptides, 8 terminally modified peptides displayed KD < 70 nM against MBP-16E6 (Peptides N1, N2, N7, N9, N10, N19, C4, C9, and C10). At the N-terminus, planar and tricyclic arenes enhanced binding by 50 to 250-fold relative to unmodified peptide 1, while bulky, hydrophobic, or aliphatic small molecules modestly improved affinity by less than 5-fold (FIG.1C). The position of fluorene ring substitution had no effect on N- or C-terminal modifications. At the N-terminus, the largest ~250-fold increase in binding affinity was observed with a fluorene modification (peptide N1). Removal of a phenyl ring from fluorene (N1, KDof 10 nM) to indane (N3, KDof 425 nM) decreased the binding by ~42-fold. Compared to fluorene-modified N1, biphenyl-containing N21 (KDof 1412 nM) decreased the binding by ~27-fold. These results suggest that N-terminal planar tricyclic aromatic scaffolds are favorable motifs to enhance binding of parent peptide 1 to 16E6 (FIG. 4A). The greatest improvements at the C-terminus, from 30 to 111-fold, were found with planar, tricyclic, and polyarenes, while bulky, non-conjugated aromatic or aliphatic small molecules did not significantly improve the binding compared to parent peptide 1 (FIG.1C and TABLE 2A). The best performing C-terminal modification was anthracene, which showed a competition KD value of 22 nM, enhancing binding affinity to MBP-16E6 by ~111-fold compared to the parent peptide 1 (FIG.4B, TABLE 2B). TABLE 2BAttorney Docket No.: CLS-035WO PATENT

[0252] Combining the N- and C-terminal modifications with the highest 16E6 binding affinity generated double-modified peptide binders that displayed improvements over single modifications–likely due to synergistic effect from binding features of the two-terminal modifications (FIG.1D). Peptide 6 (SEQ ID NO: 2) contained a N-terminal fluorene, and a C- terminal anthracene and showed the lowest competition KD of 3.7 nM to MBP-16E6 and was selected for further terminal modifications. Additional optimization was achieved by substituting Ser5 to Ala, as previous Ala scan studies revealed a 2-fold increase in binding affinity (FIG.3), resulting in peptide 6’ (SEQ ID NO: 3) with a competition KDof 2.7 nM.

[0253] To determine the binding affinity and selectivity of 6', biotinylated peptide 6’-biotin and its 3L3A derivative 6’-3L3A were synthesized.6’-3L3A displayed no observable binding towards MBP-16E6 (FIG.1E).6’-biotin showed a direct binding KD of 3.0 ± 1.8 nM to MBP- 16E6(FIG.1F). These results demonstrate that peptide 6' binding to MBP-16E6 is sequence- specific and requires the tri-leucine hotspot motif LXXLL. To assess target selectivity, peptide 6’-biotin (FIG.5A) was immobilized and its affinity towards the unrelated proteins murine double minute 2 (MDM2) and an LXXLL motif-binding protein thyroid hormone receptor alpha (THRA) were measured by BLI. No appreciable signal during the association or dissociation steps was observed by BLI (FIG.5B and 5C), indicating that peptide 6' selectively binds MBP- 16E6.Attorney Docket No.: CLS-035WO PATENT EXAMPLE 7 - Development of modified E6 binding peptides for crosslinking to MBP- 16E6

[0254] This example describes the design of E6 binding peptides that are modified with a warhead for covalent binding. Irreversible covalent inhibition is an effective strategy to increase drug potency and selectivity toward inhibiting ‘undruggable’ targets.

[0255] Briefly, the binding interface of 16E6 contains the nucleophilic Cys58 residue in proximity to the E6AP LXXLL peptide binding pocket (FIG.6A). To exploit this finding, peptide reactides E1–E11 (FIG.7A) comprising a warhead for covalent binding, were based on peptide E0, a truncated version of peptide 1. Reactides E1 -11 were synthesized, with a Cys- reactive acrylamide replacing Gly9, Glu10, or Glu11 of E0 as shown (FIG.6B) and FIG.7A). Multiple electrophiles including phenylacrylamide (Ph-acr), Dap-acrylamide (dap-acr), Dab- acrylamide (dab-acr), Dap-propiolamide (dap-ppa), and dehydroalanine Dha (FIG.7B) were tested as a warhead. Dehydroalanine (Dha) Formula I is a cysteine targeting, monocrosslink with irreversible reaction and relatively mild chemistry. The resulting bond is stable to reducing in vivo environment.

[0256] Formula I

[0257] Liquid chromatography-mass spectrometry (LC-MS) monitored conjugation between MBP-16E6 protein and the reactides E1-E11 was measured, and the area under the total ion peak was used to estimate crosslink yield.

[0258] The results show, that Gly9 was found to be the closest residue to Cys58 and its substitution with Dha gave the highest crosslink yield of 74% (peptide E3, or ELT-G9Dha) (FIG.6C).

[0259] Additional kinetic studies were performed with reactide peptide E3 to estimate the binding constant Ki and first-order rate constant (kinact) involved in the two-stage bind-and- react strategy. Crosslink yield was monitored over time by LC-MS (FIG.6D and FIG.6E). Kinetic parameters were estimated assuming a steady-state approximation, and a kinact value of 0.022 s-1 and Ki value of 50 μM were obtained. The kinact / Ki ratio was calculated to be 446 M-Attorney Docket No.: CLS-035WO PATENT 1 s-1, indicating that improvements in binding affinity will produce a more efficient covalent inhibitor. For comparison, the FDA-approved small molecular drug nirmatrelvir showed a kinact / Ki value of 55 mM-1 s-1.

[0260] Additional longer reactide peptides based on peptide 6' (Example 6) were designed. First Dha was installed onto peptide 6' to obtain reactide 7, which has a net charge of í3 that may be detrimental to cell permeability (FIG.8). To increase positive charge, improve solubility, retain binding affinity to 16E6, and further reduce molecular weight, additional residue substitutions and truncations on 7 were performed (peptides 8-13, FIG.8). The LXXLL proximal RRNKK [residues 417-421] segment from the E6AP protein was appended to the C-terminus of reactide 7 to increase the total charge from í3 to í1 (reactide 8). Next, Glu3Gln, Glu15Gln, and Glu16Ala mutations were applied to increase the total charge to +2, resulting in peptides 9 and 10 (FIG.8). To reduce molecular weight, Ala13, Gln14, and Asn19 residues were omitted from peptide 10 to generate 11, 12, and 13 (Fluorene- IPQSAELTLQELL(DHA)RRKKK(Anthracene) (SEQ ID NO: 4)) (FIG.8). Reactides were evaluated by the BLI competition assay against 1-biotin to estimate their inhibitory constant, reported here as an apparent Ki value. TABLE 2CAttorney Docket No.: CLS-035WO PATENT TABLE 2DTABLE 2E

[0261] The results show that peptides 8 - 13 have comparable binding affinity to the parent reactide 7, with an apparent Ki ranging from 11 nM to 39 nM (FIG.8). Peptide reactide 13 was selected for further characterization since it has the smallest molecular weight, highest net charge, and relatively low apparent Ki = 17 ± 3.9 nM (FIGS.9A, 9B and 9C). The negative control derivative of peptide 13, (13-3L3A) showed no observable binding to MBP-16E6 protein. Kinetic study was performed with peptide 13 and revealed a kinact of 0.027 s-1, a KiAttorney Docket No.: CLS-035WO PATENT value of 120 nM and a kinact / Ki ratio of 270 mM-1 s-1 (FIGS.9D and 9E). Reactide 13 represents a 504-fold improvement over E3.

[0262] Despite increasing numbers of reports describe peptide-based covalent inhibitors it is under-exploited to target challenging protein-protein interactions. One major drawback for this strategy is off-target crosslinking which can be mitigated through a bind-and-react strategy. With this approach, the risks of non-specific crosslinking and non-selective inhibition are reduced through specific binding of the base peptide sequence which brings the reactive warhead and its target residue in close proximity. This may allow for the use of less reactive groups to alleviate non-selective modification.

[0263] Dha is the simplest dehydroamino acid is found in some microbial peptides. Due to its electrophilic nature and lack of geometric isomers resulting from the methylidene group, it has been utilized as a reactive probe targeting enzymatic mechanisms. Dha is used as an electrophilic warhead to generate a reactide for targeting a cancer-relevant PPI. Reactivity of the reactide was fine-tuned by adjusting the electrophile and distance between the warhead and peptide backbone.

[0264] Crosslinking specificity of the reactides was further tested in cell media. High crosslinking efficiency was achieved with Dha placed directly onto the peptide backbone to fit in the tight binding pocket at the target protein surface containing Cys58, as suggested by the co- crystal structure. The reactide was stable and not reactive to 0.4 mM free Cys and GST in cell media. EXAMPLE 8 – Crosslinking of Reactide 13 to MBP-16E6

[0265] This example described the crosslinking of reactide 13(SEQ ID NO: 4) to MBP-16E6.

[0266] Briefly, a solution of cross-linker (250 μM solution in 10% DMSO / H2O, 2 to 10 equiv, 0.66 μL) was diluted with H2O (30 μL) and 10× PBS (4.1 μL, pH = 7.4). A 30 μM solution of MBP-6 (2.4 μL, 1 equiv) and then incubated at room temperature for 2 h. An 8 μL aliquot of the reaction mixture was removed and quenched with 92 μL 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 determined using total ion count. %yield =^^^^ା^బ× 100 where Pc is the peak area of the peptide-protein conjugate, and P0 is the peak area of the unmodified proteinAttorney Docket No.: CLS-035WO PATENT

[0267] When peptide 13 (3 μM) and MBP-16E6 (1 μM) were incubated in PBS at 37 °C for two hours, protein deconvolution mass spectra revealed >99% mono-crosslinking of 13 to MBP- 16E6 (FIG.10A). This was achieved despite the presence of 10 cysteine residues on MBP-16E6. To confirm site-selectivity, 13 was incubated with the MBP-16E6 mutant C58S for 12 h in PBS at 37 °C. No crosslinking was observed between 13 and MBP-16E6 C58S (FIG.10B), which supports Cys58 as the sole site of MBP-16E6 modification.13 showed no crosslink to THRA (FIG.11) Additionally, the negative control 13-3L3A showed no observable crosslinking to MBP-16E6 protein (FIG.10C), supporting peptide 13 specificities. Therefore, 13-3L3A was used as a negative control for subsequent peptides. Crosslinking of Reactide 13 to MBP-16E6 in a protein mixture

[0268] To investigate whether peptide 13 selectively crosslinks to 16E6 in a protein mixture modification of Sumo-MDM2 which contains one cysteine and bovine serum albumin (BSA) which has 35 cysteines were monitored. A mixture of three proteins, including 16E6 (1 μM), BSA (1 μM), and Sumo-MDM2 (1 μM) was incubated with peptide 13 (10 μM) at 37 °C for 12 h and resolved by LC-MS (FIG.10D). The results indicate the 16E6 is mono-crosslinked with >95% conversion with no observable modifications to BSA or MDM2. These results demonstrate crosslinking of peptide 13 is specific to MBP-16E6 and depends on the hotspot motif LXXLL. Assessment of 13-biotin and 13-TAMRA specificity in HT1080 cells

[0269] HT1080 cells (CCL-121, ATCC) were cultured until they reached ~80% confluency, at which point they were harvested using trypsinization and subsequent centrifugation at 500 x g for 5 minutes. Resulting cell pellets were washed with 1x PBS and lysed via the introduction of cold Pierce IP lysis buffer (catalog no.87787), supplemented with 1x Halt Protease Phosphatase inhibitor cocktail (ThermoFisher, catalog no.1861284). This lysis process was conducted on ice for 10 minutes, with periodic pipetting to ensure adequate mixing. Post-lysis, the supernatant was cleared by centrifuging at maximum speed using a bench-top Eppendorf centrifuge (model #5425R) for 10 minutes at 4°C. After removing the supernatant, the resulting lysates were quantified using the Pierce 660 nm Protein Assay (catalog no.22660), with BSA standards serving as a reference.

[0270] HT1080 lysate (50 μg) was supplemented with either MBP-16E6 (2 μM) or MBP-16E6 C58S (2 μM), followed by an overnight incubation at 4°C with the presence of 13-TAMRA (10 μM), 13-biotin (10 μM), or 13-3L3A-biotin (10 μM). Each reaction was diluted in 1 x PBS supplemented with 1 mM DTT and 5% glycerol to a final volume of 20 μL while maintainingAttorney Docket No.: CLS-035WO PATENT DMSO concentration constant at 0.5%. Resulting reactions were subjected to 4-20% Criterion TGX polyacrylamide gel electrophoresis (BioRad). Samples containing 13-TAMRA were visualized on an Amersham ImageQuant 800 using the Cy2 settings. Protein ladder (LI-COR; catalog no.928-60000) was also imaged using IR short and IR long settings. Following this, all gels underwent transfer onto 0.2 ^m nitrocellulose (catalog no.1704159, BioRad) using the ‘Mixed Mw Turbo’ mode of the Trans-Blot Turbo Transfer System (catalog no.1704150, BioRad). Post-transfer, membranes were rinsed with ultrapure water, stained with Ponceau S staining solution (Thermo Scientific; catalog no. A40000278) for 5 minutes, washed again, and imaged on a BioRad ChemiDoc MP Imaging System with visible light.

[0271] Membranes were then blocked with Intercept (TBS) blocking buffer (catalog no. 9276001, LI-COR), followed by overnight incubation at 4°C with shaking in Intercept (T20, TBS) Antibody Diluent buffer (catalog no.92765001, LI-COR) incubated with Į-HPV16E6 antibody (catalog no. GTX132686, Genetex, lot no.44601) and Į-GAPDH (catalog no. MA5- 15738, lot no. WL332983), each diluted to 1:1,000. Blots were then washed four times for 10 minutes each with TBS-T, and incubated with goat-anti-mouse (680 nm conjugate; catalog no. 925-68070, lot no. D10901-11) and goat-anti-rabbit (800 nm conjugate; catalog no.926-32211, lot no. D20322-15) secondary antibodies for 2 hours at room temperature with shaking. This was followed by an additional series of four 10-minute washes with TBS-T.

[0272] 13-biotin and 13-3L3A-biotin blots were visualized using the ODYSSEY CLx LI-COR instrument with Image Studio software (version no.5.2). The scanning parameters included automated intensity, 169 ^m pixel size, and 0.0 mm offset. After imaging for GAPDH and MBP- 16E6, the 13- biotin and 13-3L3A-biotin blots were incubated with IRDye 680 RD streptavidin (LI-COR, catalog no.926-68079, lot no. D20803-07), at a 1:2,000 dilution in Intercept antibody diluent for 30 minutes. The blots were then washed four times for 10 minutes each with TBS-T and subsequently imaged. These initial conditions led to signal saturation, prompting the blots to be stripped using the NewBlot Nitrocellulose stripping buffer (LI-COR, catalog no.928-40030, lot no. D30111-04) for 5 minutes, following the manufacturer’s instructions. After stripping, the blots were re-imaged as previously described, resulting in an unsaturated signal.

[0273] Blots containing 13-TAMRA were visualized using the ODYSSEY M LI-COR instrument paired with LI-COR acquisition 1.1 software. Scanning was carried out in the 700, 800, and 488 nm channels using the ‘membrane’ assay settings and 100 ^m resolution. To ensure reproducibility, all experiments were conducted at least three times. The figures herein represent typical gels and blots from these repeated experiments.Attorney Docket No.: CLS-035WO PATENT

[0274] To investigate selectivity of peptide 13 in the context of the proteome, the HPV negative lysate was first incubated from HT1080 cells supplemented with recombinant MBP–16E6 or MBP–16E6 C58S then spiked in 10 ^M of 13-TAMRA at 4 °C overnight. After resolution of the lysate by SDS-PAGE, the gel was scanned in the TAMRA channel which displayed selective covalent binding for MBP–16E6 over MBP–16E6 C58S (FIGS.37A-37D). Despite this high concentration used in the assay (>500-fold higher than 13's binding affinity to 16E6), only a modest degree of fluorescent signal was distributed across the proteome with a long exposure time suggesting that 13-TAMRA has a minor degree of non-specific covalent modifications (FIG.37B). As an orthogonal approach, 13-biotin (10 ^M) or 13-3L3A-biotin (10 ^M) were incubated with 50 ^g of lysate and resolved by SDS-PAGE followed by western blot analysis probing with fluorescently labeled streptavidin which recognizes biotin (FIGS.38A-38D). A signal at ^60 kDa corresponding to MBP–16E6 was detected in the active 13-biotin reactide lane, but not in any of the other conditions, supporting 13-biotin's selectivity (FIGS.38A–38C). However, general background levels of signal were also observed in both 13-biotin and 13- 3L3A-biotin at 10 ^M, both of which contain the reactive Dha warhead, suggesting that 13- biotin and 13-3L3A-biotin at 10 ^M have a modest degree of non-specific covalent modifications. Background signal was not observed in peptide-free conditions suggesting that the signal originates from the peptide (FIG.38D). LC-MS / MS analysis of pull-down by biotinylated peptides from cells

[0275] To gain insight into the identity of the reactome, 13-biotin, 13-3L3A-biotin (5 ^M) or DMSO was incubated in 2 mg of HPV16+ CaSki or HPV-HT1080 lysate overnight at 4°C. Subsequently, the biotinylated peptides were enriched using streptavidin-conjugated magnetic beads followed by trypsin digestion and analysis by LC-MS / MS to identify peptide-associated proteins.

[0276] HT1080 and CaSki (CRL-1550, ATCC) cells were grown to 70-80% confluency and harvested by trypsinization and centrifugation (500 x g, 5 min). Pellets were subsequently washed with 1 x PBS and lysed by the addition of cold Pierce IP lysis buffer (cat. no.87787) supplemented with 1x Halt protease phosphatase inhibitor cocktail (ThermoFisher, cat. no. 1861284) for 10 minutes on ice, with intermittent pipetting to mix. The supernatant was cleared by centrifugation at max speed on a bench-top centrifuge (10 min, 4°C). Supernatant was removed and lysates quantified using a 660 nm protein assay (Pierce, cat. no.22660). Each lysate replicate (2 mg) was incubated with either DMSO (final conc.0.5%), 5 μM of 13-biotin or 5 μM of 13-3L3A-biotin in a final volume of 200 μL in IP lysis buffer (Pierce) overnight at 4°C.Attorney Docket No.: CLS-035WO PATENT For each condition, 200 μL of streptavidin magnetic beads (Pierce, cat. no.88816) were prepared according to the manufacturer’s instructions and incubated with the biotin-peptide treated lysate for 2 h at RT with rotation. Beads were subsequently washed 3× 1 mL with RIPA buffer (ThermoScientific; cat. no.8990) with the addition of 1× Halt Protease Phosphatase inhibitor cocktail, and 3× 1 mL with 50 mM HEPPS, pH 8.5 buffer. After the final wash, beads were resuspended in 50 mM HEPPS (pH 8.5) for on-bead reduction, alkylation and trypsin digestion. The samples were reduced with 5 mM BondBreaker TCEP (1h, RT, shaking), then alkylated with 10 mM iodoacetamide (30 min in the dark,RT, with shaking). Trypsin was added at a final concentration of 0.5 mg / ml for overnight digestion at 37°C with shaking. Peptides were collected in new tubes and placed on ice until ready for Tandem mass tag (TMT) labeling.

[0277] TMTpro reagents were resuspended in anhydrous acetonitrile to a concentration of 20 mg / mL, then 15 μl of each TMTpro reagent (126, 127n, 127c, 128n, 128c, 129n, 129c, 130n, 130c, 131n, 131c, 132n) was used to label the individual samples. Following incubation at 25°C for 1 hour, the reaction was quenched with 5% hydroxylamine to a final concentration of 0.3% hydroxylamine. The samples were acidified to a final concentration of 0.5% TFA and then all samples combined. The combined sample was desalted with StageTip as previously described and dried under vacuum

[0087] .

[0278] Peptides were analyzed on an Orbitrap Fusion Lumos mass spectrometer coupled to an EASY-nLC 1200 (Thermo Fisher Scientific). Peptides were separated on an IonOpticks Aurora Ultimate C18 column (1.7 μm particle size, 120 Å pore size, 25 cm length × 75 μm internal diameter) with the system operating at a flow rate of 300 nL / min and the column heated to 60°C. Peptides were eluted into the mass spectrometer using a 180-minute method with acetonitrile increasing over a 165-minute linear gradient from 8 to 30% in 0.125% formic acid.

[0279] Data-dependent acquisition (DDA) mode was used for mass spectrometry data collection. A high resolution MS1 scan was collected in the Orbitrap (500-1200 m / z range, 60,000 resolution, AGC 5×105, 30% RF lens, 100 ms max. injection time), and the top 10 precursors were selected for MS2 followed by MS3 analysis. For MS2, ions were isolated using a 0.5 m / z window. The MS2 scan was performed in the quadrupole ion trap (CID, AGC 1x104, 34% fixed collision energy, 35 ms max. injection time). The MS3 scan was analyzed in the Orbitrap (HCD, 60k resolution, max. AGC 5×104, 45% normalized collision energy, 250 ms max. injection time). For TMT reporter ion quantification, up to 6 fragment ions from each MS2 spectra were selected for MS3 analysis using synchronous precursor selection (SPS).Attorney Docket No.: CLS-035WO PATENT

[0280] An in-house software pipeline was used to process all proteomics data

[0088] . Raw files were converted to mzXML files and searched against a composite human UniProt database containing forward and reverse sequences with HPV16 and HPV18 E6 sequences added (P03126 and P06463) using the Sequest algorithm. Database searching matched MS / MS spectra with fully tryptic peptides from this composite dataset with a precursor ion tolerance of 20 ppm and product ion tolerance of 0.6 Da. TMTpro modification of peptide N-termini and lysine residues (+304.207146 Da) and carbamidomethylation of cysteine residues (+57.021464 Da) were set as static modifications. Oxidation of methionine residues (+15.994914 Da) was set as a differential modification. Peptide spectral matches were filtered to a 1% false discovery rate (FDR) using linear discriminant analysis (LDA) as previously described

[0088] . Non-unique peptides that matched to multiple proteins were assigned to proteins that contained the largest number of matched redundant peptide sequences using the principle of Occam’s razor

[0088] . TMT reporter ion intensities were quantified by extracting the most intense ion within a 0.003 m / z window at the predicted m / z value for each reporter ion. Peptide intensities and signal-to-noise ratios were exported and analyzed using the msTrawler software package. Default settings were used with the exception of turning off the column / TMT channel normalization since differences in abundance between samples was to be expected

[0089] .

[0281] The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD044352.

[0282] Approximately 372 proteins were identified across all tested conditions in both the HT1080 and CaSki cells, with 13-biotin showing the highest magnitude of enrichment (FIG. 39A). Subsequently, pairwise analysis was conducted across the conditions for both HT1080 (FIGS.39B–39D) or HPV16+ CaSki cells (FIGS.39E-39G) which did not reveal clear enrichment patterns; however, a major signal was observed for Rpn1 which has been reported to interact with E6AP.79Of note, HPV16 E6 was not identified in CaSki cells, which may be attributed to the low abundance of this protein or inaccessibility of the peptide binding site in a cellular environment. Nevertheless, 13-biotin or 13-3L3A-biotin had only modest nonspecific enrichment of other proteins in both cell lines. Taken together, these data suggest that 13- biotin recognizes supplemented MBP–16E6 but not MBP–16E6 C58S with a minor to modest degree of non-specific conjugation to other proteins from lysate.Attorney Docket No.: CLS-035WO PATENT EXAMPLE 9 – Disruption of 16E6 / E6AP Interaction by Reactide 13.

[0283] This example describes the determination of the disruption of 16E6 / E6AP interaction by reactide 13, by a 16E6 / E6AP BLI binding assay using immobilizing biotinylated E6AP protein onto streptavidin tips.

[0284] Briefly, E6AP protein was biotinylated by a commercial BirA kit. In brief, 40 ^L of E6AP was added directly into a tube containing 20 μl of 1 mg / ml BirA biotin-protein ligase. Then the solution was added with 5 μL of BiomixB (10× concentration: 100 mM Adenosine 5ƍ- triphosphate, 100 mM MgOAc, 500 ^M d-biotin), followed by the addition of 5 μL of BIO200 (d-biotin; 10× concentration: 500 ^M). The whole mixture was incubated at 4°C for 16 hrs, followed by a spinning filter through 0.2 ^m. Excess biotin was purified away on Superdex200 or AdvanceBio 150mm (Agilent, MA) equilibrated with PBS + 1mM DTT. Biotin-E6AP fractions were collected and frozen at -80°C before use.50 μL of 12 μM MBP-E6 in PBS was added with 0.5 μL of 10 mM reactive peptide of interest (final concentration 100 μM). The mixture was incubated at 4°C for 16 hrs. One μM of Biotin-E6AP protein was immobilized onto streptavidin sensors soaked in a blocking buffer. The sensors were then dipped into the mixture of MBP-E6 and reactive peptide. The signal response was recorded.

[0285] The 16E6 / E6AP interaction was evaluated as the response (in nm) during the association step when dipped into recombinant MBP-16E6 solution (1 μM). MBP-16E6 conjugated with reactide 13 had a significantly decreased BLI response signal to the immobilized E6AP protein, while the control reactide 13-3L3A had no impact (FIG.10E). The decrease in BLI response signal indicates that the 16E6-13 conjugate loses its E6AP-binding ability, as its E6AP binding pocket was occupied by 13. EXAMPLE 10 – Structural and Molecular Modeling of E6-Peptide-13 Complex

[0286] This example describes molecular modeling of the mechanism by which reactide 13 disrupts the 16E6 / E6AP interaction using molecular docking and molecular dynamics (MD) simulations.

[0287] The MBP tag may affect the conformation of the isolated E6AP-based peptide from the ternary complex structure with 16E6 and p53 (PDB: 4XR8). Therefore, the MBP tag and p53 protein was removed from the structure and a 1.1 ^s simulation of the 16E6-bound E6AP- LXXLL peptide was performed.

[0288] Briefly, peptide-13 consists of a flexible N-terminal region (9-fluorenyl acetamido- IPQSA), the native E6AP core (ELTLQEELL), and flexible C-terminal (Dha-RRKK-K (1-Attorney Docket No.: CLS-035WO PATENT anthracenyl acetamido)) segments, as shown by its sequence (FIG.9B). The E6AP peptide, which interacts with the E6 protein and functions as a ubiquitin ligase, has an alpha-helical conformation according to the x-ray E6-MBP(E6AP)-P53 ternary complex (PDB ID: 4XR8). To investigate whether the native core sequence of E6AP in Peptide-13 also adopts an alpha helix fold, the x-ray complex was used as a starting point, with removed MBP and P53 proteins, and the E6-bound E6AP peptide was kept. A MD simulation in a box (60.0Å * 60.0Å * 60.0Å) of water (~6K water molecules) and ions was performed and found that the E6AP peptide remained stable (FIG.14), with an r.m.s.d. of 0.4 Å (measured using the Cα atoms) compared to the E6AP peptide in the ternary complex.

[0289] Additional modeling using Molecular Operating Environment (MOE) software (MOE, Canada), MOE docking module for molecular docking, and MD simulations to further refine the E6-Peptide-13 complexes in the presence of water and ions were used. Briefly, using the MOE software, the alpha helix E6AP core was expanded upon by incorporating the flexible N-terminal and C-terminal sequences to construct the Peptide 13 molecule (FIG.15B). To optimize the Peptide 13 structure, the LowModeMD algorithm

[0090] , which is integrated into MOE, was employed. The sampling was limited by imposing the following parameters defined in the MOE package: {Rejection Limit=100; Iteration Limit=10000; RMS Gradient=0.005, MM Iteration Limit=500; RMSD limit=0.75 Å; Energy Window =10.0 kcal / mol; Conformational Limit=10,000}. Additionally, the backbone atoms of the E6AP peptide were treated as a rigid segment to maintain the alpha-helix conformation during the calculations.

[0290] To determine how Peptide 13 binds to the 16E6 protein (FIGS.15C and 15D), the MOE docking module was used for molecular docking, which is known to be reliable for capturing peptide / receptor interactions[91,92]. To ensure the accuracy of the approach, molecular docking was first performed to determine the binding mode of the native E6AP peptide to the crystallographic 16E6 protein, using the X-ray E6-bound native E6AP peptide (PDB ID: 4XR8) as the starting point after eliminating the MBP and p53 from the ternary complex. A docking protocol was employed that started with the triangular matcher algorithm to quickly generate 1,000 poses of docked peptides based on the receptor shape. From the resulting docked poses, the London dG scoring function was used to screen among the resulting poses to keep the top 100 structures for further refinement. Then, the rigid receptor replacement method combined with the GBVI / WSA dG scoring function was used to refine the resulting poses, obtaining an E6-bound E6AP peptide that closely matched the crystallographic binding mode of E6AP (FIG. 16). Similarly, the same docking procedure was followed to obtain top 3 docking poses of 16E6-Attorney Docket No.: CLS-035WO PATENT Peptide-13 complexes using the pre-generated conformations of peptide 13 (described above) to dock to the binding pocket of the native E6AP peptide in the 16E6.

[0291] Next, ~60 ns of constrained MD simulations was performed to further refine the E6- Peptide-13 complexes in the presence of water and ions. The goal was to push the peptide-13 warhead (Dha) toward the 16E6 Cys58 hotspot and create a covalent link between the two. To achieve this, a harmonic restraint was placed at 3.0 Å between Cys58 (S atom) and Dha (C atom), with a force constant of 0.36 kcal mol-1 Å-2. These simulations allowed to prepare the 16E6-Peptide-13 constructs for the formation of the covalent link between Dha and Cys58.

[0292] Subsequently, Cys58 and Dha were covalently linked using MOE followed by energetically minimizing the covalent link and the 16E6-Pepitde-13 complexes. Each of three complexes were then subjected to a ~120 ns classical MD simulation to characterize the binding mode of Peptide-13 covalently linked to the 16E6 protein. The resulting final complexes closely match the crystallographic E6AP binding peptide (FIG.17). For further analysis, the structure featuring the largest binding affinity between the peptide-13 core sequence and E6 while exhibiting the lowest RMSD of the peptide-13 core sequence compared to the crystallographic native E6AP peptide was selected. For the results presented in FIGS.13A-13F, GROMOS algorithm

[0093] with RMSD cutoff=0.18Å was used to cluster the entire MD trajectory of the best 16E6-Peptide-13 complex.

[0293] A model of peptide 13 was built by incorporating the flexible N- and C-terminal sequences into the alpha helix of the E6AP LXXLL peptide and extensive conformational sampling calculations to optimize the reactide 13 model were performed. The covalent bond formed between 13 and 16E6 Cys58 was not considered when initiating the molecular docking to avoid any biases in the calculations. The docking result suggested that the primary driver of 13 binding to 16E6 are electrostatic and van der Waals interactions (FIG.13A). By refining the top candidates obtained from the docking calculations using MD simulations, the Dha (C atoms) resides within 3.7 Å from Cys58 (S atoms), confirming optimal placement for 13 to cross-link with 16E6. The thioester bond between 16E6 and Dha acts as a covalent lock, securing 13 at the 16E6 / E6AP interface. Trajectory r.m.s.d analysis (FIG.13B) for the 16E6 and 13 complex and 13 alone indicated that the covalent bond between Cys58 and Dha is instrumental in stabilizing 13 and effectively restricts the flexibility of the E6AP-LXXLL core sequence with an r.m.s.d of 1.5 ± 0.3 Å. The molecular simulations reveal that reactide 13 occupies the same position as the parent E6AP LXXLL peptide (r.m.s.d = 1.9 Å, measured for Cα atoms), leading to the blocking of E6AP / 16E6 binding (FIG.13C).Attorney Docket No.: CLS-035WO PATENT

[0294] The modeling suggests important side chain interactions between 13 and 16E6 basic residues, including E6AP Glu6 -16E6 Arg138 and E6AP Glu11 -16E6 Arg11.16E6 Arg138 is crucial as Arg138 mutation reduces E6AP recruitment and interactions substantially. In addition, 16E6 Arg17 forms a salt bridge with the C-terminus of the E6AP core peptide. The Glu6 carboxylate group is essential for 13 binding and establishes an additional network of hydrogen bonding with Ser81 and His85 from 16E6 (FIG.13D).16E6 Arg136 forms a hydrogen bond with Gln10 in the E6AP core. The keystone 16E6 Arg109 interacts with Leu13 through hydrophobic interactions, consistent with crystallographic observations. Notably, the alanine substitutions of both Arg136 and Arg109 led to impaired peptide binding, highlighting their crucial roles in stabilizing the 16E6 / E6AP complex, though R136 exhibited conformational disorder and was oriented away from the peptide towards MBP. Collectively, 13 selectively targets all residues appearing in the binding pocket of E6 to disrupt the binding interface of 16E6 and E6AP.

[0295] The N- and C-terminal groups are located on flexible loops and dynamically bind to 16E6, making it challenging to identify static interactions similar to those found for the core motif. However, despite their dynamic binding, the N- and C-terminal groups still interact with important surfaces of E6. This flexibility and ability to cover essential surfaces of 16E6 may prevent 16E6 from recruiting E6AP (FIG.13E and 13F). Notably, the binding interface of the N and C-terminal groups includes several key residues that are essential in the 16E6-E6AP complex. Therefore, these results suggest that hydrophobic modifications contribute to the inhibition of 16E6. MD Simulation protocol

[0296] Before running molecular docking and MD simulations, the MOE preparation module was used to refine the crystallographic E6 protein and the missing sidechains / residues were added. The 16E6- pepitde complexes were then immersed in a water / ion (150 mM excess ions) box and underwent 500 steps of energy minimization using the steepest descents algorithm in GROMACS

[0094] . The refinement was followed by an MD simulation in a canonical ensemble, where the system was heated gradually from 0 K to 310 K in 20 ps, followed by MD simulations in an isobaric-isothermal ensemble for an aggregated 80 ps, during which the pressure was maintained at 1 bar to relax the simulation box. Throughout these pre-equilibration steps, the positional restraints were placed on all heavy atoms, gradually reduced to 0 kcal.mol-1Å2 for the final equilibration step. Finally, the 16E6-pepitde constructs were optimized by removing the positional restraints.Attorney Docket No.: CLS-035WO PATENT

[0297] In all simulations, the AMBER-14 force field parameter set

[0095] was used to describe 16E6, Peptide-13, E6AP peptide, and ions. Non-canonical amino acid residues were parameterized with the Antechamber program

[0096] to ensure compatibility with the AMBER force field, while the force field parameters for two tagged molecules were borrowed from the Generalized AMBER force field (GAFF)

[0097] . The TIP3P model was utilized to describe water.

[0298] The temperature was maintained at 310 K using a velocity-rescale

[0098] thermostat with a damping constant of 1.0 ps for temperature coupling and the pressure was controlled at 1 bar using a Parrinello-Rahman barostat algorithm

[0099] with a 5.0 ps damping constant for the pressure coupling. Isotropic pressure coupling was used during this calculation. The Lennard-Jones cutoff radius was 12 Å, where the interaction was smoothly shifted to 0 after 10 Å. Periodic boundary conditions were applied to all three directions. The Particle Mesh Ewald algorithm

[0100] was used to calculate long-range coulombic interactions with a real cutoff radius of 10 Å and a grid spacing of 1.2 Å. A compressibility of 4.5 ×10-5 bar-1 was used to relax the box volume. In all the above simulations, water OH bonds were constrained by the SETTLE algorithm

[0101] . The remaining H-bonds were constrained using the P-LINCS algorithm

[0102] . All MD simulations were carried out using GROMACS, with constrained MD simulations aided by PULMED

[0103] . EXAMPLE 11 – Identification Of Reactive Peptides Mimicking E6AP

[0299] This example describes the design and identification of reactive peptides based on E6AP that target HPV16 E6 (E6).

[0300] Briefly, the structure of the complex formed between E6, E6AP, and p53 was analyzed (PDB: 4XR8) and a truncation study on E6-E6AP interaction sites revealed the main recognition domain–a peptide fragment of E6AP (residues 406-417), termed ELT peptide: ELTLQELLGEER (SEQ ID NO: 5).

[0301] Binding was measured with BLI assay as described above and fluorescent polarization (FP) assay. Fluorescent polarization(FP) direct binding assay

[0302] Direct FP binding assay of FITC-ELT (FITC-ELTLQELLGEER) and FITC-IPESS (FITC-IPESSELTLQELLGEER) (FIG.21 and FIG.22). FP was recorded after incubating various concentration of MBP 16E6 with 100nM FITC- ELTLQELLGEER. FP was performed with a BioTek H1 plate reader. Excitation: 485 / 20, Emission: 528 / 20. (Green) Filter set. Read Height: 10.5 mm. Gain sets to 50. Temperature 27ÛC.Attorney Docket No.: CLS-035WO PATENT

[0303] The results show, that ELT peptide had a mid-micro molar binding affinity to 16E6, about 6 μM by direct biolayer interferometry (BLI) assay with biotin-ELT, and about 3.7 μM by direct fluorescent polarization (FP) assay with FITC-ELT to the recombinant MBP-16E64C4S protein (MBP-16E6) (FIG.21). The MBP-16E6 was stabilized and solubilized by a maltose- binding protein (MBP) tag and four cysteine to serine. The peptide fragment contains a common binding motif LXXLL that are critical for binding (see also Example 7).

[0304] To overcome the fast koff rate and disrupt the 16E6-E6AP interaction, E3 (ELT-G9Dha) was developed, a cysteine reactive peptide containing dehydroalanine (Dha) that mimics E6AP. This peptide showed an apparent Ki of 10 μM by FP and apparent Ki of 2μM by BLI (FIG.21).

[0305] To dissociate the peptide-protein complex, the Dha warhead that forms irreversible bonds was replaced with a cysteine that is capable of forming reversible disulfide bonds. Disulfide dimers ELT-G9DS and ELT-E10DS, were prepared which showed crosslink yields of 75% and 45%, respectively. Despite not having the highest crosslink yield, ELT-G9DS was chosen to match with ELT-G9Dha (ELT with Dha substitution).

[0306] Three different small molecule-peptide mix disulfides were prepared: cysteine (Cys), cysteamine (ȕME) and N-acetyl cysteamine (NACȕME) . Using an on-resin synthesis, a mix cysteamine-peptide disulfide was produced on resin with quantitative yield. Among the disulfides, ȕME disulfide (ȕME-DS) showed the fastest crosslink yield of 86%, while NACȕME disulfide (NACȕME-DS) yielded 37%, which was twice that of the free cysteine yield of 16%.

[0307] After screening the ELT-G9DS and ELT-E10DS disulfides mix with small molecules, cysteine peptides were identified. In the resynthesis, Cys was replaced with Dha substituted Gly13. EXAMPLE 12 –Design and Screening of a 1.42-Million-Member Peptide Library

[0308] 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.

[0309] Briefly, a library with 1.42 million members was designed based on the ELT peptide ELTLQELLGEER.

[0310] Results from the Alanine scan showed that alanine mutations in the residues E1 (E1A) and E6 (E6A) of the ELT peptide decreased binding affinity to MBP-16E6 by 9-fold and 16-fold respectively(FIG.23). 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. TheAttorney Docket No.: CLS-035WO PATENT four Leu residues were mutated to aliphatic and aromatic sidechains. Thr3 position was replaced by Thr analogs and β-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 3L3A) were used as negative control peptides. The library had the consensus sequence X1X2X3X2QX1X2X2CEER (SEQ ID NO: 6) with X1, X2, X3, and X4chosen from FIG.24 (X1(Nva, Leu, Aoc, Cpa, Cba, Cha, Phg, Hof, and Naf), X2(Aad, Glu, and Cya), X3(Thr, Asn, Hyp, Cpg, Cbg, and Ceg), and X4(d-Ala, Aib, Gly, and Gln)). Residue G9 replaced with a Cys.

[0311] The ELT peptide library was screened with the ReAct-ASMS method (FIG.25). 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 μM. This platform enables affinity maturation on covalent PPI peptide inhibitors using non-canonical amino acids.

[0312] To mature the E6AP-mimicking peptide, ReAct-ASMS libraries were prepared with fast crosslinker -βME-DS and slow crosslinker NACβME-DS. Library I-βME and library I- NACβME were individually screened against MBP-16E6. With the same amount of incubation time, library 1-βME generate an average of 7 hits with ALC>90, 7-fold more than library I- NACβME 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.

[0313] ReAct-ASMS with MBP-16E6 identified peptides from the X1X2X3X2QX1X2X2CEER 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-βME 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 stringentAttorney Docket No.: CLS-035WO PATENT 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.26A).

[0314] Additional peptide libraries screened are listed in TABLES 3 to 31. ASMS of MBP 16E6 TABLE 3Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 4 Quality control of peptide E6 library IIdentified binders from peptide E6 library I-bME (ALC>80) TABLE 5 Condition I-1.1(1)Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 6 Condition I-1.2(1)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 7 Condition I-1.3(1)Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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-1.1(2)TABLE 9 Condition I-1.3(2)Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 10 Condition I-1.3(2)Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 11 Condition I-1.1(3)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (Aoc): k, Homo phenylalanine (Hof): m, PhenylAttorney Docket No.: CLS-035WO PATENT 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 12 Condition I-1.2(3)Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 13 Condition I-1.3(3)Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 14 Condition I-2.1Attorney Docket No.: CLS-035WO PATENTAttorney Docket No.: CLS-035WO PATENTAttorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 15 Condition I-2.2Attorney Docket No.: CLS-035WO PATENTAttorney Docket No.: CLS-035WO PATENTAttorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 16 Condition I-2.3Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 17 Condition I-2.4Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 18 Condition I-3.1Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (Aoc): k, Homo phenylalanine (Hof): m,Attorney Docket No.: CLS-035WO PATENT 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 19 Condition I-3.2Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 20 Condition I-4.1Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 21 Condition I-4.2Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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.Attorney Docket No.: CLS-035WO PATENT TABLE 22 Condition I-4.3Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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. Condition I-4.4 No hit TABLE 23 Condition I-3.3Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 24 Condition I-3.4Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 25 Condition I-4.5Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 26 Condition I- 4.6Attorney Docket No.: CLS-035WO PATENTCyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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. Identified binders from peptide E6 library I-NACbME (ALC>80) TABLE 27 Condition I-5.1TABLE 28 Condition I-5.2TABLE 29 Condition I-5.3Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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.Attorney Docket No.: CLS-035WO PATENT TABLE 30 Condition I-5.4Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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 31 Condition I-5.5Cyclobutylglycine (Cbg): b, Cyclopropylglycine (Cpg): d, Cyclopentylglycine (Ceg): x, Cyclopropylalanine (Cpa): i, Cyclobutylalanine (Cba): h, Cyclohexylalanine(Cha) :j, Octanonic (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.Attorney Docket No.: CLS-035WO PATENT EXAMPLE 13 – Analysis of Peptide 18 HPV16E6 binding in Crosslink Assays

[0315] To compare crosslink efficiency of peptides identified in the library screen, eleven identified peptides were synthesized as Dha peptides replacing the Cys with a DHA group. The peptides (Fig.26A) and the parent ELT-DHA9G were mixed one to one with MBP-16E6 protein for 2 h, at 37 ^. The mono-crosslinked MBP-16E6-peptide 18 conjugate was the only observed conjugate with a yield of 83%, indicating that peptide 18 outcompeted other peptides in the mixture to react with MBP-16E6 (FIG.26B).

[0316] The DHA reactides were further evaluated by the BLI competition assay against peptide 1-biotin to estimate their inhibitory constant, which is report here as an apparent Ki value. Six peptides showed an improved apparent Ki, peptide 3 (Ki of 466 nM), peptide 6 (Ki of 288 nM), peptide 8 (Kiof 302 nM), peptide 10 (Kiof 815nM), peptide 18 (Kiof 177 nM) and peptide 23 (Kiof 179 nM). The other peptides showed comparable binding affinity to the parent reactide ELT-DHA9G (FIGs. 27A-D).

[0317] Orthogonally, a fluorescent polarization assay was performed to ELT-DHA9G (IC50 of 10 μM), 14 (IC50 of 766 nM), 18 (IC50 of 330 nM), and 23 (IC50 of 389 nM). In both competitions based binding assays, peptide 18 demonstrated the highest inhibitory effect to MBP-16E6, therefore, was selected for further validation.

[0318] Peptide 18 and its structural analog peptide 23 showed improved 50-fold improved affinity compared to parent reactide ELT-DHA9G.18 and 23 only differ in their aliphatic side chains in position 4 and 7, where Leu was replaced with cyclobutyl-alanine or cyclopropyl- alanine (FIG.27A). The negative control derivative of 18, (18-3L3A) showed no observable binding to MBP-16E6 protein. Three major structural changes, Leu2Naf, Thr3Ceg and Gln5Aib were observed with 18, which make 18 distinct from the parent peptide ELT-DHA9G. Noticeably, substitution Leu8Hof in peptide 14 showed decrease 2-fold lower binding affinity compared to peptide 18, may showed that the position 8 has lower tolerance for aromatic modification. Compared to the Thr3Ceg in peptide 18, Thr3Cbg in 14, position 3 may require a larger ring in the cyclic sidechain to improve affinity (FIGs.27-B-D).Attorney Docket No.: CLS-035WO PATENT

[0319] TABLE 40

[0320] Reactide 18 was further evaluated for crosslinking MBP-16E6. Reactide 18 uses a two- stage bind-and-react strategy to covalently crosslink to MBP-16E6 and block the E6AP binding site. Reactide 18 (10 μM) and MBP-16E6 (1 μM) were incubated in PBS at 37 °C for two hours, and subsequent protein deconvolution mass spectra revealed >95% mono-crosslinking of 18 to MBP-16E6 (FIG.28A). This was achieved despite the presence of 10 cysteine residues on MBP-16E6. with selectively crosslinks to MBP-16E6. The negative control 18-3L3A showed no observable crosslinking to MBP-16E6 protein (FIG.28B), supporting reactide 18 specificities. Therefore, 18-3L3A was used as a negative control for subsequent peptides. To confirm site- selectivity, reactide 18 was incubated with the MBP-16E6 mutant C58S for 12 h in PBS at 37 °C. No crosslinking was observed between reactide 18 and MBP-16E6 C58S (FIG.28C), which supports Cys58 as the sole site of MBP-16E6 modification. L57A mutant is a critical residue that are important for full-lengthen E6AP / 16E6 PPI (cite), no crosslinking was observed between reactide 18 and MBP-16E6 L57A (FIG.28D), indicating that E6AP binding activity is required for reactide 18 to crosslink.Attorney Docket No.: CLS-035WO PATENT

[0321] A kinetic study was performed with reactide 18 to estimate the binding constant Ki and first-order rate constant (kinact) involved in the two-stage bind-and-react strategy. Crosslink yield was monitored over time by LC-MS (FIG.28E and FIG.28F). Kinetic parameters were estimated assuming a steady-state approximation, and a kinact of 0.018 s-1, a Ki value of 792 nM and a kinact / KI ratio of 22.7 mM-1 s-1. Reactide 18 represents a 50-fold improvement over ELT-DHA9G (kinact of 0.022 s-1, Ki of 50 μM and kinact / Ki ratio of 446 M-1 s-1). EXAMPLE 14 – N-terminally modified libraries of peptides

[0322] A library of E6AP-mimicking peptides (IPESS peptide) with N-terminal small molecule modifications was further synthesized and screened for binding of 16E6.

[0323] Exemplary small molecule moieties for N-terminal modifications are shown in FIG. 29A-29B. Exemplary small molecule moieties comprised: 2-Phenyl-4-quinolinecarboxylic acid, 1-(Phenylsulfonyl)-1H-indole-2-carboxylic acid, 6-Fluoro-2-naphthoic acid, 4- phthalimidobenzoic acid, Xanthene 9-carboxylic acid, 1-Pyrenebutyric acid, 5- Acenaphthenecarboxylic acid, 1-Phenyl-1H-indole-2-carboxylic acid, Indole-2-carboxlic acid, quinoline-4-carboxylic acid, and 10-(Carboxymethyl)-9(10H)acridone.

[0324] Among these moieties, fluorene showed the highest improvement. An analog campaign of fluorene reveals that N-terminal carbazole maintained the binding affinity improvement (FIG. 30). As carbazole derivatives are a common pharmacophore seen in FDA-approved drugs, i.e., Carbazomycin A, Staurosporinone, and Alectini46. Carbazole was thereafter, chosen as the small molecule to modify peptide 18.

[0325] A library of N-terminally modified peptides based on peptide 18 was further synthesized and screened for binding of 16E6. The results show, that N-term small molecule modification improve 18’s binding to MBP-16E6 (FIG.30). N-term carbazole modification improved peptide 18’s binding to 16E6 by 3-fold (18-Car showed an apparent Ki of 96 ± 33 nM). Substitution of the Naf2 position with Lys (Carbazole acetamido) improved binding, for example 18-Naf2Cab showed an apparent Ki of 51 ± 11 nM.

[0326] N-term elongated peptides of the E6 binder were previously shown to have a binding KDof 1–10nM. Reactide 18 with the N-terminal Carbazole-IPQSA modification (18-Car-IPQSA) showed an apparent Ki of 25 ± 5.5 nM. This N-term extension improved reactide 18’s binding to 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 residuesAttorney Docket No.: CLS-035WO PATENT from the peptide do not significantly affect binding where 18-Car-IPQSA-truncEER showed an apparent Ki of 44 ± 11 nM.

[0327] Crosslink assay was performed with peptide 18 analogs. (FIG.31A-31C).18-Car, 18- Naf2Cab, 18-Car-IPQSA and 18-Car-IPQSA-truncEER achieved quantitatively mono-crosslink in 2 h at 37 ^, 10 eq. to protein 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 molecule modifications can be viable approach to improve binding affinity and binding specificity. The structure-activity relationship (SAR) study expands the molecular diversity of peptide 18.

[0328] To further modify peptide 18, a peptide 18 based E6 library II-βME was designed (Aad- X1-X2-Leu-Aib-Aad-Cba-Leu-Cys-X3-X3-X3) (SEQ ID NO: 20) composed of 80,000 members (FIG.32, Tables 32-39). The X1 Naf2 position was mutated to Naphthalene analogs (PhF, Naf, Qua, Clw, and Trp) based on the SAR findings. The X2 Ceg3 position was mutated to Chg and Ceg. The last three residues positions X3 were mutated to d-Ala, Aib, Gly, Ser, Aad, Glu, Gln, Hoc, Val, Leu, Met, Pro, Dap, Lys, Arg, His, Trp, Phe, Tyr, and Null. The library was designed with a focus on the C-term EER residues, which were 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 ReAct-ASMS were performed on 16E6 with five conditions. Only conditions with more than 1 ng per member enriched binders, 2–20 unique hits with ALC >90 were identified. TABLE 32 Quality control of E6 library IIAttorney Docket No.: CLS-035WO PATENTPosition 3 Cyclopentylglycine (Ceg): x, Cyclohexylglycine (Chg): i, position 7 Cyclobutylalanine (Cba): x, 4-phenyl phenylalanine(Phf): j, 6-Cl tryptophan (ClW): m, QuinolylAlanine (Qua): n, Napthylalanine(Naf): o, Aminoadipic acid (Aad): b, Homocysteic acid (Hoc): z, Hydroxyproline (Hyp): p, Amino isobutyric (Aib): d TABLE 33 Identified binders from E6 library II330-091-01ASMS: Condition II-1: 40μg library (0.5ng / member), MBP-E640μg (No hit)Attorney Docket No.: CLS-035WO PATENT 330-091-02ASMS: Condition II-2: 80μg library (1ng / member), MBP-E640μg 330-092-01ASMS: Condition II-3.1: 100μg library (1.25 ng / member), MBP-E620μg 330-092-02ASMS: Condition II-3.2: 100μg library (1.25 ng / member), MBP-E620μg 330-092-03ASMS: Condition II-3.3: 100μg library (1.25 ng / member), MBP-E620μg 330-092-05ASMS: Condition II-4: 200μg library (2.5 ng / member), MBP-E620μg 330-092-04ASMS: Condition II-5: 300μg library (3.75 ng / member), MBP-E620μg TABLE 34 Condition II-2 Scan Peptide 12-mer ALC (%) m / z z RT Mass ppm Uniqueness 9211 bWiLdExLCbHp 97 774.4008 2 37.49 1546.774 8.4 UNIQUE 9069 bWiLdExLCdRE 91 762.9122 2 37.18 1523.801 6.2 UNIQUE 12509 WjxLdExLCAKK 88 774.4422 2 43.54 1546.854 10.6 UNIQUEScan Peptide 11-mer ALC (%) m / z z RT Mass ppm Uniqueness 10770 boxLdExLCRH 87 722.8878 2 40.28 1443.752 6.2 UNIQUE 11931 bjxLdExLCKR 84 731.4149 2 42.35 1460.806 6.5 UNIQUE 11284 RnxLdExLCRK 80 725.413 2 41.19 1448.816 -3.5 UNIQUE Position 3 Cyclopentylglycine (Ceg): x, CycloHexylglycine (Chg): i, position 7 Cyclobutylalanine (Cba): x, 4-phenyl phenylalanine(Phf): j, 6-Cl tryptophan (ClW): m, QuinolylAlanine (Qua): n, Napthylalanine(Naf): o, Aminoadipic acid (Aad): b, Homocysteic acid (Hoc): z, Hydroxyproline (Hyp): p, Amino isobutyric (Aib): d TABLE 35 Condition II-3.1 Scan Peptide 12-mer ALC (%) m / z z RT Mass ppm Uniqueness 7898 bWiLdExLCKbp 99 769.9179 2 36.73 1537.81 7.3 UNIQUE 10213 boiLdExLCVAK 97 732.4222 2 40.88 1462.812 12.1 UNIQUE 9661 boxLdExLCVGK 94 718.3994 2 39.91 1434.777 5.3 UNIQUE 8506 boxLdExLCQRd 93 760.9122 2 37.84 1519.799 7.5 UNIQUE 7767 bWiLdExLCQRd 92 762.4196 2 36.52 1522.816 5.4 UNIQUE 8651 boxLdExLCKbp 90 768.4146 2 38.16 1534.792 14.5 UNIQUE 9083 boxLdExLCGRd 90 725.3954 2 38.86 1448.762 10.2 UNIQUE 7502 bWxLdExLCKFH 89 776.9178 2 36 1551.812 5.9 UNIQUE 8603 boxLdExLCdRG 88 725.3952 2 38.08 1448.762 9.9 UNIQUE 10546 boiLdExLCAVK 87 732.4175 2 41.5 1462.812 5.6 UNIQUE 7990 bWiLdExLCRGp 86 740.9042 2 36.95 1479.775 12.9 UNIQUE 7914 bWxLdExLCdMY 84 760.385 2 36.82 1518.741 9.7 UNIQUE 9029 boiLdExLCKEp 81 768.4141 2 38.81 1534.792 14.1 UNIQUE 9110 boiLdExLCKbp 81 775.4219 2 38.96 1548.812 11.1 UNIQUEScan Peptide 11-mer ALC (%) m / z z RT Mass ppm Uniqueness 9688 boiLAEiLCQR 83 725.3954 2 38.86 1448.782 -3.6 UNIQUE 9838 boiLAExLCRR 81 732.4131 2 40.21 1462.804 5.2 UNIQUE Position 3 Cyclopentylglycine (Ceg): x, CycloHexylglycine (Chg): i, position 7 Cyclobutylalanine (Cba): x, 4-phenyl phenylalanine(Phf): j, 6-Cl tryptophan (ClW): m, QuinolylAlanine (Qua): n, Napthylalanine(Naf): o, Aminoadipic acid (Aad): b, Homocysteic acid (Hoc): z, Hydroxyproline (Hyp): p, Amino isobutyric (Aib): dAttorney Docket No.: CLS-035WO PATENT TABLE 36 Condition II-3.2 Scan Peptide 12-mer ALC (%) m / z z RT Mass ppm Uniqueness 8113 bWiLdExLCGKb 99 741.8891 2 38.09 1481.789 -16.8 UNIQUE 8633 bWiLdExLCAbR 98 762.918 2 38.97 1523.811 7.2 UNIQUE 6980 bWxLdExLCKQA 97 734.4079 2 35.94 1466.78 14.3 UNIQUE 8011 boxLdExLCbKQ 96 775.9196 2 37.89 1549.808 10.9 UNIQUE 8987 bWiLdExLCKVb 95 762.9294 2 39.64 1523.836 5.6 UNIQUE 9600 bWiLdExLCbVR 95 776.933 2 40.71 1551.842 6.2 UNIQUE 7306 bWiLdExLCQRd 94 762.4192 2 36.19 1522.816 4.9 UNIQUE 8404 boxLdExLCbQH 94 780.4025 2 38.6 1558.772 11.8 UNIQUE 10027 boiLdExLCVAK 94 732.4146 2 41.5 1462.812 1.7 UNIQUE 8459 boxLdExLCbHQ 93 780.4025 2 38.6 1558.772 11.8 UNIQUE 8699 boxLdExLCbGH 91 744.8862 2 39.12 1487.735 15.5 UNIQUE 8877 boiLdExLCREE 91 790.4153 2 39.52 1578.798 11.4 UNIQUE 9188 boiLdExLCVWb 91 797.4235 2 39.68 1592.822 6.5 UNIQUE 8480 boiLdExLCLFH 90 781.9266 2 38.74 1561.823 9.8 UNIQUE 8880 boxLdExLCKEF 90 778.4177 2 39.45 1554.798 14.8 UNIQUE 9013 boiLdExLCRbE 87 797.4235 2 39.68 1592.818 9 UNIQUE 9187 boiLdExLCLWb 87 804.4321 2 39.99 1606.838 7.4 UNIQUE 10876 boiLdExLCWHH 85 813.4177 2 43.06 1624.809 7.3 UNIQUE 8133 boxLdExLCMVp 83 747.8912 2 38.48 1493.743 16.3 UNIQUE 8233 boxLdExLCbKG 82 740.4008 2 38.31 1478.771 11 UNIQUE 8060 boxLdExLCdRG 80 725.3955 2 37.99 1448.762 10.3 UNIQUE 12918 bWiLdExLCGVE 80 720.3849 2 46.32 1438.742 9.1 UNIQUE Position 3 Cyclopentylglycine (Ceg): x, CycloHexylglycine (Chg): i, position 7 Cyclobutylalanine (Cba): x, 4-phenyl phenylalanine(Phf): j, 6-Cl tryptophan (ClW): m, QuinolylAlanine (Qua): n, Napthylalanine(Naf): o, Aminoadipic acid (Aad): b, Homocysteic acid (Hoc): z, Hydroxyproline (Hyp): p, Amino isobutyric (Aib): d TABLE 37 Condition II-3.3 Scan Peptide 12-mer ALC (%) m / z z RT Mass ppm Uniqueness 6562 boiLAExLCKVp 92 746.4188 2 39.59 1490.808 10.1 UNIQUE 6680 bjxLdExLCKbp 90 781.4214 2 39.64 1560.81 11.6 UNIQUE 6363 boxLdEiLCdKQ 88 753.9178 2 38.98 1505.813 5.7 UNIQUE 6385 boxLdEiLCdKQ 87 753.9267 2 39.04 1505.813 17.5 NOT UNIQUE 6245 boiLdExLCdRp 85 760.4169 2 38.33 1518.803 10.8 UNIQUE 6433 boxLdExLCKAG 83 704.3904 2 39.12 1406.745 14.9 UNIQUE 6117 boxLdExLCdRG 78 725.3954 2 38.17 1448.762 10.3 UNIQUE 8532 boiLdExLCVpQ 68 753.4082 2 46.14 1504.782 13.5 UNIQUE 8104 boiLdExLCHHH 63 788.9081 2 44.76 1575.789 8.4 UNIQUE 6326 boxLVExLCtYE 58 772.403 2 38.97 1542.784 4.8 UNIQUEScan Peptide 11-mer ALC (%) m / z z RT Mass ppm Uniqueness 6403 boxLdExLCKQ 95 704.3729 2 39.09 1406.745 -10.1 UNIQUEAttorney Docket No.: CLS-035WO PATENT Position 3 Cyclopentylglycine (Ceg): x, CycloHexylglycine (Chg): i, position 7 Cyclobutylalanine (Cba): x, 4-phenyl phenylalanine(Phf): j, 6-Cl tryptophan (ClW): m, QuinolylAlanine (Qua): n, Napthylalanine(Naf): o, Aminoadipic acid (Aad): b, Homocysteic acid (Hoc): z, Hydroxyproline (Hyp): p, Amino isobutyric (Aib): d TABLE 38 Condition II-4Position 3 Cyclopentylglycine (Ceg): x, CycloHexylglycine (Chg): i, position 7 Cyclobutylalanine (Cba): x, 4-phenyl phenylalanine(Phf): j, 6-Cl tryptophan (ClW): m, QuinolylAlanine (Qua): n, Napthylalanine(Naf): o, Aminoadipic acid (Aad): b, Homocysteic acid (Hoc): z, Hydroxyproline (Hyp): p, Amino isobutyric (Aib): d, C-term amide: U.Attorney Docket No.: CLS-035WO PATENT TABLE 39 Condition II-5Attorney Docket No.: CLS-035WO PATENT Position 3 Cyclopentylglycine (Ceg): x, CycloHexylglycine (Chg): i, position 7 Cyclobutylalanine (Cba): x, 4-phenyl phenylalanine(Phf): j, 6-Cl tryptophan (ClW): m, QuinolylAlanine (Qua): n, Napthylalanine(Naf): o, Aminoadipic acid (Aad): b, Homocysteic acid (Hoc): z, Hydroxyproline (Hyp): p, Amino isobutyric (Aib): d EXAMPLE 15– Cell toxicity Assays with Reactide 13

[0329] Cell toxicity of Reactide 13 was determined in a cell toxicity assay using the HPV16 E6 positive cell lines SiHa and CaSki.

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[0331] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0332] 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

Attorney Docket No.: CLS-035WO PATENT What is claimed is 1. A synthetic peptide comprising an amino acid sequence IPESSELTLQELLGEER (SEQ ID NO: 1) or ELTLQELLGEER (SEQ ID NO: 5).

2. The synthetic peptide of claim 1, wherein the N- or C- terminus of the peptide is modified with a chemical moiety.

3. The synthetic peptide of claim 2, wherein 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-1-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.

4. The synthetic peptide of claim 2, wherein 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-Indanecarboxamido, 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, triphenylacetamido, diphenylacetamido, 1-naphthyl carboxamido, and Exo-norbornene-carboxamido.

5. The synthetic peptide of claim 2, wherein 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-1-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.

6. The synthetic peptide of claim 2, wherein 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.Attorney Docket No.: CLS-035WO PATENT 7. The synthetic peptide of claim 2, wherein 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.

8. The synthetic peptide of claim 2, wherein the peptide comprises Fluorene- IPESSELTLQELLGEERRAA-K(1-Anthracene (SEQ ID NO:2) or Fluorene- IPESAELTLQELLGEERRAA-K(1-Anthracene (SEQ ID NO:3).

9. A synthetic peptide modified with a warhead, wherein the peptide is selected from TABLE 2C, TABLE 2D, or TABLE 2E.

10. The synthetic peptide of claim 9, wherein the warhead is at position 9 with respect to SEQ ID NO:

3.

11. The synthetic peptide of claim 9, wherein the warhead is selected from the group of phenylacrylamide (Ph-acr), Dap-acrylamide (dap-acr), Dab-acrylamide (dab-acr), Dap- propiolamide (dap-ppa), and dehydroalanine Dha.

12. The synthetic peptide of claim 9 or 10, wherein the peptide comprises Fluorene- IPQSAELTLQELL(DHA)RRKKK(Anthracene) (SEQ ID NO: 4).

13. The synthetic peptide of claim 9, wherein the warhead is selected from the group of phenyl fluorosulfate (FS), phenyl sulfonyl fluoride(SF), phenyl Carbamate (p-PhC), phenyl Carbamate (m-PhC), and disulfide (DS).

14. A synthetic peptide comprising an amino acid sequence X1X2X3X2QX1X2X2CEER (SEQ ID NO: 6), wherein X1is Nva, Leu, Aoc, Cpa, Cba, Cha, Phg, Hof, or Naf; X2is Aad, Glu, or Cya; X3 is Thr, Asn, Hyp, Cpg, Cbg, or Ceg; and X4 is d-Ala, Aib, Gly, or Gln.

15. The synthetic peptide of claim 13, wherein the peptide is selected from TABLES 3-31.

16. The synthetic peptide of claim 14, wherein the peptide is selected from SEQ ID NO: 7- 19.

17. The synthetic peptide of claim 14, wherein the peptide comprises SEQ ID NO:

17.

18. A synthetic peptide comprising an amino acid sequence Aad-X1-X2-Leu-Aib-Aad-Cba- Leu-Cys-X3-X3-X3 (SEQ ID NO: 20) wherein X1 is PhF, Naf, Qua, Clw, or Trp; X2 isAttorney Docket No.: CLS-035WO PATENT Chg or Ceg; and X3 is d-Ala, Aib, Gly, Ser, Aad, Glu, Gln, Hoc, Val, Leu, Met, Pro, Dap, Lys, Arg, His, Trp, Phe, Tyr, or Null.

19. The synthetic peptide of claim 18, wherein the peptide is 11 or 12 amino acids long.

20. The synthetic peptide of any one of claims 18-19, wherein the N-terminus of the peptide is further modified with a modifier selected from 2-Phenyl-4-quinolinecarboxylic acid, 1- (Phenylsulfonyl)-1H-indole-2-carboxylic acid, 6-Fluoro-2-naphthoic acid, 4- phthalimidobenzoic acid, Xanthene 9-carboxylic acid, 1-Pyrenebutyric acid, 5- Acenaphthenecarboxylic acid, 1-Phenyl-1H-indole-2-carboxylic acid, Indole-2-carboxlic acid, quinoline-4-carboxylic acid, and 10-(Carboxymethyl)-9(10H)acridone.

21. The synthetic peptide of claim 13, wherein the peptide is selected from TABLES 32-39.

22. A synthetic peptide comprising the formula.

23. The synthetic peptide of any one of claims 14-22, further comprising a warhead.

24. The synthetic peptide of claim 23, wherein the warhead is selected from the group of phenylacrylamide (Ph-acr), Dap-acrylamide (dap-acr), Dab-acrylamide (dab-acr), Dap- propiolamide (dap-ppa), and dehydroalanine Dha.

25. The synthetic peptide of any one of claims 1-24, wherein the synthetic peptide is an HPV16E6 / E6AP specific inhibitor.

26. A synthetic peptide of any one of claims 1-25 comprising one or more additional modifications selected from: a) acetylated, formylated, propanoylated, hexanoylated, or myristoylated N-terminus; b) amidated C-terminus;Attorney Docket No.: CLS-035WO PATENT c) substitution of one or more L-amino acid with a D-amino acid; d) substitution of one or more amino acid with a methyl-amino acid; and e) substitution of an Į-amino acid with a ȕ-amino acids.

27. A synthetic peptide / HPV16E6 complex, wherein the peptide is selected from any one of the synthetic peptides of claims 1-26.

28. The complex of claim 27, wherein the synthetic peptide and the HPV16E6 in the complex are covalently linked.

29. The complex of claim 28, wherein the synthetic peptide is covalently linked to amino acid residue Cys-58, Arg-84, or Arg-13 of HPV16E6.

30. The peptide / HPV16E6 complex of any one of claims 27-29, wherein the complex is inhibited in binding of human E6AP.

31. A pharmaceutical composition, comprising the synthetic peptide of any one of claims 1- 27 and a pharmaceutically acceptable salt or carrier.

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