IL300815A - Proteins or peptides that have been modified for directed covalent binding - Google Patents

Description

P-623495-IL 1 MODIFIED PROTEINS OR PEPTIDES FOR COVALENT TARGETING SEQUENCE LISTING [001] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 20, 2023, is entitled "P-623495-IL _ST26.xml", and is 45,293 bytes in size. FIELD OF THE INVENTION[002] This invention is directed to modified proteins or modified peptides (modified by an electrophile, such as acrylamide or acrylate) for covalent binding with target proteins. BACKGROUND OF THE INVENTION[003] Covalent tool compounds and chemical probes have been established as a powerful technology with diverse applications in chemical biology. These applications range from inhibitors used for therapeutic applications to covalent probes used to study the function and properties of target proteins. Covalent compounds have been developed targeting a large variety of proteins including kinases, G-protein coupled receptors, hydrolases and others, have found important uses as probes for proteomics and microscopy, and are also used in emerging applications such as targeted degradation. The advantages of covalent compounds in chemical biology stem from several aspects: first, the irreversible binding to the target achieves prolonged potent inhibition with short systemic exposure as in the case of covalent proton pump inhibitors, which maintain potent target inhibition long after the drug has been cleared from the body. For chemical probes, covalent binding to the target facilitates downstream processes involving denaturation and proteolysis of the target without loss of the bound probe, making them especially useful in proteomics and microscopy. Second, covalent compounds frequently show enhanced selectivity by targeting non-conserved nucleophilic residues. This is exemplified by the recently approved Sotorasib and Adagrasib, which selectively target the G12C mutant of KRAS. [004] A surge in the development of novel warheads for small molecules has expanded the targetable scope of amino acids to include lysine, tyrosine, acidic residues, histidine and others. These chemistries have greatly expanded the spectrum of protein targets. However, the synthetic installation of a reactive group on a peptide or full protein is not trivial. The large number of nucleophilic amino acids on a protein or the relatively harsh deprotection conditions required for solid phase peptide synthesis complicates electrophile installation. Recently genetic code expansion to incorporate a fluorosulfate-tyrosine followed by chemical transformation to afford the reactive group has proven P-623495-IL 2 effective in protein modifications. However, effective genetic code expansion remains technically challenging, and may be limited in the scope of amino acids that could be incorporated. [005] Despite the surge in research into covalent compounds, targets such as transcription factors and protein-protein interaction interfaces are difficult to target with small molecules due to their broad and shallow binding surfaces. The use of peptide or peptidomimetics has emerged as a powerful approach to address these issues. Peptide binders can cover a large surface area, can bind protein targets with high affinity and can frequently be derived from known protein-protein interactions. [006] Potent covalent peptide binders have been developed for targets such as the bacterial divisome, E3 ubiquitin ligases, the anti-apoptotic protein BFL-1, and others. Due to the size of peptides and their conformational flexibility, computational modeling can aid in the design and placement of the electrophile. SUMMARY OF THE INVENTION[007] In some embodiments, provided herein a modified protein or a modified peptide comprising a recombinant protein or a synthetic peptide modified by an electrophile, wherein the electrophile is covalently bound to a thiol group of a cysteine within the protein or peptide; wherein the electrophile is represented by the structure of Formula I; (I) wherein, R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; X comprises O, NR or substituted or unsubstituted alkylene; and R is hydrogen, alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; wherein the modified protein or modified peptide is capable of covalently binding a target protein. [008] In some embodiments, the modified peptide comprises the following peptides: Ac-RSApSmCPSL-NH2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH2 ( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ); wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine. In other embodiments, the modified peptide of Ac-RSApSmCPSL-NH2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH2( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ); wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine-covalently binds 14-3-3 P-623495-IL 3 target protein. In other embodiments, the peptide of Ac-RSApSmCPSL-NH2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH2( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ); covalently binds Cys38, Lys 122 or Lys 49 of 14-3-3σ target protein. [009] In some embodiments, the modified protein comprises a modified immunity (Im9) protein ( SEQ ID 27 ). In other embodiments, the Im9 ( SEQ ID 27 ) binds an E9 target protein. [0010] In some embodiments, provided herein a method of preparing a modified protein or a modified peptide of any one of claims 1-12, wherein the method comprises; a. identifying a target protein; b. designing a peptide or protein covalent binder candidate based on previously characterized noncovalent binders of the target protein; c. synthesizing a modified peptide or a modified protein, to allow recognition with the target protein at the recognition site; wherein said modified protein or modified peptide comprise a cysteine which is reacted with an electrophile of Formula I-IV. [0011] In some embodiments, provided herein a protein-protein covalent conjugate comprising a modified protein described herein, covalently bound to a target protein, wherein the modified protein and the target protein possess non-covalent recognition prior to the covalent binding with the target protein. [0012] In some embodiments, provided herein a peptide-protein covalent conjugate comprising a modified peptide described herein, covalently bound to a target protein, wherein the modified peptide and the target protein possess non-covalent recognition prior to the covalent binding with the target protein. [0013] In some embodiment, provided herein a modified protein or modified peptide provided herein for use in selectively label, fluorescent label, inhibition, drug conjugation or conjugation to a target protein. BRIEF DESCRIPTION OF THE DRAWINGS[0014] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which: [0015] Figure 1. Scheme for generating thioether-based modified protein or modified peptide of this invention. A cysteine residue can be introduced into peptides (pink ribbon) or recombinant proteins (light green), which are then modified using an ethyl 2-(bromomethyl)acrylate. The resulting P-623495-IL 4 modified protein or modified peptide reacts with lysine or cysteine side chains proximal to the binding site on the receptor (light blue), to yield covalent adducts. [0016] Figures 2A-2C. Generating peptide covalent reagents for 14-3-3 proteins. Figure 2A:Structure of the complex of 14-3-3σ (white) with phosphorylated peptide from YAP (cyan; PDB: 3MHR). The non-conserved cysteine 38 and the conserved lysines 49 and 122 are highlighted. Figure 2B: Scheme for synthesis of electrophilic peptides. a) 20% piperidine in DMF, 3 x 4 minutes; b) 4 equ. Fmoc-AA-OH/HATU/HOAT, 8 equ. DIPEA, 30 minutes RT, c) 92.5% TFA, 2.5% water, 2.5% TIPS, 2.5% DODT, 3 hours. Figure 2C: HPLC chromatograms and MS spectra of the crude peptide 8 (red) and the crude peptide after reaction with (3-Bromo)methacrylate (blue). [0017] Figures 3A-3B. Designed methacrylate peptides bind 14-3-3σ. Figure 3A. Peptides (2µM) were incubated with the 14-3-3σ protein (2 µM; overnight; 4°C) and analyzed using intact protein LC/MS. See Table 1 for peptide structures. Figure 3B. Selected peptides (5 µM) were incubated with 14-3-3σ protein (2 µM, room temperature) for different times and analyzed. [0018] Figures 4A-4B: Methacrylate peptides bind conserved 14-3-3 lysine residue. Figure 4A:Overlay of the CovPepDock prediction (Light brown) and co-crystal structure of peptide 8 (blue sticks) bound to 14-3-3σ (white surface; left), and a close-up view on the methacrylate residue and the electron density of the peptide (right). Similarly, Figure 4B:docking overlay (left) and electron density close-up view (right) of peptide 3 . Figure 4C: Binding of electrophilic peptides to 14-3-isoforms. Peptides were incubated with the various indicated isoforms overnight at room temperature, diluted and analyzed using intact protein LC/MS. [0019] Figure 5. Labeling of 14-3-3 proteins by Bodipy-modified peptides in A549 lysates and in medium. Lysates or concentrated media were incubated for 24 hours at room temperature with the peptides, followed by SDS-PAGE and western blot. Left Panel: Detection of 14-3-3β by western blot; Middle panel - peptide fluorescence; right panel - overlaid images. [0020] Figures 6A-6B. Generation of an Im9 protein that can irreversibly bind E9 . Figure 6A:Model of the C23A/E41C mutant of Im9 (gold) covalently bound to Lys97 in E9 (white), compared to the wild type complex (with Im9 in blue). Figure 6B: Deconvoluted MS spectra of purified Im methacrylate, purified E9 and the covalent complex formed after their incubation. C. Chromatograms of samples of Im methacrylate incubated with wild type E9 and several E9 mutants. Incubation with the K97R mutant abolishes covalent bond formation. [0021] Figure 7. Covalent complex formation significantly stabilizes complex thermal stability. Average derivative data from 3 samples is shown for each condition. measured melting temperature (calculated from maximum derivative) for different constructs. Error bars represent 3 standard deviations from 3 samples.

P-623495-IL id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22"

[0022] Figures 8A-8C. Methacrylate peptides label different 14-3-3σ residues. Figure 8A: Peptide SEQ ID 3 was incubated with 2 mM of either TCEP or DTT for 130 minutes at room temperature and the products were characterized using LC/MS. Figure 8B: 14-3-3σ was incubated with peptide SEQ ID 3 until fully labeled, followed by incubation with TCEP or DTT for 1minutes at room temperature and analysis by LC/MS. Figure 8C: 14-3-3σ was incubated with Bodipy-modified peptides until fully labeled. The samples were then denatured using Lithium Dodecyl Sulfate buffer in different conditions (with or without heating and with or without DTT) and analyzed by SDS-PAGE. (See Example 2). [0023] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. DETAILED DESCRIPTION OF THE PRESENT INVENTION[0024] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention. Modified protein or modified peptide [0025] In some embodiments, provided herein a modified protein or a modified peptide comprising a synthetic/recombinant protein or a synthetic peptide modified by an electrophile, wherein the electrophile is covalently bound to a thiol group of a cysteine within the synthetic/recombinant protein or synthetic peptide and the electrophile is represented by the structure of Formula I: (I) wherein, R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; and P-623495-IL 6 X comprises O, NR or substituted or unsubstituted alkylene; and R is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; wherein the modified protein or modified peptide is capable of covalently binding a target protein. [0026] In other embodiments, the electrophile is represented by the structure of formula II (methacrylate): (II); wherein, R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, carbocycle, aryl, heteroaryl or heterocyclic. [0027] In other embodiments, the electrophile is: (ethyl methacrylate) [0028] In other embodiments, the electrophile is represented by the structure of formula III (methacrylamide): (III); wherein, each of R or R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl or heterocyclic. [0029] In other embodiments, the electrophile is represented by the structure of formula IV: (IV); N R O R Alk R O P-623495-IL 7 wherein, R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl or heterocyclic; and Alk refers to substituted or unsubstituted alkylene. [0030] In some embodiments, the modified protein or modified peptide comprise: (i) an electrophile installation on unprotected peptides or proteins ;(ii) the electrophile reacts with both thiols and primary amines. As such the electrophile of formula I-IV is selected that react with both cysteine, and lysine residues via aza-Michael addition. As a nonlimiting example ethyl 2-(bromomethyl)acrylate (Figure 2B) is used for its selective reactivity with cysteine residues via its -bromo-methylene functional group. [0031] In some embodiments, provided herein a modified protein or a modified peptide comprising a synthetic/recombinant protein or a synthetic peptide modified by an electrophile of Formula I-IV, wherein the electrophile is covalently bound to a thiol group of a cysteine within the synthetic/recombinant protein or synthetic peptide. The cysteine residues represent an attractive anchoring residue due to their low proteomic abundance and their high reactivity at physiological pH, enabling rapid and selective modification on unprotected peptides and proteins. [0032] In some embodiments, provided herein a modified protein or a modified peptide comprising a synthetic/recombinant protein or a synthetic peptide modified by an electrophile of Formula I-IV, forming a thioether bond between the electrophile and the synthetic protein or synthetic peptide. [0033] In some embodiments, provided herein a modified protein comprising a synthetic protein modified by an electrophile of Formula I-IV, forming a thioether bond between the electrophile and the synthetic protein. [0034] In some embodiments, provided herein a modified protein comprising a recombinant protein modified by an electrophile of Formula I-IV, forming a thioether bond between the electrophile and the recombinant protein. [0035] In some embodiments, provided herein a modified peptide comprising synthetic peptide modified by an electrophile of Formula I-IV, forming a thioether bond between the electrophile and the synthetic peptide. [0036] In some embodiments, the modified protein or modified peptide is capable of covalently binding a target protein, having a noncovalent recognition with the target protein prior to the covalent binding with the target protein. [0037] In some embodiments, the modified protein or modified peptide is capable of covalently binding a target protein, wherein the electrophile is in close proximity to a target residue of the target protein prior to the covalent binding with the target protein. [0038] In some embodiments, the modified protein or modified peptide is capable of covalently binding a target protein, wherein the modified protein or modified peptide has a non-covalent P-623495-IL 8 recognition with the target protein; and the electrophile is in close proximity to a target residue of the target protein; prior to the covalent binding with the target protein. In other embodiments, the target residue within the target protein is a cysteine residue (SH) or a lysine residue (NH2). In other embodiments, the close proximity between the electrophile and the target residue within the target protein refers to a distance being less than 15 Å . In other embodiments less than 14 Å, 13 Å, 12 Å, Å, 10 Å, 7 Å, or 5 Å. [0039] In some embodiments, the modified protein or modified peptide described herein covalently binds a target protein via the double bond (C=CH2) of the electrophile of Formula I-IV to a thiol or an amine within the target protein. In other embodiments, the modified protein or modified peptide described herein covalently binds the thiol (SH) side chain of a cysteine within the target protein. In other embodiments, the modified protein or modified peptide described herein covalently binds the amine side chain (NH2) of a lysine within the target protein. In other embodiments, the modified protein or modified peptide covalently binds the double bond (C=CH2) of the electrophile of Formula I-IV and a lysine residue within the target protein via aza-Michael addition. [0040] The electrophilic peptides or proteins (i.e the modified proteins or modified peptides) described herein provide a versatile approach to convert native peptide sequences or native proteins into covalent binders that can target a broad range of residues, and can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chain on the target protein. [0041] In some embodiments, the modified peptide provided herein is Ac-RSApSmCPSL-NH ( SEQ ID 3 ), wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine. In some embodiments, the modified peptide provided herein is Ac-RAHpSmCPASLQ-NH2 ( SEQ ID 8 ), wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine. In some embodiments, the modified peptide provided herein is Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ), wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine. [0042] In other embodiments, the modified peptide Ac-RSApSmCPSL-NH 2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH2 ( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ), covalently binds 14-3-3 as a target protein. In other embodiments, the modified peptide Ac-RSApSmCPSL-NH 2 (SEQ ID 3), Ac-RAHpSmCPASLQ-NH2 ( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ), covalently binds a Cys 38, Lys 122 or Lys 49 of the 14-3-3σ target protein, depending on the position of the electrophile. [0043] The modified peptide Ac-RSApSmCPSL-NH 2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH ( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ), covalently bound 14-3-3 as a target protein targeting a conserved lysine residue exhibiting broad reactivity against 14-3-3 proteins, and efficiently labeled 14-3-3 proteins in lysates, as well as secreted 14-3-3 extracellularly. The P-623495-IL 9 irreversible binding to the predicted target lysines were confirmed by proteomics and X-ray crystallography of the complexes. [0044] In some embodiments, the modified protein provided herein is a modified immunity protein MELKHSISDYTEAEFLQLVTTI A NADTSSEEELVKLVTHF(mC)EMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQGLEHHHHHH ( Im9, SEQ ID 27 ), wherein mC is Mehtacrylate-modified cysteine. The Alanine at position 23 was a cysteine in the original sequence of Im. The mC was a glutamate in the original sequence of Im. In other embodiments, the modified immunity protein ( Im9, SEQ ID 27 ), covalently binds the E9 target protein. [0045] The immunity protein irreversibly bound to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. [0046] The approach provided herein offers a simple and versatile route to convert peptides and proteins into potent covalent binders. [0047] Provided herein a new approach to the development of covalent protein binders based on thioether-modified proteins or modified peptides. The modified protein or modified peptide can react with both lysine and cysteine side chains via Michael addition of a target protein. The electrophile of Formula I-IV is installed by direct, selective modification of a cysteine side chain, enabling synthesis of binders from unprotected peptides and even recombinant proteins (Figure 1). Preparation of the modified protein or modified peptide [0048] In some embodiments, provided herein a method of preparing a modified protein or a modified peptide described herein, wherein the method comprises; a. identifying a target protein; b. designing a peptide or protein covalent binder candidate based on previously characterized noncovalent binders of the target protein; c. synthesizing a modified peptide or a modified protein, to allow recognition with the target protein at the recognition site; wherein said modified protein or modified peptide comprise a cysteine which is reacted with an electrophile of Formula I-IV. [0049] In some embodiments, the design of the peptide or the protein covalent binder candidate is done by computational modeling, structure based rational design, random screening of possible positioning for modification, or any combination thereof. In other embodiments, the designed covalent binder candidate has molecular recognition with a target protein. The "peptide or protein covalent binder candidate" is prepared and modified with an electrophile of Formula I-IV to obtain the modified protein or modified peptide described herein. [0050] The design of the peptide or protein covalent binder candidate is based on previously characterized noncovalent binders with a target protein. "Previously characterized noncovalent P-623495-IL binders of the target protein refer to peptide-protein or protein-protein interactions known in the art, for example : PDB entries for known interactions. [0051] In some embodiments, based on the computer modeling, a synthetic protein is synthesized having only one cysteine amino acid which is further modified with the electrophile of Formula I-IV. In some embodiments, based on the modeling calculation, a synthetic peptide is synthesized having only one cysteine amino acid. [0052] In some embodiments, the modified peptide is prepared according to Figure 2B. [0053] An example of a computational modeling system is the CovPepDock, Rosetta-based framework for modeling covalent protein-peptide interactions and for the design and virtual screening of potential covalent peptide binders (Tivon, B.; Gabizon, R.; Somsen, B. A.; Cossar, P. J.; Ottmann, C.; London, N. Covalent Flexible Peptide Docking in Rosetta. Chem. Sci. 2021 , 12 (32), 10836–10847). [0054] Using CovPepDock the modified peptides Ac-RSApSmCPSL-NH 2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH2 ( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ), displayed highly potent and selective detection of 14-3-3 in cell lysates, a difficult task for noncovalent binders due to the high sequence homology within the family. [0055] In some embodiments, the modified protein or modified peptide is prepared by reacting a synthetic protein or synthetic peptide with an electrophilic reagent , wherein the synthetic/recombinant protein or synthetic peptide possess only one cysteine amino acid, and the electrophilic reagent reacts via the R group (of the electrophilic reagent) with the thiol of the cysteine to form a thioether bond; wherein the electrophilic reagentis represented by the following structure if Formula IA, IIA, IIIA or IVA: wherein, P-623495-IL 11 R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; R comprises Br, Cl or tosyl; R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; and X comprises O, NR or substituted or unsubstituted alkylene. [0056] In some embodiments, the electrophilic reagent is ethyl 2-(bromomethyl)acrylate. [0057] In some embodiments, R of Formula I, II, III, IV, IA, IIA, IIIA or IVA is substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic. In other embodiments R is substituted or unsubstituted alkyl. In other embodiments R is substituted or unsubstituted alkenyl. In other embodiments R is substituted or unsubstituted alkynyl. In other embodiments R is substituted or unsubstituted carbocycle. In other embodiments R is substituted or unsubstituted aryl. In other embodiments R is substituted or unsubstituted heteroaryl. In other embodiments R is substituted or unsubstituted heterocyclic. [0058] In some embodiments, R of Formula IA, IIA, IIIA or IVA is Br, Cl or tosyl. In other embodiments, R is Cl. In other embodiments, R is Br. In other embodiments, R is tosyl. [0059] In some embodiments, X of Formula I or IA is O, NR or substituted or unsubstituted alkylene. In other embodiments X is O. In other embodiments X is NR. In other embodiments X is NH. In other embodiments X is substituted or unsubstituted alkylene. In other embodiments X is methylene (-CH2-). [0060] In some embodiments, R of Formula I, IA, III or IIIA is substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic. In other embodiments R is substituted or unsubstituted alkyl. In other embodiments R is substituted or unsubstituted alkenyl. In other embodiments R is substituted or unsubstituted alkynyl. In other embodiments R is substituted or unsubstituted carbocycle. In other embodiments R is substituted or unsubstituted aryl. In other embodiments R is substituted or unsubstituted heteroaryl. In other embodiments R is substituted or unsubstituted heterocyclic. [0061] As used herein, "alkyl" refers to a linear or branched alkyl. In certain embodiments linear or branched alkyl having from 1 to about 20 carbon atoms, in another embodiment having from 1 to carbons. In a further embodiment the alkyl includes lower alkyl, having 1 to 6 carbons. In a further embodiment the alkyl includes lower alkyl, having 1 to 3 carbons. There may be optionally inserted along the alkyl group one or more oxygen, sulfur, including S(=O) and S(=O)2 groups, or substituted or unsubstituted nitrogen atoms including -NR- and -N+RR- groups, where the nitrogen substituent(s) P-623495-IL 12 is(are) alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl or COR, wherein each R is independently selected from alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, -OY or -NYY, wherein each Y is independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. The alkyl groups include, but are not limited to, methyl, ethyl, propyl, methoxy, ethoxy, isopropyl, isobutyl. The alkyl group may be substituted or unsubstituted. In other embodiments the substituted alkyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, oxo, aryl, nitro, azide, aryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, carbonyl, amine or amide. [0062] As used herein, "alkylene" refers to a linear, branched or cyclic, in certain embodiments linear or branched, divalent aliphatic hydrocarbon group, in one embodiment having from 1 to about carbon atoms, in another embodiment having from 1 to 12 carbons. In a further embodiment alkylene includes lower alkylene having 1 to 6 carbons. In a further embodiment alkylene includes lower alkylene having 1 to 3 carbons. There may be optionally inserted along the alkylene group one or more oxygen, sulfur, including S(=O) and S(=O)2 groups, or substituted or unsubstituted nitrogen atoms including -NR- and -N+RR- groups, where the nitrogen substituent(s) is(are) alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl or COR, wherein each R is independently selected from alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, -OY or -NYY, wherein each Y is independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. Alkylene groups include, but are not limited to, methylene (-CH2), ethylene (-CH2CH2-), propylene (-(CH2)3), methylenedioxy (-O-CH2-O-) and ethylenedioxy (-O-(CH2)2-O-). [0063] As used herein, "alkenyl" refers to a linear or branched alkenyl. In one embodiment straight or branched alkynyl having from 2 to about 20 carbon atoms and at least one double bond, in other embodiments 1 to 12 carbons. In further embodiments, alkenyl groups include lower alkenyl having to 6 carbons. In further embodiments, alkenyl groups include lower alkenyl having 3 to 4 carbons. There may be optionally inserted along the alkenyl group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl. Alkenyl groups include, but are not limited to, -CH=CH-CH=CH2 and -H=CH-CH3. The alkenyl group may be substituted or unsubstituted. In other embodiments the substituted alkenyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, oxo, aryl, nitro, azide, aryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, carbonyl, amine or amide. [0064] As used herein, "alkynyl" refers to a straight or branched alkynyl. In certain embodiments straight or branched alkynyl, in one embodiment having from 2 to about 20 carbon atoms and at least one triple bond, in another embodiment 1 to 12 carbons. In a further embodiment, alkynyl includes lower alkynyl having 2 to 6 carbons. In a further embodiment, alkynyl includes lower alkynyl having P-623495-IL 13 3 to 4 carbons. There may be optionally inserted along the alkynyl group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl. Alkynyl groups include, but are not limited to, -C≡C-C≡CH, -C≡CH and -C≡C-CH3. The alkynyl group may be substituted or unsubstituted. In other embodiments the substituted alkynyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, oxo, aryl, nitro, azide, aryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, carbonyl, amine or amide. [0065] A "carbocycle" group refers to a saturated on unsaturated all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of carbocycle groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cycloheptane, and adamantane. A cycloalkyl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, hydrazide, thiohydrazide, and amino. When a carbocycle group is unsaturated, it may comprise at least one carbon-carbon double bond and/or at least one carbon-carbon triple bond. An unsaturated carbocycle include cyclohexene, cycloheptene, cyclohexadiene, cycloheptatriene. [0066] An "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) end groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, hydrazide, thiohydrazide, and amino. [0067] A "heteroaryl" group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) end group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, P-623495-IL 14 alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino. [0068] A "heterocyclic" group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heterocyclic may be substituted or non-substituted. When substituted, the substituted group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, hydrazide, thiohydrazide, and amino. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine and the like. Uses of the Covalent binding of the modified protein or modified peptide with a target protein [0069] The covalent binding of the modified protein or modified peptide with a target protein provides high potency and specificity can be used to modulate, inhibit or measure the activity of the protein, as well as measure other properties such as its localization and turnover rate. [0070] In some embodiments, provided herein a protein-protein covalent conjugate comprising a modified protein provided herein, covalently bound to a target protein, wherein the modified protein and the target protein possess non-covalent recognition prior to the covalent binding with the target protein. In other embodiments, the protein-protein covalent conjugate comprises a modified protein of Im9 and a target protein E9. In other embodiments, the protein-protein covalent conjugate (of the Im9 and E9) displayed a higher thermal stability than the noncovalent complex. [0071] In some embodiments, provided herein a peptide-protein covalent conjugate comprising a modified peptide provided herein, covalently bound to a target protein, wherein the modified peptide and the target protein possess non-covalent recognition prior to the covalent binding with the target protein. In other embodiments, the modified peptide is Ac-RSApSmCPSL-NH2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH2( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ); wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine; and the target protein is 14-3-3. [0072] Peptides derivatized with covalent warheads can greatly expand the repertoire of targets and the possible effects of modification. They can be used to selectively label target proteins, enabling P-623495-IL modifications such as fluorescent labeling, drug conjugation or conjugation to other proteins. Genetic code expansion extends this further and enables installing of warheads on large proteins as well. [0073] Since the approach is applicable to peptides in their native form, it can be used to prepare electrophile-modified proteins directly from native, recombinant proteins. The resulting probes can efficiently and selectively label target proteins on cysteines and lysines. Furthermore, the R group of the electrophile of Formula I-IV group can function as a point of diversification and screening either for the development of more potent binders or for the functionalization of the probes in various ways, for example via the attachment of E3 ligase binders for targeted degradation, for the attachment of fluorophores for imaging or detection purposes, for targeting proteins to particular cell types or locations in the cell and more. [0074] In some embodiments, provided herein a modified protein or modified peptide for use in selectively label, fluorescent label, inhibition, drug conjugation or conjugation to a target protein. [0075] In some embodiments, provided herein a method of diagnosing a disease by a known biomarker wherein the method comprises covalently binding a modified peptide or modified protein described herein with a known biomarker, thereby identifying the biomarker by fluorescence or immunoassays. [0076] The potency and specificity of the modified peptides or modified proteins can be used as versatile chemical probes. For example, while most known functions of 14-3-3 proteins are intracellular, they also serve some extracellular functions and the presence of extracellular 14-3-proteins can serve as a biomarker for various diseases . Using BDP (fluorescent dye Bodipy) -modified methacrylate peptides a 14-3-3 was detected in extracellular medium with higher sensitivity than western blot, illustrating the power of this method. (See Example 3). Furthermore, when targeting extracellular proteins, issues such as membrane permeability and proteolytic stability have less impact on the activity of the probe, opening the door for peptide and protein-based covalent probes. [0077] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

P-623495-IL 16 EXAMPLES EXAMPLE 1 Design and synthesis of Modified Peptides covalent binders to 14-3-3 proteins [0078] The objective was to design binders that would target conserved lysines in the phoshopeptide binding groove of 14-3-3σ: lysines 49 and 122 (Figure 2A). Specifically, Lys122 has been previously shown to react with aldehydes to form a reversible covalent imine bond, with high selectivity over other lysine residues, which is attributed to a lower pKa of the Lys122 side-chain (Wolter, M., et al., Fragment-Based Stabilizers of Protein-Protein Interactions through Imine-Based Tethering. Angew. Chem. Int. Ed Engl. 2020 , 59 (48), 21520–21524.; Cossar, P. J. et al., Reversible Covalent Imine-Tethering for Selective Stabilization of 14-3-3 Hub Protein Interactions. J. Am. Chem. Soc. 2021 , 143 (22), 8454–8464.; Wolter, M.; Valenti, D.; Cossar, P. J. et al., An Exploration of Chemical Properties Required for Cooperative Stabilization of the 14-3-3 Interaction with NF-κB-Utilizing a Reversible Covalent Tethering Approach. J. Med. Chem. 2021 , 64 (12), 8423–8436.) [0079] CovPepDock was first used to design a series of methacrylate-based peptides based on the non-covalent complex between 14-3-3σ and YAP1 phosphopeptide (Protein data bank (PDB): 3MHR). [0080] The residues were identified in the YAP1 peptide that were within Cα-Cα distance < 14Å from the target lysine and mutated each of these residues to the methacrylate modified side-chain; this included residues 126-131 for targeting Lys49, and residues 126-133 for targeting Lys122. Four peptides were selected that were predicted to bind either Lys49 or Lys122 with high score and low RMSD to the original peptide binding mode. To further expand the scope of series, a second set of peptides were designed based on other known peptide binders of 14-3-3σ. Four such structures were selected, with peptides derived from Raf1 (Rapidly Accelerated Fibrosarcoma 1) (PDB: 3IQU and 4IEA), TASK-3 (TWIK-related acid-sensitive K+ channel 3) (PDB: 3P1N) and SNAI1 (PDB: 4QLI). For these peptides, Lys122 was focused on, which was more reactive towards electrophiles. [0081] CovPepDock was used to model Lys122-targeting peptides based on each of these structures and selected seven additional peptides with high scores and low RMSD from this set for synthesis and testing. The structures of the peptides are detailed in Table 1.

P-623495-IL 17 Table 1: Sequences and structures of the peptides * Peptide PDB Sequence Source Protein (Protein data bank) SEQ ID 1 SNAI1 4QLI_4 Ac-SH pTmC PS-NH2 SEQ ID 2 SNAI1 4QLI_5 Ac-SH pT L mC S-NH2 SEQ ID 3 Raf1 4IEA_5 Ac-RSA pSmC PSL-NH SEQ ID 4 Raf1 4IEA_7 Ac-RSA pS EP mC L-NH SEQ ID 5 Raf1 4IEA_8 Ac-RSA pS EPS mC -NH SEQ ID 6 Raf1 3IQU_6 Ac-QRST pSmC -OH SEQ ID 7 TASK-3 3P1N_6 Ac-KRRK pSmC -NH SEQ ID 8 YAP-1 3MHR_5 Ac-RAH pSmC PASLQ-NH SEQ ID 9 YAP-1 3MHR_7 Ac-RAH pS SP mC SLQ-NH2 SEQ ID 10 YAP-1 3MHR _8 Ac-RAH pS SPA mC LQ-NH2 SEQ ID 11 YAP-1 3MHR_9 Ac-RAH pS SPAS mC Q-NH SEQ ID 12 YAP-1 3MHR_Cl Ac-RAH pS SPASL X -NH * pS = Phoshoserine; pT = Phosphothreonine; mC = Mehtacrylate-modified cysteine (figures 1,2); X = γ-chloroacetamido-diaminobutyric acid [0082] To prepare peptide methacrylate adducts, the peptides were synthesized using standard Solid Phase Peptide Synthesis (SPPS) procedures and N-terminally acetylated the peptides (Figure 2B). After cleavage from the resin, the crude peptides were reacted with 3 equivalents of 2-(bromomethyl)acrylate, resulting in efficient conversion of the peptide to the methacrylate (i.e. modified peptide) within 1-2 hours at room temperature (Figure 2C). [0083] The peptides were incubated at 200 µM with 2 µM 14-3-3σ at 4°C overnight and monitored the binding using intact protein LC/MS (Figure 3A). Peptide SEQ ID 12 , which contains a chloroacetamide warhead that reacts with 14-3-3σ via Cys38, was used as a positive control. Significant covalent labeling of 14-3-3σ with the expected adduct mass was observed for the peptides SEQ ID 3 , SEQ ID 8 and SEQ ID 11 , all of which were predicted to bind Lys122. These peptides were analyzed further using time course labeling experiments at lower peptide concentrations (5 µM) at room temperature. Peptides SEQ ID 3 and SEQ ID 8 reached 60% and 80% labeling within hours, respectively. Interestingly, when incubated with SEQ ID 11 , non-labelled 14-3-3σ disappeared rapidly – within 2.5 hours less than 5% free 14-3-3σ remained. However, the reaction initially yielded a mixture of full peptide-labeled protein (+1275 Da) and protein modified with only the methacrylate group (+112 Da). The methacrylate-labeled protein was gradually converted to full peptide labeled protein (Figure 3B).

P-623495-IL 18 Methods:Preparation of recombinant 14-3- 3σ [0084] A pPROEX HTb expression vector encoding the human 14-3-3σ with an N-terminal His6-tag was transformed by heat shock into NiCo21 (DE3) competent cells. Single colonies were cultured in 50 mL LB medium (100 mg/ml ampicillin). After overnight incubation at 37 °C, cultures were transferred to 2 L TB media (100 mg/ml ampicillin, 1 mM MgCl2) and incubated at 37 °C until an OD600 nm of 0.8–1.2 was reached. Protein expression was then induced with 0.4 mM isopropyl-β-d-thiogalactoside (IPTG), and cultures were incubated overnight at 18 °C. Cells were harvested by centrifugation (8600 rpm, 20 minutes, 4 °C) and resuspended in lysis buffer (50 mM Hepes, pH 8.0, 300 mM NaCl, 12.5 mM imidazole, 5 mM MgCl2, 2 mM βME) containing cOmplete™ EDTA-free Protease Inhibitor Cocktail tablets (1 tablet/100 mL lysate) and benzonase (1 μl/100 mL). After lysis using a C3 Emulsiflex-C3 homogenizer (Avestin), the cell lysate was cleared by centrifugation (20 000 rpm, 30 minutes, 4 °C) and purified using Ni2+-affinity chromatography (Ni-NTA superflow cartridges, Qiagen). Typically two 5 mL columns (flow 5 mL/min) were used for a 2 L culture in which the lysate was loaded on the column washed with 10 CV wash buffer (50 mM Hepes, pH 8.0, 300 mM NaCl, 25 mM imidazole, 2 mM βME) and eluted with several fractions (2–4 CV) of elution buffer (50 mM Hepes, pH 8.0, 300 mM NaCl, 250 mM imidazole, 2 mM bME). Fractions containing the 14-3-3σ protein were combined and dialyzed into 25 mM HEPES pH 8.0, 100 mM NaCl, 10 mM MgCl2, 500 μM Triscarboxyethylphosphine (TCEP). Finally, the protein was concentrated to ∼mg/ml, analyzed for purity by SDS-PAGE and Q-Tof LC/MS and aliquots flash-frozen for storage at −80 °C. Peptide Synthesis [0085] Reagents for peptide synthesis were purchased from Chem-Impex. Peptides were synthesized on Rink Amide resin using standard Fmoc chemistry on a 0.025 mmol scale. The resin was swelled for 30 minutes in dichloromethane (DCM), then washed with dimethylformamide (DMF). Fmoc deprotections were carried out using 20% piperidine in DMF (3 × 3 minutes), and couplings were performed as follows: 4 equivalents of amino acid were mixed with 4 equivalents of HATU(Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium) / HOAT (1-Hydroxy-7-azabenzotriazole) and 8 equivalents of DIPEA (Diisopropylethylamine) in DMF and added to the resin with mixing for 45 minutes. For phosphoserine, propargylglycine and amino acids used for introducing the electrophile, 2 equivalents were used and reaction times were extended to 2 hours. After the last Fmoc deprotection, the peptides were acetylated at the N terminus using acetic anhydride (10 equivalents) and DIPEA (20 equivalents) in DMF for 30 minutes. Finally the resins P-623495-IL 19 were washed with DCM, dried in a dessicator, and cleaved using 92.5% TFA /2.5% DODT(3,6-dioxa-1,8-octanedithiol )/2.5% TIPS(triisopropylsilane)/2.5% water for 2 hours with tumbling. The cleaved peptides were precipitated in cold diethyl ether: hexane, washed once with ether, dried, dissolved in 50% acetonitrile and lyophilized. [0086] The electrophile was introduced directly to the crude peptides as follows: Crude peptides were dissolved in 100 mM NaPi pH = 7.5 at a concentration of 25 mM. Ethyl 2-(bromomethyl)acrylate was dissolved in acetonitrile to 200 mM and 3 equivalents were added to the peptide solution. Reactions were monitored using LCMS and were typically complete within 1-hours at room temperature. Reacted peptides were then purified using reverse phase HPLC. [0087] To prepare fluorescently labeled peptides, a residue of propargylglycine was coupled to the peptide at the N-terminus prior to N-terminal acetylation, cleavage and reaction with Ethyl 2-(bromomethyl)acrylate. The pure peptide was then labeled as follows using copper-catalyzed azide–alkyne cycloaddition (CuAAC): 5 ul of 20 mM peptide was mixed with 15 μL of BDP-TMR azide (150 nmol). Water was added to 100 μL and about 50 μL tBuOH was added to dissolve the dye. At this point, CuSO4:THPTA 100 mM (1 μL), and 200 mM sodium ascorbate (200 nmol, freshly dissolved) were added and the reaction continued for 1 hour and the product was purified using HPLC. The purity of all peptides was confirmed using LCMS. LC/MS instrumentation and runs [0088] The LC/MS runs for 14-3-3σ were performed on a Waters ACQUITY UPLC class H instrument, in positive ion mode using electrospray ionization. UPLC separation used a C4-BEH column (300 Å, 1.7 μm, 21 mm × 100 mm). The column was held at 40 °C and the autosampler at °C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The run flow was 0.4 mL/min. The gradient used was 1% B for 2 min, increasing linearly to 80% B for 2.5 min, holding at 80% B for 0.5 min, changing to 20% B in 0.2 min, and holding at 1% for 0.8 min. The MS data were collected on a Waters SQD2 detector with an m/z range of 2–3071.98 at a range of 900–1900 m/z. The desolvation temperature was 500 °C with a flow rate of 800 L/h. The voltages used were 1.00 kV for the capillary and 24 V for the cone. Raw data were processed using openLYNX and deconvoluted using MaxEnt with a range of 28000 : 34000 Da and a resolution of 1 Da/channel. [0089] The LS/MS runs for peptides were performed using the same instrument with a C18-CSH column (300 Å, 1.7 μm, 21 mm × 100 mm) using a gradient starting from 1% B for 1 minute, rising to 95% B in 4.5 minutes, holding at 95% B for 0.75 minutes, then decreasing to 1% B in 0.75 minutes and holding at 1% B for 1 minute. MS data were collected at a range of 80-2500 m/z, using identical conditions for ionization as with the protein.

P-623495-IL EXAMPLE 2 Modified Peptides can target both cysteine and lysine, controlling 14-3-3 isoform selectivity. [0090] To elucidate the binding sites of the peptides on 14-3-3σ, the peptides SEQ ID 3 , SEQ ID 8 and SEQ ID 11 were incubated with the protein and performed trypsin digestion followed by LC/MS/MS. ( The peptides and 14-3-3σ were prepared as disclosed in Example 1) irect identification and quantification of modified peptides was proved challenging due to long peptide chain lengths, fragmentation from multiple directions and weak relative signals. To characterize peptide ligation sites, we switched strategy and measured the relative change in the signal of non-modified peptides on 14-3-3σ relative to a DMSO-treated control. Specifically, we looked at the peptides containing or immediately following the residues Cys38, Lys49 and Lys122. Peptide SEQ ID 11 specifically reduced the signal of the Cys38-containing peptides, with little effect on the signals for other peptides, indicating specific Cys38 binding. In contrast, peptides containing or following Lys122 were significantly depleted by peptides SEQ ID 3 and SEQ ID 8 , albeit not uniformly, while the signals of Lys49 containing peptides were slightly increased. This result pointed to Lys122 as the likely binding site for peptides SEQ ID 3 and SEQ ID 8 . The non-uniform reduction in signal for Lys122-containing peptides may be due to incomplete labeling of 14-3-3σ by SEQ ID 3 and SEQ ID 8 . The possibility that adducts to lysine are unstable in the presence of reducing agents used before tryptic digest was considered. To test this, the peptides were incubated with TCEP (Triscarboxyethylphosphine) or DTT (Dithiothreitol), which caused rapid release of the peptide from the methacrylate within two hours (Figure 8A). However, under the same conditions the peptide-protein adducts were stable (Figure 8B). The protein was incubated with fluorescently labeled methacrylate peptides, followed by denaturation in DTT-containing sample buffer and SDS-PAGE. Here as well, reduction did not affect the intensity of the fluorescent band, indicating that after aza-Michael addition, the protein-peptide adduct is stable (Figure 8C). [0091] To further confirm that Lysine 122 is the target residue for peptides SEQ ID 3 and SEQ ID 8 , 14-3-3σ was co-crystallized with both peptides. The crystal structures (Figures 4A, 4B) clearly showed the covalent bond formed between the amine of Lys122 with the methacrylate, with Lysremaining unmodified. Comparison of the crystal structure with the prediction from CovPepDock indicated the model correctly predicted the binding pose around the phosphate and the N-terminal part of the peptide, but less so for the C-terminal region. More specifically, compared to the prediction, Lys122 adopts a more relaxed conformation, the C-terminal residues are not as tightly packed in the binding groove, and in peptide SEQ ID 8 (Figure 4A) the C-terminal glutamine could not be modeled due to insufficient density. The measured structures of the covalent complexes were compared with the known structures of the noncovalent complexes. For peptide SEQ ID 8 , the C P-623495-IL 21 terminal part of the peptide was displaced outwards due to the space occupied by the methacrylate ester moiety. The conformation of the peptide SEQ ID 3 complex was far less affected by covalent binding due to the shorter C terminal part of the peptide. In contrast, both peptides exhibited only a minor effect of covalent binding on the structure of the N terminal region. These results indicate that noncovalent interactions with the C-terminal part of the peptide play a minor role in the binding. [0092] Since Lys122 is highly conserved in all 14-3-3 isoforms (in contrast to cysteine 38 which is unique in 14-3-3σ), peptides SEQ ID 3 and SEQ ID 8 were incubated with other isoforms, together with the Cys38-targeting peptide SEQ ID 12 . While peptide SEQ ID 12 labeled only the sigma isoform, peptides SEQ ID 3 and SEQ ID 8 labeled all isoforms with similar efficiencies. Taken together these result conclusively validate that peptides SEQ ID 3 and SEQ ID 8 specifically bind to Lys122 via aza-Michael addition. Methods:Binding experiments to 14-3- 3σ id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"

[0093] Peptide 100X stocks were prepared by dissolving in DMSO + 5 mM acetic acid and storing at -80°C. Binding of peptides to 14-3-3σ was performed in 25 mM HEPES pH = 7.5, 100 mM NaCl, mM MgCl2. The protein was diluted to 2 μM in assay buffer, and the diluted protein was added to the peptide stock at 100:1 ratio and incubated in various conditions. For analysis, 24 μl sample was mixed with 6 μl of 2.4% formic acid in water and then 10 μl were injected to intact protein LCMS. LC/MS/MS characterization of labeling sites of methacrylate peptides in 14-3- 3σ [0094] 14-3-3σ was diluted to 2 µM in HEPES 25 mM pH 7.5, 100 mM NaCl, 10 mM MgCl2, and incubated with 5 µM peptide in samples of 50 µl. The samples were incubated for 48 hours at room temperature, resulting in ~75% labeling by 4IEA-5 ( SEQ ID 3 ), 90% labeling by 3MHR-5 ( SEQ ID 8 ) and 100% labeling by 3MHR-9 ( SEQ ID 11 ). At this point 50 µl of 10% SDS in HEPES 25 mM pH =7.5 was added and DTT was added to 5 mM, followed by incubation at 65°C for 45 minutes. This was followed by addition of iodoacetamide to 10 mM and incubation of 40 minutes at room temperature in the dark. The samples were then processed using S-trap (Protify) according to the manufacturer's instructions, followed by desalting using Oasis plate (Waters). [0095] Each sample was dissolved in 50 µl of 3% acetonitrile + 0.1% formic acid, and 0.5 µl was injected to the column. Samples were analyzed using EASY-nLC 1200 nano-flow UPLC system, using PepMap RSLC C18 column (2 μm particle size, 100 Å pore size, 75 μm diameter × 50 cm length), mounted using an EASY-Spray source onto an Exploris 240 mass spectrometer. uLC/MS-grade solvents were used for all chromatographic steps at 300 nL/min. The mobile phase was: (A) H2O + 0.1% formic acid and (B) 80% acetonitrile + 0.1% formic acid. Peptides were eluted from the P-623495-IL 22 column into the mass spectrometer using the following gradient: 1–40% B in 60 min, 40–100% B in min, maintained at 100% for 20 min, 100 to 1% in 10 min, and finally 1% for 5 min. Ionization was achieved using a 1900 V spray voltage with an ion transfer tube temperature of 275 °C. Initially, data were acquired in data-dependent acquisition (DDA) mode. MS1 resolution was set to 120,0(at 200 m/z), a mass range of 375–1650 m/z, normalized AGC of 300%, and the maximum injection time was set to 20 ms. MS2 resolution was set to 15,000, quadrupole isolation 1.4 m/z, normalized AGC of 100%, and maximum injection time of 22 ms, and HCD collision energy at 30%. 3 injections were performed for each sample. The DDA data was analyzed using MaxQuant 1.6.3.4. The database contained the sequence of the 14-3-3σ construct used in the study, and contaminants were included. [0096] Methionine oxidation and N terminal acetylation were variable modifications, and carbamidomethyl was a fixed modification in the analysis, with up to 4 modifications per peptide. Digestion was defined as trypsin/P with up to 2 missed cleavages. PSM (peptide spectrum match) FDR (false discovery rate) was defined as 1 and Protein FDR/Site Decoy fraction were defined as 0.01. Second Peptides were enabled and Match between runs was enabled with a Match time window of 0.7 minutes. The data was imported into skyline and precursors from 9 peptides containing or following the residues Cys38, Lys49 and Lys122 were selected for parallel reaction monitoring (PRM). In every acquisition cycle, on full MS was taken at a range of 350-1000 Da, 300% AGC (automatic gain control) target, maximum injection time 20 ms at a resolution of 120,000. Data for each precursor was measured during a 4-5 minute window around the retention time measured in the DDA run, with Q1 resolution of 2 Da, orbitrap resolution of 15,000, 300% AGC target and maximum injection time of 160 ms. The acquired data was then analyzed in skyline using a spectral library generated from the DDA runs. The 3 most intense product ions were used for quantitation relative to the DMSO control. Crystallization of 14-3- 3σ-peptide complexes: [0097] 14-3-3s was C-terminally truncated (DC=delta C) after T231 (Threonine 231 in the protein) to enhance crystallization. 14-3-3 and 4IEA_5 (SEQ ID 3)/3MHR-5 ( SEQ ID 8 ) peptides were dissolved in complexation buffer (20 mM HEPES pH 7.5, 100mM NaCl, 10 mM MgCl2) and mixed in a 1:2.5 or 1:5 molar stoichiometry (protein:peptide) at a final protein concentration of 10, 11, and 12.5 mg/mL. The complex was set up for sitting-drop crystallization after overnight incubation at 4 °C, in a custom crystallization liquor (0.095 M HEPES (pH7.1, 7.3, 7.5, 7.7), 0.19 M CaCl2, – 29 % (v/v) PEG 400 and 5% (v/v) glycerol). Crystals grew within 5 - 10 days at 4 °C. [0098] Crystals were fished and flash-cooled in liquid nitrogen. X-ray diffraction (XRD) data were collected at the Deutsches Elektronen-Synchrotron (DESY) PETRA III beamline P11, Hamburg, Germany.

P-623495-IL 23 id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"

[0099] Initial processing of datasets was done using CCP4i from the CCP4 suite. First, XIA2/DIALS was run for data indexing and integration, and AIMLESS for scaling. The structures were phased by molecular replacement, using protein data bank (PDB) entry 5N75 as a template, in MOLREP. REFMAC5 was used for initial structure refinement. Correct peptide sequences were modeled in the electron density in Coot. The presence of the covalent interaction of the peptides with Lysine 1was verified by visual inspection of the Fo-Fc and 2Fo-Fc electron density maps in Coot and build in via AceDRG. Finally, REFMAC5 and Coot were used in alternating cycles for model building and refinement. See Table 2 for data collection and refinement statistics. Table 2: Data collection and refinement statistics for 14-3-3σ bound to 4IEA_5 ( SEQ ID 5 ) and 3MHR_5 ( SEQ ID 8 ) PDB SEQ ID 5 SEQ ID 8Protein 14-3-3σDc 14-3-3σDc Peptide 4IEA_5 3MHR_ Beam DESY p11 DESY p11 Data CollectionWavelength (Å) 1.03322 1.033Space Group C 2 2 21 C 2 2 Cell Dimensionsa, b, c (Å) 82.5, 111.7, 63.1 82.8, 112.2, 63.0 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 55.92 – 2.20 (2.27-2.20) 56.14-2.30 (2.38-2.30) I / σ(I) 12.1 (6.6) 8.2 (5.0) Completeness (%) 99.7 (99.4) 99.9 (100) Redundancy 12.8 (10.4) 13.1 (13.1) CC1/2 0.991 (0.895) 0.980 (0.720) Refinement Number of Reflections 15133 133Rwork / Rfree 0.175/0.221 0.201/0.2 Number of Atoms Protein 3516 35Ligand/Ion 105/4 150/Water 110 B-factors Protein 18.98 17.Ligand/ion 18.01/43.35 14.18/36.Water 23.72 18.83 P-623495-IL 24 R.M.S deviationsBond Lengths (Å) 0.0091 0.00Bond Angles (°) 1.515 1.3 RamachandranFavored (%) 98.69 97.Outliers (%) 0.00 0. EXAMPLE 3 Modified Peptides detect 14-3-3 proteins in lysates and extracellular media with high sensitivity. [00100] In Example 2 it was demonstrated that methacrylate peptides can react with all 14-3-isoforms, BODIPY-labeled derivatives of peptides SEQ ID 3 , SEQ ID 8 and SEQ ID 12 were prepared and tested if they can function as pan-reactive 14-3-3 probes in cell lysates and in extracellular media. A549 cells were grew in serum-free media and incubated the peptides both with lysates prepared from the cells, and with the media following filtration, buffer exchange and concentration (Figure 5). The methacrylates SEQ ID 3 and SEQ ID 8 formed multiple bands, with the bottom band displaying a slight mass shift compared to 14-3-3β as found by western blot. In contrast, medium contained considerably less 14-3-3β, but the peptides still detected it with high sensitivity, and the 14-3-3β in the medium samples was efficiently converted to a higher MW band corresponding perfectly to the band observed by fluorescence. This may indicate that in lysates only a small fraction of 14-3-3β is labeled, either due to very high abundance of the protein, the presence of reducing agents in the lysate (such as cellular glutathione) that may react with some of the peptide, or competing binding proteins. In both lysates and media, the methacrylate peptides SEQ ID 3 and SEQ ID 8 generated multiple bands, which most likely indicates binding to other 14-3-3 isoforms. In contrast to the results with peptides SEQ ID 3 and SEQ ID 8 , 14-3-3σ was not detected by peptide SEQ ID 12 in the medium. Therefore, methacrylate peptides are powerful tools for the detection and quantitation of 14-3-3 isoforms in lysates and extracellular media. Methods:Binding of 14-3-3 proteins to peptides in extracellular media and lysates [00101] A549 cells were grown in DMEM + FBS and then transferred to DMEM without FBS, followed by incubation for either 24 hours or 48 hours. After the incubation, 7.5 ml of the medium P-623495-IL was filtered through a 0.2 µm filter, concentrated using a centrifugal concentrator (vivaspin, cutoff 8000-10000 Da) to 300 µl, and diluted to 8 ml using HEPES 20 mM pH = 7.5, 10 mM MgCl2, 1mM NaCl. This was followed by additional concentration to 300 µl, followed by another round of dilution and concentration. Finally the samples were diluted to 400 µl and used for experiments. [00102] The cells were washed with PBS, scraped from the plate and centrifuged 200 g for minutes. The cells were lysed in HEPES 20 mM pH = 7.5, 10 mM MgCl2, 100 mM NaCl with the addition of a protease inhibitor cocktail (Roche 11836170001). Cells were sonicated with 10 pulses of 2 seconds at 22% amplitude using a microprobe, followed by centrifugation at 21000 g for minutes at 4°C. The protein concentration was estimated using BCA, and the lysate was diluted to 1.97 in the lysis buffer. [00103] For incubation with the peptides, 38 µl of either lysate or medium was mixed with 2 µl of X stock of the peptide (for 3MHR_5 ( SEQ ID 8 ) and 4IEA_5 ( SEQ ID 3 ): 20 µM in 20% DMSO/buffer; for 3MHR_Cl ( SEQ ID 12 ): 5 µM in 20% DMSO/buffer; for no peptide: 20% DMSO/buffer), and incubated at room temperature for 24 hours in the dark. Then 13.3 µl of 4×LDS (Lithium dodecyl sulfate) samples buffer with 20 mM DTT was added and the samples were heated for 10 minutes at 70 °C. The samples were loaded on Bis-Tris gradient gels (4-20%, Genscript) and run using Tris-MOPS buffer at 55 mA/200 V. The gels were transferred to nitrocellulose membrane and the membrane was blocked with 5% BSA/TBST (Bovine serum albumin/Tris buffer saline + Tween20) for 1 hour RT. The membrane was incubated overnight at 4°C with 1:500 diluted anti-14-3-3β antibody (abcam ab15260) in 5% BSA/TBST. The membrane was washed thrice with TBST and incubated with 1:2000 diluted anti-rabbit Horseradish Peroxidase (HRP) antibody (CST 7074S) for 1 hour RT in 5% BSA/TBST. The membrane was washed thrice with TBST and imaged as followed: fluorescence using 532 nm excitation was measured using typhoon with Photomultiplier Tube (PMT) = 530 and 50 µm resolution. Chemiluminescence was measured using GelDoc (Biorad) with exposure times of 10 seconds for lysate samples and 300 seconds for medium samples. Chemiluminescence and fluorescence images were aligned using Photoshop and composite images were generated using Image. EXAMPLE 4 Modified proteins (Im9) covalent binders[00104] A recombinant protein was modified into a covalent binder using with 2-(bromomethyl)acrylate. As a model system the bacterial Colicin E9 toxin/anti-toxin system was selected. This system is composed of a highly toxic nuclease (E9) which is bound in the cell by an inhibitory partner termed the immunity protein (Im9). The complex can be excreted and internalized P-623495-IL 26 by target cells while displacing Im9, leading to E9-induced toxicity. The affinity of the Im9/Ecomplex is very high and is well characterized structurally. [00105] A computational pipeline using Rosetta Relax application (Tyka, M. D. et al., Alternate States of Proteins Revealed by Detailed Energy Landscape Mapping. J. Mol. Biol. 2011 , 405 (2), 607–618.; Leman, J. K.; et al., Macromolecular Modeling and Design in Rosetta: Recent Methods and Frameworks. Nat. Methods 2020 , 17 (7), 665–680.) was done which performed all-atoms refinement using relatively small moves that sample the local conformational space, while applying covalent constraints between the methacrylate side-chain and the target lysine to enforce the covalent bond between them. Based on the non-covalent complex of colicin E9 and Im9 (PDB: 1EMV), we mutated 20 positions of Im9 that are within Cα-Cα distance < 14Å from the target Lys97 to our methacrylate side-chain, and found five mutations that yielded sub-angstrom models (interface backbone RMSD < 1Å) with particularly good interface and constraint scores (Supplementary Figure 3). From these designs, an Im9 mutant (C23A/E41C) was selected onto which a methacrylate warhead was installed to react with Lysine 97 in the E9 nuclease (Figure 6A). E9 and the Im9 mutants were expressed and purified. Preparation of methacrylate-modified Im9 mutant under native buffer conditions was impractical as modification of the cysteine was slow and was competed by modification of other sites, as observed by the appearance of multiply-labeled species before full formation of the mono-labeled protein was observed, possibly indicating the cysteine was not fully exposed. Preparation under denaturing conditions (such as GuHCl 6M or 50% acetonitrile) was far more efficient, with rapid and selective modification in the time scale of minutes up to an hour, and combined with HPLC purification we obtained >95% single labeled protein. [00106] To assess ligation of methacrylate-modified Im9 to Lys97 of E9, modified Im9 mutant and E9 were incubated and the crosslinked complex was monitored via intact protein LCMS. The results show about 50% conversion to the covalent complex within 5 hours and near quantitative conversion with 16 hours (Figure 6B). To validate Lys97 ligation, a point mutation experiment was performed by individually mutating lysines 55, 81, 89, 97 and 125 to arginine. For the mutants K81R and K125R, bacterial growth was dramatically inhibited, possibly indicating reduction in the binding affinity leading to E9-mediated toxicity. We tested the binding K55R, K89R and K97R to the methacrylate-modified Im9 mutant (Figure 6C). While the K55R and K89R mutations had no effect of ligation efficiency, mutation of Lys97 abolished the formation of the covalent complex almost completely, indicating Lys97 as the target binding site, in agreement with the model. [00107] The covalent binding affects was studied, by measuring the stability of the complex. To this end, a scanning differential scanning fluorimetry (DSF) was used to monitor the thermal stability of the complex (Figure 7). Im9 protein in its free and methacrylate-modified form exhibit unfolding around 50°C, while the E9 protein does not show any discernible transition. While the noncovalent P-623495-IL 27 complex shows only minimal differences compared to free Im9, the covalent complex is considerably more stable, unfolding at 72°C. (Figure 7). Methods: Cloning of Im9 mutant and E9 mutants [00108] pET21d plasmids encoding for either E9 + wild type Im9 or for Im9 were used. For mutation of E41 into cysteines, PCR was performed using the plasmids as a template using the following primers: ImFor: GAAATGACTGAGCACCCTAGT (SEQ ID 13) ImRev: ACAAAAGTGTGTAACCAATTTAACCAGTTC (SEQ ID 14) [00109] The PCR product was purified and 1 µg was phosphorylated using 10 units T4 PNK (NEB) in 20 µl of T4 ligase buffer (NEB) for 1 hours at room temperature. This was followed by addition of 400 units of T4 ligase (NEB) for 2 hours at room temperature. The product was transformed to DH5α and plated on ampicillin plates. After pick of colonies and identification of correct sequences, this step was repeated with the following primers to introduce the second mutation (C23A): Im2For: GCTAATGCGGACACTTCCAGTG (SEQ ID 15) Im2Rev: AATTGTTGTTACAAGCTGTAAAAATTCAG (SEQ ID 16) For mutations of E9 we used the same procedure with the following sets of primers: Lys55for: CGGGCTGTATGGGAAGAGGTGTC (SEQ ID 17) Lys55rev: CCGAAAATCGTCGAAGCTTTTAAATTC (SEQ ID 18) Lys81for: CGAGGTTATTCTCCGTTTACTCCAAAG (SEQ ID 19) Lys81rev: TGAAACACTAGACTTATTGCTTGGG (SEQ ID 20) Lys89for: CGGAATCAACAGGTCGGAGGG (SEQ ID 21) Lys89rev: TGGAGTAAACGGAGAATAACCTTTTG (SEQ ID 22) Lys97for: CGAGTCTATGAACTTCATCATGACAAG (SEQ ID 23) Lys97rev: TCTCCCTCCGACCTGTTG (SEQ ID 24) Lys125for: CGGCGACATATCGATATTCACCG (SEQ ID 25) Lys125rev: AGGTGTAGTCACTCGGATATTATC (SEQ ID 26) Expression and Purification of E9 and Im9 mutants [00110] The plasmids were transformed into BL21(DE3) bacteria. The bacteria were grown in 2YT + NPS + 1 mM MgSO4 at 37°C to OD = 0.6, cooled rapidly on ice to 16°C, and induced using mM IPTG for 16 hours.

P-623495-IL 28 id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111"

[00111] For purification of Im9, the cells were dispersed in 30 ml of lysis buffer (Tris 25 mM pH = 7.5, 50 mM NaCl, 10 mM imidazole) + protease inhibitors, and sonicated (55%, one minute, second pulses). After this, MgCl2 was added to 1 mM and 5 µl of benzonase nuclease (Fischer) were added. The lysates were spun (20000 rpm for 20 minutes), and the lysates were filtered 0.45 µm. Then, each lysate was loaded on Ni-NTA column (5 ml) preequilibrated with lysis buffer, and the column was washed with 4 CV of lysis buffer. Im9 was eluted with Tris 25 mM pH = 7.5, 50 mM NaCl, 500 mM imidazole, dialyzed extensively against NaPi 20 mM pH = 7.2, 100 mM NaCl (times), filtered 0.2 µm and flash frozen in -80°C. [00112] Purification of E9 was performed using an identical procedure, except that elution was performed using 6 M GuHCl. Some precipitation was observed during dialysis. Methacrylate labeling of Im9 mutant [00113] Labeling was performed in protein storage buffer (NaPi 20 mM pH = 7.2, 100 mM NaCl). For labeling in native conditions, ethyl-(3-bromomethacrylate) was dissolved in DMSO to a concentration of 705 mM, and 2 µl were added to a samples of 312.5 µl of 1.13 mM protein (equivalents). Reaction was performed at room temperature. Reactions were stopped by desalting µl samples into NaPi 20 mM pH = 7.2, 100 mM NaCl using biospin columns and kept on ice. Protein was then diluted with storage buffer to 2 µM and 10 µl were injected to LCMS. For reactions in 6 M GuHCl, solid GuHCl was added to the protein sample to give 6 M, and ethyl-(3-bromomethacrylate) was added as before. Reaction was against stopped by desalting into native buffer as described. In GuHCl the reaction was extremely rapid and had to be stopped within 5-10 minutes to avoid over labeling. [00114] Samples of Im9 mutant methacrylate used in later experiments were prepared by reaction in 50% acetonitrile as described here: the protein was diluted X2 in acetonitrile (leading to some precipitation), and 1.1 equivalents of ethyl-(3-bromomethacrylate) (dissolved beforehand in acetonitrile) was added. After 1 hour at room temperature, 70% labeling was observed, and another 0.7 equivalents were added. After an hour the sample was diluted in 0.1% TFA in water, filtered and purified using HPLC. Reaction between Im9 methacrylate and E [00115] The purified proteins were diluted to 20 µM in NaPi 20 mM pH = 7.2, 50 mM NaCl. Then the Im methacrylate solution was mixed in a 1:1 ratio with the E9 solution, giving 10 µM complex. The reactions were incubated at room temperature for 4.5 hours, and then stopped by diluting the complex 5-fold in 0.1% TFA/water, followed by LC-MS analysis.

P-623495-IL 29 Differential Scanning Fluorimetry for Im-E9 complexes [00116] 50 µl samples of E9:Im complexes in a concentration of 50 µM were prepared and incubated overnight at room temperature in NaPi 20 mM pH = 7.2, 50 mM NaCl. SYPRO Orange (X5000 stock) was diluted 200-fold in buffer, and from this stock 13 µl were added to each sample, diluting the protein to 40 µM. Each sample were split into 3 technical replicates and heated in a thermal cycler over 1.5 hours to 95°C while measuring the fluorescence. Introducing New Residues to Rosetta [00117] Our methacrylate side-chain was introduced to Rosetta using the protocol described in Renfrew et al. (Renfrew et al. Incorporation of Noncanonical Amino Acids into Rosetta and Use in Computational Protein-Peptide Interface Design. PLoS One 2012 , 7 (3), e32637.) As the reaction between the methacrylate warhead and the lysine amine forms two different stereoisomers, they were implemented as different residues. The GaussView interface was used to draw each stereoisomer, and then used the Gaussian software to optimize the structures, with the following options: HF/6-31G(d) scf = tight test. Each optimized structure was converted to a mol file using OpenBabel toolbox (http://openbabel.org), and then to a Rosetta residue ‘params file’ using the molfile_to_params_polymer.py script provided in Rosetta. To allow the residue to form a covalent bond to another residue, we added a CONNECT record to each stereoisomer params file, specifying which atom participates in the inter-residue covalent bond, as described in Drew et al. (Drew et al. Adding Diverse Noncanonical Backbones to Rosetta: Enabling Peptidomimetic Design. PLoS One 2013 , 8 (7), e67051.) for oligooxopiperazines. A virtual atom was added to each params file, and defined its internal coordinates according to the optimal position of the lysine NZ atom as predicted by the Gaussian optimization. These virtual atoms were used during the modeling process to favor the correct covalent bond geometry. Rotamer libraries were generated using the Rosetta MakeRotLib application. [00118] A suitable covalently-linked variant of lysine was implemented through the residue patch system, to utilize the existing definitions and rotamer libraries that have been optimized for use in Rosetta. The reacted lysine was modeled as described above, and created a patch file that deletes the 3HZ atom of lysine, and adds a CONNECT record and a virtual atom with internal coordinates that match the Gaussian optimized structure. A PROTON_CHI record was added to allow sampling of the new rotamers around the bond CE-NZ bond. Design of Colicin E9 Protein Binders [00119] PDB ID: 1EMV was used as a template structure. Similar to the peptide design protocol, Rosetta fixed backbone design application (fixbb) was used to mutate Lys97 of colicin E9 to our P-623495-IL covalently-linked variant, and to mutate positions 30-41 and 48-55 of Im9 to each stereoisomer of our methacrylate side-chain. The RosettaScripts interface was then used and the FastRelax mover to generate 200 models of each complex (100 for each stereoisomer), while applying similar constraints to these described in the peptide design method section. To select a construct for synthesis and testing, the 10 top-interface-scoring models were manually inspected of each mutated complex, focusing on near-native models with constraint score < 2. [00120] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (21)

1. P-623495-IL 31

2. CLAIMSWhat is claimed is: 1. A modified protein or a modified peptide comprising a recombinant protein or a synthetic peptide modified by an electrophile, wherein the electrophile is covalently bound to a thiol group of a cysteine within the protein or peptide; wherein the electrophile is represented by the structure of Formula I;

3. (I) wherein, R comprises substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; X comprises O, NR or substituted or unsubstituted alkylene; and R is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocyclic; wherein the modified protein or modified peptide is capable of covalently binding a target protein. 2. The modified protein or modified peptide of claim 1, having a non-covalent recognition with the target protein; and the electrophile is in close proximity to a target residue of the target protein; prior to the covalent binding with the target protein. 3. The modified protein or modified peptide of claim 2, wherein the target residue within the target protein is a cysteine residue (SH) or a lysine residue (NH2).

4. The modified protein or modified peptide of claim 2 or claim 3, wherein the close proximity comprises a distance being less than 15 Å between the electrophile and the target residue.

5. The modified protein or modified peptide of any one of claims 1-4, wherein the electrophile is represented by the structure of Formula II: P-623495-IL 32

6. The modified protein or modified peptide of any one of claims 1-5, wherein the electrophile is ethyl methacrylate.

7. The modified peptide of any one of claims 1-6 comprising the following peptides:Ac-RSApSmCPSL-NH2 ( SEQ ID 3 ), Ac-RAHpSmCPASLQ-NH2 ( SEQ ID 8 ) or Ac-RAHpSSPASmCQ-NH2 ( SEQ ID 11 ); wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine.

8. The modified peptide of claim 7, wherein the peptide covalently binds 14-3-3 target protein.

9. The modified peptide of claim 8, wherein the peptide covalently binds Cys38, Lys 122 or Lys 49 of 14-3-3 σ target protein.

10. The modified protein of any one of claims 1-7 comprising a modified immunity (Im9) protein ( SEQ ID 27 ).

11. The modified protein of claim 10, wherein the Im9 protein ( SEQ ID 27 ) binds an E9 target protein.

12. A method of preparing a modified protein or a modified peptide of any one of claims 1-12, wherein the method comprises; a. identifying a target protein; b. designing a peptide or protein covalent binder candidate based on previously characterized noncovalent binders of the target protein; c. synthesizing a modified peptide or a modified protein, to allow recognition with the target protein at the recognition site; wherein said modified protein or modified peptide comprise a cysteine which is reacted with an electrophile of Formula I.

13. The method of claim 12, wherein the design of the peptide is based on computational modeling comprising Rosetta CovPepDock.

14. The method of claim 12, wherein the modified peptide is synthesized from unprotected peptides.

15. The method of claim 12, wherein the modified protein is synthesized from recombinant proteins.

16. A protein-protein covalent conjugate comprising a modified protein of any one of claims 1-11, covalently bound to a target protein, wherein the modified protein and the target protein possess non-covalent recognition prior to the covalent binding with the target protein.

17. The protein-protein covalent conjugate of claim 16, wherein the modified protein is Improtein and the target protein is E9.

18. The protein-protein covalent conjugate of claim 17, wherein the conjugate displayed a higher thermal stability than the noncovalent complex. P-623495-IL 33

19. A peptide-protein covalent conjugate comprising a modified peptide of any one of claims 1-11, covalently bound to a target protein, wherein the modified peptide and the target protein possess non-covalent recognition prior to the covalent binding with the target protein.

20. The peptide-protein covalent conjugate of claim 19, wherein the modified peptide is Ac-RSApSmCPSL-NH2 (SEAQ ID 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID 8) or Ac-RAHpSSPASmCQ-NH2 (SEQ ID 11); wherein pS is Phoshoserine and mC is Mehtacrylate-modified cysteine; and the target protein is 14-3-3.

21. The modified protein or modified peptide of any one of claims 1-11 for use in selectively label, fluorescent label, inhibition, drug conjugation or conjugation to a target protein.