Modified proteins or peptides for covalent targeting

Abstract

The present invention is directed to modified proteins or peptides (modified with electrophiles, such as acrylamide or acrylate) for covalent attachment to target proteins.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy, created on February 19, 2024, has the filename "P-623495-PC_ST26.xml" and is 56,897 bytes in size.

[0002] The present invention is directed to modified proteins or peptides (modified with electrophiles, such as acrylamide or acrylate) for covalent attachment to target proteins. [Background technology]

[0003] 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 to target a wide variety of proteins, including kinases, G-protein-coupled receptors, and hydrolases, and have found important applications as probes for proteomics and microscopy, as well as in emerging applications such as targeted degradation. The advantages of covalent compounds in chemical biology arise from several aspects. Irreversible binding to the target achieves sustained and potent inhibition with short systemic exposures. Covalent binding to the target facilitates downstream processes, including target denaturation and proteolysis, without loss of the bound probe, making them particularly useful in proteomics. Finally, covalent binders often exhibit enhanced selectivity by targeting non-conserved nucleophilic residues. This is particularly evident in Kras G12C This is exemplified by the recently approved sotorasib and adagrasib, which selectively target

[0004] The rapid development of novel warhead groups for small molecules has expanded the targetable range 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 introduction of reactive groups on peptides or full-length proteins is not straightforward. The large number of nucleophilic amino acids on proteins or the relatively harsh deprotection conditions required for solid-phase peptide synthesis make the introduction of electrophiles difficult. Various approaches have emerged in recent years for functionalizing natural sequences and preparing protein-based covalent binders. These include genetic code expansion to incorporate reactive groups into protein sequences via unnatural amino acids, approaches utilizing enzymatic activation of proteins, and site-selective chemical approaches. However, these techniques remain technically challenging, and there remains a need for simple approaches to expand the range of targetable residues with high selectivity.

[0005] Despite the proliferation of research into covalent compounds, targets such as transcription factors and protein-protein interaction interfaces are difficult to target with small molecules due to their large and shallow binding surfaces. The use of peptides or peptidomimetics has emerged as a powerful approach to address these challenges. Peptide binders can cover large surface areas, bind protein targets with high affinity, and can frequently be derived from known protein-protein interactions.

[0006] Strong covalent peptide binders have been developed for targets such as bacterial division complexes, E3 ubiquitin ligases, and the anti-apoptotic protein BFL-1. Due to the size of peptides and their structural flexibility, computational modeling can aid in the design and placement of electrophiles. Summary of the Invention

[0007] In some embodiments, provided herein are modified proteins or peptides, including recombinant proteins or synthetic peptides modified with an electrophile, wherein the electrophile is covalently attached to a thiol group of a cysteine ​​within the protein or peptide, and the electrophile has Formula I; [ka] (In the formula, R 1 includes substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle; X is O, NR 3 or substituted or unsubstituted alkylene; and R 3 is hydrogen, alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle) is represented by the structure The modified protein or modified peptide is capable of covalently binding a target protein.

[0008] In some embodiments, the modified peptides include the following peptides: Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8), or Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11), where pS is phosphoserine and mC is methacrylate-modified cysteine. In other embodiments, the modified peptides Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8), or Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11), where pS is phosphoserine and mC is methacrylate-modified cysteine, covalently bind 14-3-3 target proteins. In other embodiments, the peptide Ac-RSApSmCPSL-NH2 (sequence ID3), Ac-RAHpSmCPASLQ-NH2 (sequence ID8) or Ac-RAHpSSPASmCQ-NH2 (sequence ID11) covalently binds Cys38, Lys122 or Lys49 of the 14-3-3σ target protein.

[0009] In some embodiments, the modified protein comprises a modified immunity (Im9) protein (SEQ ID 27). In other embodiments, Im9 (SEQ ID 27) binds the E9 target protein.

[0010] In some embodiments, provided herein are methods for preparing a modified protein or peptide provided herein, the methods comprising: a. Identifying the target protein; b. Designing candidate covalent binders of a peptide or protein based on previously characterized non-covalent binders of the target protein; c. synthesizing a modified peptide or protein that allows recognition of the target protein at the recognition site, wherein the modified protein or peptide contains a cysteine ​​that reacts with an electrophile of Formula I-IV; Includes.

[0011] In some embodiments, provided herein is a protein-protein covalent conjugate comprising a modified protein described herein covalently linked to a target protein, wherein the modified protein and the target protein have non-covalent recognition prior to covalent linkage with the target protein.

[0012] In some embodiments, provided herein is a peptide-protein covalent conjugate comprising a modified peptide described herein covalently attached to a target protein, wherein the modified peptide and the target protein have non-covalent recognition prior to covalent attachment to the target protein.

[0013] In some embodiments, provided herein are modified proteins or modified peptides provided herein for use in selective labeling, fluorescent labeling, inhibition, drug conjugation, or conjugation to a target protein.

[0014] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. However, the invention, together with its objects, features, and advantages, both as to organization and method of operation, may best be understood by reference to the following detailed description when read in connection with the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] Scheme for generating thioether-based modified proteins or peptides of the present invention. Cysteine ​​residues can be introduced into peptides (ribbon-like lines) or recombinant proteins (marked with *), which are then modified using ethyl 2-(bromomethyl)acrylate. The resulting modified protein or peptide reacts with lysine or cysteine ​​side chains near the binding site on a receptor to form a covalent adduct. [Figure 2A] Generating peptide covalent binding reagents for 14-3-3 proteins. Figure 2A: Structure of the complex of 14-3-3σ with a phosphorylated peptide derived from YAP (; PDB: 3MHR). The nonconserved cysteine ​​38 and the conserved lysines 49 and 122 are highlighted. Figure 2B: Scheme for the synthesis of electrophilic peptides. a) 20% piperidine in DMF, 3 × 4 min; b) 4 equivalents of Fmoc-AA-OH / HATU / HOAT, 8 equivalents of DIPEA, 30 min at RT; c) 94% TFA, 2.5% water, 2.5% TIPS, 1% DODT, 3 h. Figure 2C: HPLC chromatogram and MS spectrum of crude peptide 3 (bottom) and the crude peptide (top) after reaction with 2-(bromomethyl)acrylate. The second peak is excess 2-(bromomethyl)acrylate. [Figure 2B]Generating peptide covalent binding reagents for 14-3-3 proteins. Figure 2A: Structure of the complex of 14-3-3σ with a phosphorylated peptide derived from YAP (; PDB: 3MHR). The nonconserved cysteine ​​38 and the conserved lysines 49 and 122 are highlighted. Figure 2B: Scheme for the synthesis of electrophilic peptides. a) 20% piperidine in DMF, 3 × 4 min; b) 4 equivalents of Fmoc-AA-OH / HATU / HOAT, 8 equivalents of DIPEA, 30 min at RT; c) 94% TFA, 2.5% water, 2.5% TIPS, 1% DODT, 3 h. Figure 2C: HPLC chromatogram and MS spectrum of crude peptide 3 (bottom) and the crude peptide (top) after reaction with 2-(bromomethyl)acrylate. The second peak is excess 2-(bromomethyl)acrylate. [Figure 2C] Generating peptide covalent binding reagents for 14-3-3 proteins. Figure 2A: Structure of the complex of 14-3-3σ with a phosphorylated peptide derived from YAP (; PDB: 3MHR). The nonconserved cysteine ​​38 and the conserved lysines 49 and 122 are highlighted. Figure 2B: Scheme for the synthesis of electrophilic peptides. a) 20% piperidine in DMF, 3 × 4 min; b) 4 equivalents of Fmoc-AA-OH / HATU / HOAT, 8 equivalents of DIPEA, 30 min at RT; c) 94% TFA, 2.5% water, 2.5% TIPS, 1% DODT, 3 h. Figure 2C: HPLC chromatogram and MS spectrum of crude peptide 3 (bottom) and the crude peptide (top) after reaction with 2-(bromomethyl)acrylate. The second peak is excess 2-(bromomethyl)acrylate. [Figure 3A]Designed methacrylate peptides bind 14-3-3σ. Figure 3A. Peptides (200 μM) were incubated with 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 various times and analyzed using intact protein LC / MS. 3 and 8 formed only full-length peptide adducts. 11 also formed a transient methacrylate-only adduct, which converted to the full adduct over time. [Figure 3B] Designed methacrylate peptides bind 14-3-3σ. Figure 3A. Peptides (200 μM) were incubated with 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 various times and analyzed using intact protein LC / MS. 3 and 8 formed only full-length peptide adducts. 11 also formed a transient methacrylate-only adduct, which converted to the full adduct over time. [Figure 4A] The methacrylate peptide binds the conserved 14-3-3 lysine residue. Figure 4A: Overlay of the CovPepDock prediction and co-crystal structure of peptide sequence ID8 bound to 14-3-3σ (white surface; left), with a zoom in on the methacrylate residue (right). The final 2Fo-Fc electron density (thin mesh, contoured at 1.0σ) is shown for peptide sequence ID8 (right). Similarly, Figure 4B: Docking overlay (left) and zoom in (right) for peptide sequence ID3. The final 2Fo-Fc electron density (thin mesh, contoured at 1.0σ) is shown for the peptide (right). [Figure 4B]The methacrylate peptide binds the conserved 14-3-3 lysine residue. Figure 4A: Overlay of the CovPepDock prediction and co-crystal structure of peptide sequence ID8 bound to 14-3-3σ (white surface; left), with a zoom in on the methacrylate residue (right). The final 2Fo-Fc electron density (thin mesh, contoured at 1.0σ) is shown for peptide sequence ID8 (right). Similarly, Figure 4B: Docking overlay (left) and zoom in (right) for peptide sequence ID3. The final 2Fo-Fc electron density (thin mesh, contoured at 1.0σ) is shown for the peptide (right). [Figure 5A] Labeling of 14-3-3 proteins with BODIPY-modified peptides in A549 lysate and medium. A549 cells were grown in serum-free medium for the indicated times. Lysate (Figure 5A) or concentrated medium (Figure 5B) was then incubated with peptides at room temperature for various times, followed by SDS-PAGE and Western blotting. Bottom panel: detection of 14-3-3β by Western blotting; middle panel - peptide fluorescence; right panel - overlay image. The Y-axis indicates molecular weight. [Figure 5B] Labeling of 14-3-3 proteins with BODIPY-modified peptides in A549 lysate and medium. A549 cells were grown in serum-free medium for the indicated times. Lysate (Figure 5A) or concentrated medium (Figure 5B) was then incubated with peptides at room temperature for various times, followed by SDS-PAGE and Western blotting. Bottom panel: detection of 14-3-3β by Western blotting; middle panel - peptide fluorescence; right panel - overlay image. The Y-axis indicates molecular weight. [Figure 6] Characterization of the selectivity of peptide sequences ID3 and ID8 using pull-down proteomics. A549 lysates were treated with biotinylated derivatives of peptide sequences ID3 or ID8 (1 μM, 22 h at 25°C). Biotinylated proteins were enriched using streptavidin beads, digested with trypsin, and analyzed using LC-MS / MS. [Figure 7A]Generation of Im9 protein capable of irreversibly binding E9. Figure 7A: Model of the C23A / E41C mutant of Im9 covalently bound to Lys97 in E9 (white) compared to the wild-type complex (with Im9). Figure 7B: Deconvoluted MS spectra of purified Im methacrylate, purified E9, and the covalent complex formed after their incubation. Figure 7C: Reverse-phase HPLC chromatograms of samples of Im methacrylate incubated with wild-type E9 and several E9 mutants. Incubation with the K97R mutant abolishes covalent bond formation. Figure 7D: Deconvoluted mass spectra of the Im C23A / E41C mutant (top) and the methacrylate-modified protein (bottom). [Figure 7B] Generation of Im9 protein capable of irreversibly binding E9. Figure 7A: Model of the C23A / E41C mutant of Im9 covalently bound to Lys97 in E9 (white) compared to the wild-type complex (with Im9). Figure 7B: Deconvoluted MS spectra of purified Im methacrylate, purified E9, and the covalent complex formed after their incubation. Figure 7C: Reverse-phase HPLC chromatograms of samples of Im methacrylate incubated with wild-type E9 and several E9 mutants. Incubation with the K97R mutant abolishes covalent bond formation. Figure 7D: Deconvoluted mass spectra of the Im C23A / E41C mutant (top) and the methacrylate-modified protein (bottom). [Figure 7C]Generation of Im9 protein capable of irreversibly binding E9. Figure 7A: Model of the C23A / E41C mutant of Im9 covalently bound to Lys97 in E9 (white) compared to the wild-type complex (with Im9). Figure 7B: Deconvoluted MS spectra of purified Im methacrylate, purified E9, and the covalent complex formed after their incubation. Figure 7C: Reverse-phase HPLC chromatograms of samples of Im methacrylate incubated with wild-type E9 and several E9 mutants. Incubation with the K97R mutant abolishes covalent bond formation. Figure 7D: Deconvoluted mass spectra of the Im C23A / E41C mutant (top) and the methacrylate-modified protein (bottom). [Figure 7D] Generation of Im9 protein capable of irreversibly binding E9. Figure 7A: Model of the C23A / E41C mutant of Im9 covalently bound to Lys97 in E9 (white) compared to the wild-type complex (with Im9). Figure 7B: Deconvoluted MS spectra of purified Im methacrylate, purified E9, and the covalent complex formed after their incubation. Figure 7C: Reverse-phase HPLC chromatograms of samples of Im methacrylate incubated with wild-type E9 and several E9 mutants. Incubation with the K97R mutant abolishes covalent bond formation. Figure 7D: Deconvoluted mass spectra of the Im C23A / E41C mutant (top) and the methacrylate-modified protein (bottom). [Figure 8A]Methacrylate peptides label different 14-3-3σ residues. Figure 8A: Peptide sequence ID3 was incubated with either 2 mM 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 sequence ID3 until fully labeled, followed by incubation with TCEP or DTT for 130 minutes at room temperature and analyzed by LC / MS. Figure 8C: 14-3-3σ was incubated with Bodipy-modified peptides (sequences ID3 and ID8) until fully labeled. Samples were then denatured using lithium dodecyl sulfate buffer under various conditions (with or without heat and with or without DTT) and analyzed by SDS-PAGE (see Example 2). Figure 8D: Following incubation with peptides (SEQ ID NO:3, SEQ ID NO:8, and SEQ ID NO:11), 14-3-3σ samples were trypsinized and tryptic peptides containing or following potential target residues (numbers 1-9, SEQ ID NO:29-37, respectively) were quantified using parallel reaction monitoring. [Figure 8B] Methacrylate peptides label different 14-3-3σ residues. Figure 8A: Peptide sequence ID3 was incubated with either 2 mM 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 sequence ID3 until fully labeled, followed by incubation with TCEP or DTT for 130 minutes at room temperature and analyzed by LC / MS. Figure 8C: 14-3-3σ was incubated with Bodipy-modified peptides (sequences ID3 and ID8) until fully labeled. Samples were then denatured using lithium dodecyl sulfate buffer under various conditions (with or without heat and with or without DTT) and analyzed by SDS-PAGE (see Example 2). Figure 8D: Following incubation with peptides (SEQ ID NO:3, SEQ ID NO:8, and SEQ ID NO:11), 14-3-3σ samples were trypsinized and tryptic peptides containing or following potential target residues (numbers 1-9, SEQ ID NO:29-37, respectively) were quantified using parallel reaction monitoring. [Figure 8C]Methacrylate peptides label different 14-3-3σ residues. Figure 8A: Peptide sequence ID3 was incubated with either 2 mM 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 sequence ID3 until fully labeled, followed by incubation with TCEP or DTT for 130 minutes at room temperature and analyzed by LC / MS. Figure 8C: 14-3-3σ was incubated with Bodipy-modified peptides (sequences ID3 and ID8) until fully labeled. Samples were then denatured using lithium dodecyl sulfate buffer under various conditions (with or without heat and with or without DTT) and analyzed by SDS-PAGE (see Example 2). Figure 8D: Following incubation with peptides (SEQ ID NO:3, SEQ ID NO:8, and SEQ ID NO:11), 14-3-3σ samples were trypsinized and tryptic peptides containing or following potential target residues (numbers 1-9, SEQ ID NO:29-37, respectively) were quantified using parallel reaction monitoring. [Figure 8D] Methacrylate peptides label different 14-3-3σ residues. Figure 8A: Peptide sequence ID3 was incubated with either 2 mM 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 sequence ID3 until fully labeled, followed by incubation with TCEP or DTT for 130 minutes at room temperature and analyzed by LC / MS. Figure 8C: 14-3-3σ was incubated with Bodipy-modified peptides (sequences ID3 and ID8) until fully labeled. Samples were then denatured using lithium dodecyl sulfate buffer under various conditions (with or without heat and with or without DTT) and analyzed by SDS-PAGE (see Example 2). Figure 8D: Following incubation with peptides (SEQ ID NO:3, SEQ ID NO:8, and SEQ ID NO:11), 14-3-3σ samples were trypsinized and tryptic peptides containing or following potential target residues (numbers 1-9, SEQ ID NO:29-37, respectively) were quantified using parallel reaction monitoring. [Figure 9]Fluorescence polarization competition experiments. Fluorescence polarization of 5 nM BODIPY-labeled noncovalent binders to 14-3-3σ was measured alone (SEQ ID NO: 38), in the presence of 0.25 μM 14-3-3σ, and in the presence of 14-3-3σ and various electrophilic peptides (SEQ ID NOs: 1-11, 5 μM and 200 μM). The sequence of the BDP-labeled binder is shown at the top (SEQ ID NO: 38); Dab = diaminobutyric acid. The control peptide is identical in sequence to the BDP-labeled binder. [Figure 10] Peptide labeling at various temperatures. Peptides sequence ID3 and sequence ID8 (5 μM) were incubated with 14-3-3σ (2 μM) at either 25° C. or 37° C., and the degree of labeling was measured using LC / MS. [Figure 11A] The formation of a covalent complex significantly stabilizes the thermal stability of the complex. Figure 11A: Average derivative data from three samples are shown for each condition. Figure 11B: Measured melting temperatures (calculated from the maximum derivative) for the various constructs. Error bars represent standard deviation (n=3). Melting temperatures could not be calculated for the E9 construct alone, which did not show a clear melting curve. [Figure 11B] The formation of a covalent complex significantly stabilizes the thermal stability of the complex. Figure 11A: Average derivative data from three samples are shown for each condition. Figure 11B: Measured melting temperatures (calculated from the maximum derivative) for the various constructs. Error bars represent standard deviation (n=3). Melting temperatures could not be calculated for the E9 construct alone, which did not show a clear melting curve. [Figure 12A] Alternative electrophiles can tune the reaction rate. Figure 12A: Labeling kinetics of 14-3-3σ (2 μM) with peptide sequence ID3 (ethyl ester-peptide 3 and phenyl ester-peptide 3a) at 25 °C and 5 μM peptide. Figure 12B: CovPepDock model of peptide 3a bound to 14-3-3σ superimposed on the structure of the parent non-covalently bound peptide. [Figure 12B]Alternative electrophiles can tune the reaction rate. Figure 12A: Labeling kinetics of 14-3-3σ (2 μM) with peptide sequence ID3 (ethyl ester-peptide 3 and phenyl ester-peptide 3a) at 25 °C and 5 μM peptide. Figure 12B: CovPepDock model of peptide 3a bound to 14-3-3σ superimposed on the structure of the parent non-covalently bound peptide. [Figure 13A] Structural insights provided by CovPepDock for covalent protein binding reagents. Figure 13A. Top-scoring model of the E9-Im9 complex when Val34 of Im9 is mutated to a methacrylate warhead group. This position is located close to the target Lys97 (Cα-Cα distance = 7.5 Å), but none of the 10 top-scoring models had a constraint score <2, suggesting a non-ideal covalent geometry. Figure 13B. Docked model of the E9-Im9 complex when Glu42 is mutated (RMSD = 0.758 Å). This position appears to be located far from the target Lys97, but CovPepDock revealed a potential shift in the helix that would allow covalent bond formation. [Figure 13B] Structural insights provided by CovPepDock for covalent protein binding reagents. Figure 13A. Top-scoring model of the E9-Im9 complex when Val34 of Im9 is mutated to a methacrylate warhead group. This position is located close to the target Lys97 (Cα-Cα distance = 7.5 Å), but none of the 10 top-scoring models had a constraint score <2, suggesting a non-ideal covalent geometry. Figure 13B. Docked model of the E9-Im9 complex when Glu42 is mutated (RMSD = 0.758 Å). This position appears to be located far from the target Lys97, but CovPepDock revealed a potential shift in the helix that would allow covalent bond formation.

[0016] 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 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 similar elements. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present 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.

[0018] Modified proteins or peptides In some embodiments, provided herein are modified proteins or peptides, including synthetic / recombinant proteins or peptides modified with an electrophile, wherein the electrophile is covalently attached to a thiol group of a cysteine ​​within the synthetic / recombinant protein or peptide, and the electrophile has a structure represented by Formula I: [ka] (In the formula, R 1 includes substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle; and X is O, NR 3 or substituted or unsubstituted alkylene; and R 3 is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle) is represented by the structure The modified protein or modified peptide is capable of covalently binding a target protein.

[0019] In other embodiments, the electrophile has Formula II (methacrylate): [ka] (In the formula, R 1 includes substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, carbocyclic, aryl, heteroaryl or heterocycle) It is represented by the structure:

[0020] In other embodiments, the electrophile is: [ka] is.

[0021] In other embodiments, the electrophile has formula III (methacrylamide): [ka] (In the formula, R 1 or R 3 each including a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl or heterocycle) It is represented by the structure:

[0022] In other embodiments, the electrophile has formula IV: [ka] (In the formula, R 1 includes substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl or heterocycle; and Alk refers to substituted or unsubstituted alkylene). It is represented by the structure:

[0023] In some embodiments, the modified protein or peptide comprises: (i) the introduction of an electrophile into an unprotected peptide or protein; and (ii) the electrophile reacts with both thiols and primary amines of the target protein. Thus, electrophiles of Formulae I-IV are selected that react with both cysteine ​​and lysine residues of the target protein via aza-Michael addition. As a non-limiting example, ethyl 2-(bromomethyl)acrylate (FIG. 2B) is used due to its selective reactivity with cysteine ​​residues via its α-bromomethylene functional group.

[0024] In some embodiments, provided herein are modified proteins or peptides, including synthetic / recombinant proteins or peptides modified with an electrophile of Formulas I-IV, where the electrophile is covalently attached to a cysteine ​​thiol group within the synthetic / recombinant protein or peptide. Cysteine ​​residues are attractive anchor residues due to their low proteome abundance and their high reactivity at physiological pH, allowing for rapid and selective modification of unprotected peptides and proteins.

[0025] In some embodiments, provided herein are modified proteins or peptides, including synthetic / recombinant proteins or peptides modified with an electrophile of Formulas I-IV, forming a thioether bond between the electrophile and the synthetic / recombinant protein or peptide.

[0026] In some embodiments, provided herein are modified proteins, including synthetic proteins modified with an electrophile of Formulas I-IV, forming a thioether bond between the electrophile and the synthetic protein.

[0027] In some embodiments, provided herein are modified proteins, including recombinant proteins modified with an electrophile of Formulas I-IV, which form a thioether bond between the electrophile and the recombinant protein.

[0028] In some embodiments, provided herein are modified peptides, including synthetic peptides modified with an electrophile of Formulas I-IV, forming a thioether bond between the electrophile and the synthetic peptide.

[0029] In some embodiments, the modified protein or modified peptide is capable of covalently binding a target protein, having non-covalent recognition with the target protein prior to covalent binding to the target protein.

[0030] In some embodiments, the modified protein or modified peptide is capable of covalently binding a target protein, and the electrophile is in proximity to a target residue of the target protein prior to covalent binding to the target protein.

[0031] In some embodiments, the modified protein or peptide can covalently bind to a target protein, and the modified protein or peptide has non-covalent recognition with the target protein; the electrophile is in proximity to a target residue in the target protein prior to covalent binding to the target protein. In other embodiments, the target residue in the target protein is a cysteine ​​(SH) residue or a lysine (NH) residue. In other embodiments, proximity between the electrophile and the target residue in the target protein refers to a distance of less than 15 Å. In other embodiments, the distance is less than 14 Å, less than 13 Å, less than 12 Å, less than 11 Å, less than 10 Å, less than 7 Å, or less than 5 Å. In other embodiments, the distance is 5-15 Å, 2-7 Å, 2-7 Å, or 5-10 Å.

[0032] In some embodiments, the modified proteins or modified peptides described herein covalently link a target protein to a thiol or amine in the target protein via the double bond (C=CH2) of an electrophile of Formulas I-IV. In other embodiments, the modified proteins or modified peptides described herein covalently link the thiol (SH) side chain of a cysteine ​​in the target protein. In other embodiments, the modified proteins or modified peptides described herein covalently link the amine side chain (NH2) of a lysine in the target protein. In other embodiments, the modified proteins or modified peptides covalently link the double bond (C=CH2) of an electrophile of Formulas I-IV to a lysine residue in the target protein via aza-Michael addition. In other embodiments, the modified proteins or modified peptides covalently link the double bond (C=CH2) of an electrophile of Formulas I-IV to a cysteine ​​residue in the target protein.

[0033] The electrophilic peptides or proteins (i.e., modified proteins or modified peptides) described herein provide a versatile approach to converting native peptide sequences or native proteins into covalent binders that can target a wide range of residues, can be easily introduced into peptides and proteins that are not protected via cysteine ​​side chains, and react efficiently and selectively with cysteine ​​and lysine side chains on target proteins.

[0034] In some embodiments, a modified peptide provided herein is Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), where pS is phosphoserine and mC is a methacrylate-modified cysteine. In some embodiments, a modified peptide provided herein is Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8), where pS is phosphoserine and mC is a methacrylate-modified cysteine. In some embodiments, a modified peptide provided herein is Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11), where pS is phosphoserine and mC is a methacrylate-modified cysteine.

[0035] In another embodiment, the modified peptide Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8), or Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11) covalently binds 14-3-3 as a target protein. In another embodiment, the modified peptide Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8), or Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11) covalently binds Cys38, Lys122, or Lys49 of the 14-3-3σ target protein, depending on the position of the electrophile.

[0036] The modified peptides Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8), and Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11) covalently bind 14-3-3 to the conserved lysine residue, show broad reactivity toward 14-3-3 proteins, effectively label 14-3-3 proteins in lysates, and secrete 14-3-3 extracellularly. The predicted irreversible binding to the target lysine was confirmed by proteomics and X-ray crystallography of the complex.

[0037] In some embodiments, the modified proteins provided herein are modified immunity protein MELKHSISDYTEAEFLQLVTTIANADTSSEEELVKLVTHF(mC)EMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQGLEHHHHHH (Im9, SEQ ID NO: 27), where mC is a methacrylate-modified cysteine. The alanine at position 23 was a cysteine ​​in the original sequence of Im. mC was a glutamate in the original sequence of Im. In other embodiments, the modified immunity protein (Im9, SEQ ID NO: 27) covalently binds the E9 target protein.

[0038] Irreversibly binding the immunity protein to E9 DNAse results in significantly increased thermal stability compared to the non-covalent complex.

[0039] The approach provided herein offers a simple and versatile means for converting peptides and proteins into strong covalent binders.

[0040] Provided herein is a novel approach to the development of covalent protein binders based on thioether-modified proteins or peptides. The modified proteins or peptides can react with both lysine and cysteine ​​side chains of target proteins via Michael addition. Electrophiles of Formulae I-IV are introduced by direct and selective modification of cysteine ​​side chains, enabling the synthesis of binders of unprotected peptides and even recombinant proteins (Figure 1).

[0041] Preparation of modified proteins or peptides In some embodiments, provided herein are methods for preparing a modified protein or peptide described herein, the methods comprising: a. Identifying the target protein; b. Designing candidate covalent binders of a peptide or protein based on previously characterized non-covalent binders of the target protein; c. synthesizing a modified peptide or protein that allows recognition of the target protein at the recognition site, wherein the modified protein or peptide contains a cysteine ​​that reacts with an electrophile of Formula I-IV; Includes.

[0042] In some embodiments, candidate peptide or protein covalent binders are designed by computational modeling, structure-based rational design, random screening of potential configurations for modification, or any combination thereof. In other embodiments, the designed candidate covalent binders have molecular recognition with the target protein. "Candidate peptide or protein covalent binders" are prepared and modified with electrophiles of Formulae I-IV to obtain the modified proteins or modified peptides described herein.

[0043] The design of candidate covalent binders for peptides or proteins is based on previously characterized non-covalent binders with the target protein. "Previously characterized non-covalent binders of the target protein" refers to peptide-protein or protein-protein interactions known in the art, such as PDB entries for known interactions. The term "non-covalent recognition" with the target protein refers to van der Waals interactions, π-π, or hydrogen bonds between the modified protein or modified peptide and the target protein.

[0044] In some embodiments, based on computer modeling, a synthetic protein is synthesized with only one cysteine ​​amino acid, which is further modified with an electrophile of Formulae I-IV. In some embodiments, based on modeling calculations, a synthetic peptide is synthesized with only one cysteine ​​amino acid.

[0045] In some embodiments, modified peptides are prepared according to Figure 2B.

[0046] Computational modeling has been widely used to model and design peptide and peptidomimetic binders for proteins. One example of a computational modeling system is CovPepDock, a 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).

[0047] Using CovPepDock, the modified peptides Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8) or Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11) showed extremely potent and selective detection of 14-3-3 in cell lysates, which has been a challenge for non-covalent binders due to the high sequence homology within the family.

[0048] In some embodiments, the modified protein or peptide is prepared by reacting a synthetic / recombinant protein or peptide with an electrophile, wherein the synthetic / recombinant protein or peptide has only one cysteine ​​amino acid and the electrophile is R 2 The electrophile reacts with the thiol of the cysteine ​​through a group (of the electrophile) to form a thioether bond, and the electrophile has the following structure: Formula IA, IIA, IIIA, or IVA: [ka] (In the formula, R1 includes substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle; R 2 contains Br, Cl or tosyl; R 3 includes substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle; and X is O, NR 3 or substituted or unsubstituted alkylene) It is expressed by:

[0049] In some embodiments, the electrophile is ethyl 2-(bromomethyl)acrylate.

[0050] In some embodiments, R of formula I, II, III, IV, IA, IIA, IIIA, or IVA 1 is a substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl, or heterocycle. 1 is substituted or unsubstituted alkyl. In other embodiments, R 1 is substituted or unsubstituted alkenyl. In other embodiments, R 1 is substituted or unsubstituted alkynyl. In other embodiments, R 1 is a substituted or unsubstituted carbocycle. In other embodiments, R 1 is substituted or unsubstituted aryl. In other embodiments, R 1 is substituted or unsubstituted phenyl. In other embodiments, R 1 is phenyl and the electrophile is a phenyl of formula II or IIA. 1 is a substituted or unsubstituted heteroaryl. In other embodiments, R 1 is a substituted or unsubstituted heterocycle.

[0051] In some embodiments, R of formula IA, IIA, IIIA, or IVA 2 is Br, Cl, or tosyl. 2 is Cl. In other embodiments, R 2 is Br. In another embodiment, R 2 is Tosyl.

[0052] In some embodiments, X of formula I or IA is O, NR 3 or substituted or unsubstituted alkylene. In other embodiments, X is O. In other embodiments, X is NR 3 In another embodiment, X is NH. In another embodiment, X is substituted or unsubstituted alkylene. In another embodiment, X is methylene (-CH-).

[0053] In some embodiments, R of formula I, IA, III, or IIIA 3 is a substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl, or heterocycle. 3 is substituted or unsubstituted alkyl. In other embodiments, R 3 is substituted or unsubstituted alkenyl. In other embodiments, R 3 is substituted or unsubstituted alkynyl. In other embodiments, R 3 is a substituted or unsubstituted carbocycle. In other embodiments, R 1 is substituted or unsubstituted aryl. In other embodiments, R 3 is a substituted or unsubstituted heteroaryl. In other embodiments, R 3 is a substituted or unsubstituted heterocycle.

[0054] As used herein, "alkyl" refers to straight or branched chain alkyl. In certain embodiments, straight or branched chain alkyl has 1 to about 20 carbon atoms, and in other embodiments, 1 to 12 carbons. In further embodiments, alkyl includes lower alkyl having 1 to 6 carbons. In further embodiments, alkyl includes lower alkyl having 1 to 3 carbons. One or more oxygen, sulfur, or -NR- and -N groups, including S(=O) and S(=O)2 groups, may be present along the alkyl group. + A substituted or unsubstituted nitrogen atom containing an RR- group may be optionally inserted, where the nitrogen substituent is alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or COR, where each R is independently selected from alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, -OY, or -NYY, and each Y is independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Alkyl groups include, but are not limited to, methyl, ethyl, propyl, methoxy, ethoxy, isopropyl, and 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 valence permits) groups independently selected from halo, hydroxy, amino, COOH, alkoxy, cyano, oxo, aryl, nitro, azido, aryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, carbonyl, amine, or amido.

[0055] As used herein, "alkylene" refers to a straight-chained, branched, or cyclic, and in certain embodiments, straight-chained or branched divalent aliphatic hydrocarbon group, in one embodiment having from 1 to about 20 carbon atoms, and in another embodiment having from 1 to 12 carbons. In a further embodiment, alkylene includes lower alkylenes having from 1 to 6 carbons. In a further embodiment, alkylene includes lower alkylenes having from 1 to 3 carbons. Along the alkylene group, one or more oxygen, sulfur, or -NR- and -N, including S(=O) and S(=O)2 groups, may be present. + A substituted or unsubstituted nitrogen atom containing an R—R— group may be optionally inserted, where the nitrogen substituent is alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or C—R, where each R is independently selected from alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, -O—Y, or -NY—Y, and each Y is independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Alkylene groups include, but are not limited to, methylene (—CH), ethylene (—CH—CH—), propylene (—(CH)), methylenedioxy (—O—CH—O—), and ethylenedioxy (—O—(CH)—O—).

[0056] As used herein, "alkenyl" refers to straight-chain or branched alkenyl. In one embodiment, a straight-chain or branched alkynyl has 2 to about 20 carbon atoms and at least one double bond, and in another embodiment, 1 to 12 carbons. In a further embodiment, an alkenyl group includes a lower alkenyl having 2 to 6 carbons. In a further embodiment, an alkenyl group includes a lower alkenyl having 3 to 4 carbons. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms may be optionally inserted along the alkenyl group, and the nitrogen substituent is alkyl. Alkenyl groups include, but are not limited to, -CH=CH-CH=CH2 and -H=CH-CH3. Alkenyl groups may be substituted or unsubstituted. In other embodiments, the substituted alkenyl can be substituted with one or more (e.g., 1, 2, 3, or more as valence allows) groups independently selected from halo, hydroxy, amino, COOH, alkoxy, cyano, oxo, aryl, nitro, azido, aryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, carbonyl, amine, or amido.

[0057] As used herein, "alkynyl" refers to straight-chain or branched alkynyl. In certain embodiments, straight-chain or branched alkynyl has, in one embodiment, 2 to about 20 carbon atoms and at least one triple bond, and 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 3 to 4 carbons. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms may be optionally inserted along the alkynyl group, and the nitrogen substituent is alkyl. Alkynyl groups include, but are not limited to, -C≡CC≡CH, -C≡CH, and -C≡C-CH3. Alkynyl groups 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 valence allows) groups independently selected from halo, hydroxy, amino, COOH, alkoxy, cyano, oxo, aryl, nitro, azido, aryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, carbonyl, amine, or amido.

[0058] A "carbocyclic" group refers to a saturated or unsaturated all-carbon monocyclic or fused ring (i.e., rings sharing adjacent pairs of carbon atoms) group in which one or more of the rings does not have a completely conjugated π-electron system. Examples of carbocyclic groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cycloheptane, and adamantane. Cycloalkyl groups can be substituted or unsubstituted. When substituted, the substituents may be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, amino, COOH, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, hydrazine, hydrazide, thiohydrazide, and amino. When the carbocyclic group is unsaturated, it may contain at least one carbon-carbon double bond and / or at least one carbon-carbon triple bond. Unsaturated carbocyclic rings include cyclohexene, cycloheptene, cyclohexadiene, and cycloheptatriene.

[0059] An "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of carbon atoms) terminal group having a completely conjugated π-electron system. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, and anthracenyl. Aryl groups can be substituted or unsubstituted. When substituted, the substituent can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, amino, COOH, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, hydrazide, thiohydrazide, and amino.

[0060] A "heteroaryl" group refers to the terminal group of a single ring or fused rings (i.e., rings that share adjacent pairs of atoms) having one or more atoms, such as nitrogen, oxygen, and sulfur, in the ring and further having a completely conjugated π-electron system. Examples of heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups can be substituted or unsubstituted. When substituted, the substituent can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, amino, COOH, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, 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.

[0061] A "heterocyclic" group refers to a monocyclic or fused ring group containing one or more atoms, such as nitrogen, oxygen, and sulfur, in the ring. The ring may also have one or more double bonds. However, the ring does not have a completely conjugated π-electron system. A heterocyclic ring may be substituted or unsubstituted. If substituted, the substituents may be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, amino, COOH, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azido, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, hydrazide, thiohydrazide, and amino. Representative examples can be piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine, and the like.

[0062] Use of covalent attachment of modified proteins or modified peptides to target proteins Covalent attachment of modified proteins or peptides to target proteins confers high efficacy and specificity, which may be useful as therapeutic agents and chemical biology tools.

[0063] In some embodiments, provided herein is 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 have non-covalent recognition prior to covalent binding to 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 Im9 and E9) exhibited higher thermal stability than the non-covalent complex.

[0064] In some embodiments, provided herein are peptide-protein covalent conjugates comprising a modified peptide provided herein covalently bound to a target protein, wherein the modified peptide and the target protein have non-covalent recognition prior to covalent bond formation with the target protein. In other embodiments, the modified peptide is Ac-RSApSmCPSL-NH2 (SEQ ID NO: 3), Ac-RAHpSmCPASLQ-NH2 (SEQ ID NO: 8), or Ac-RAHpSSPASmCQ-NH2 (SEQ ID NO: 11), where pS is phosphoserine and mC is methacrylate-modified cysteine, and the target protein is 14-3-3.

[0065] In some embodiments, the target protein is 14-3-3.

[0066] In some embodiments, the target protein is a 14-3-3 sigma represented by the following sequence: SYYHHHHHHDYDIPTTENLYFQGAMGSMERASLIQKAKLAEQAERYEDMAAFMKGAVEKGEELSCEERNLLSVAYKNVVGGQRAAWRVLSSIEQKSNEEGSEEKGPEVREYREKVETELQGVCDTVLGLLDSHLIKEAGDAESRVFYLKMKGDYYRYLAEVATGDDKKRIIDSARSAYQEAMDISKKEMPPTNPIRLGLALNFSVFHYEIANSPEEAISLAKTTFDEAMADLHTLSEDSYKDSTLIMQLLRDNLTLWTADNAGEEGGEAPQEPQS (SEQ ID 28).

[0067] Peptides derivatized with covalent warhead groups can greatly expand the repertoire of addressable targets. Provided herein are electrophilic reagents of formula IA, IIA, IIIA, or IVA that can be conjugated to peptides or proteins to yield modified peptides / proteins (electrophile peptides / proteins). Such electrophilic peptides / proteins can be repurposed to covalently and selectively bind target proteins and further enable modifications such as fluorescent labeling, drug conjugation, or conjugation to other proteins. Provided herein is an approach that enables the modification of native peptides or proteins with electrophiles of formula IA, IIA, IIIA, or IVA. This approach is distinguished by two features. First, it is a facile synthesis that does not require non-standard amino acids and introduces the electrophile to a native cysteine ​​residue in a single step (Figures 2A-2C). Second, non-standard amino acids are not required, and "universal" SPPS (solid phase peptide synthesis) can be used; peptides are synthesized with a "standard" cysteine ​​at the electrophilic site, and thus this approach can be used to synthesize recombinant proteins. Furthermore, the modified proteins and peptides of the present invention react with lysines of target proteins in addition to cysteines.

[0068] The advantage of this property is that such chemistry can be incorporated into covalent phage display platforms for the efficient discovery of electrophilic peptides. Second, very few peptides have previously been designed to irreversibly target lysine residues, and those that did utilized arylsulfonyl fluorides. Provided herein are the first electrophilic peptides to use methacrylates to irreversibly label lysine target residues, as verified by crystallography (Figures 4A-B) and mass spectrometry (Figures 8A-D).

[0069] The efficacy and specificity of peptides make them versatile chemical probes. While the majority of known functions of 14-3-3 proteins are intracellular, they also perform several extracellular functions, and the presence of extracellular 14-3-3 proteins could serve as biomarkers for various diseases. Therefore, the ability to bind and detect these proteins in the extracellular medium is of great interest. The use of the BDP-modified methacrylate peptide 14-3-3β resulted in detection in the extracellular medium with sensitivity comparable to or even greater than Western blot (Figures 5A-B), demonstrating the potential of these probes. Furthermore, when targeting extracellular proteins, issues such as membrane permeability and proteolytic stability have less of an impact on probe activity, providing opportunities for peptide- and protein-based covalent probes.

[0070] Because this 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 cysteine ​​and lysine residues. Furthermore, the R of electrophiles in Formulae I-IV groups can be easily modified. 1 The groups can serve in a variety of ways as diversification and screening points, either for the development of stronger binders or for the functionalization of probes.

[0071] Genetic code expansion previously enabled the introduction of fluorosulfates into proteins to target histidine residues. The approach provided herein offers several advantages. First, genetic code expansion requires specialized bacterial expression systems and conditions that are not yet widely available, somewhat limiting its broad applicability. Second, for future industrial applications, such modified genetic systems may limit production scale, whereas standard recombinant proteins with chemical modifications (e.g., antibody-drug conjugates) have already proven applicable. Third, genetic code expansion requires extensive research to enable the introduction of new types of amino acids. Therefore, optimizing the characteristics of electrophiles is limited. In the system described herein, many electrophiles of formulas IA-IVA can be synthesized and conjugated to the same recombinant protein, allowing for significantly higher optimization throughput. Finally, the ethyl ester group can serve not only as a diversification and screening point for the development of better binders, but also for various functionalization of probes, e.g., via attachment of E3 ligase binders for targeted degradation, for attachment of fluorescent dyes for imaging or detection purposes, targeting of proteins to specific cell types or locations within a cell, etc.

[0072] Another aspect that contributes to the practicality of the approach provided herein is computational modeling assistance. While manual inspection and selection of positions for electrophile introduction may work well for some peptides, automated modeling and selection can cover many more possibilities. Furthermore, as the number of available electrophiles expands with various side chains, modeling must address combinatorial logic (Figures 12A-12B). Finally, for protein covalent binding reagents (compared to peptides), manual inspection can be more challenging (Figures 13A-13B).

[0073] Provided herein is a simple and versatile method for the preparation of a novel class of covalent protein and peptide probes or covalent proteins and protein probes suitable for binding a wide variety of biological targets, and thus will support novel applications in chemical biology and covalent drug discovery.

[0074] In some embodiments, provided herein are modified proteins or peptides for use in selective labeling, fluorescent labeling, inhibition, drug conjugation or conjugation to target proteins.

[0075] In some embodiments, provided herein are methods for diagnosing disease by a known biomarker, the method comprising covalently linking a modified peptide or modified protein described herein to a known biomarker, thereby identifying the biomarker by fluorescence or immunoassay.

[0076] The efficacy and specificity of modified peptides or proteins allow their use as versatile chemical probes. For example, while the known functions of most 14-3-3 proteins are intracellular, they also perform several extracellular functions, and the presence of extracellular 14-3-3 proteins may serve as biomarkers for various diseases. Using BDP (fluorescent dye Bodipy)-modified methacrylate peptides, 14-3-3 was detected in the extracellular medium with greater sensitivity than Western blot, demonstrating the power of this method (see Example 3). Furthermore, when targeting extracellular proteins, issues such as membrane permeability and proteolytic stability have less of an impact on the activity of the probe, providing opportunities 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, but they should not be construed as limiting the broad scope of the invention.

[0078] Example Example 1 Design and synthesis of modified peptide covalent binders to 14-3-3 proteins The goal was to design binders that target the conserved lysines in the phosphopeptide binding groove of 14-3-3σ: lysines 49 and 122 (FIG. 2A). Specifically, Lys122 has previously been shown to react with aldehydes to form reversible covalent imine bonds with higher selectivity than other lysine residues, due to the 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, PJ 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, PJ 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] Residues in the YAP1 peptide within a Cα-Cα distance of <14 Å from the target lysine were identified, and each of these residues was mutated to a methacrylate-modified side chain; this included residues 126–131 to target Lys49 and residues 126–133 to target Lys122. Four peptides were selected that were predicted to bind either Lys49 or Lys122 with high scores and low root mean squared errors (RMSDs) relative to the binding mode of the original peptide (i.e., predicted to bind well and maintain the binding pose of the original peptide). To further expand the scope of the series, a second set of peptides was designed based on other known peptide binders of 14-3-3σ. Four such structures were selected using peptides from Raf1 (rapidly progressive fibrosarcoma 1) (PDB: 3IQU and 4IEA), TASK-3 (TWIK-related acid-sensitive K+ channel 3) (PDB: 3P1N), and SNAI1 (PDB: 4QLI). For these peptides, we focused on Lys122, which was more reactive to electrophiles.

[0081] CovPepDock was used to model Lys122-targeting peptides based on each of these structures, and seven additional peptides with high scores and low RMSD were selected from this set for synthesis and testing. The peptide structures are detailed in Table 1.

[0082] [Table 1]

[0083] We used the covalent peptide docking pipeline CovPepDock to design candidate peptides based on known binding partners of 14-3-3σ (Table 1). This yielded three irreversible binding peptides from 11 candidates. The hit peptide sequence ID11 reacted with Cys38 instead of the predicted Lys122. The electrophile in sequence ID11 is significantly closer to Cys38 in the binding pocket compared to the lysine-targeting sequence ID8 (Figure 2A). The reaction of sequence ID11 with cysteine ​​appeared to occur via two distinct mechanisms—addition and substitution—which was also observed when the peptide was incubated with cysteine. Furthermore, cysteine ​​added to the methacrylate via Michael addition, whereas in substitution, the cysteine ​​displaced the peptide, which was released as a free thiol, while the methacrylate remained on the protein. The methacrylate-labeled protein could later react with the free thiol peptide via addition, ultimately converting all proteins to stable addition products (Figure 3B). Reaction of methacrylate peptides with cysteine ​​and thiols such as DTT also releases free peptides (Figure 8A). These results may be due to the high nucleophilicity of cysteine, which also reacts very rapidly with chloroacetamide-based peptides. Nevertheless, methacrylate-based peptides are not indiscriminate and show high specificity for target proteins and residues, as demonstrated by single labeling (Figures 5A-5B and 6), even when an excess of peptide is used (100-fold excess; Figure 3A). No peptides reacted with Lys49, which may be due to the low predicted nucleophilicity of this residue.

[0084] To prepare the peptide methacrylate adduct, the peptide was synthesized using standard solid-phase peptide synthesis (SPPS) procedures and acetylated at the N-terminus (Figure 2B). After cleavage from the resin, the crude peptide was reacted with three equivalents of 2-(bromomethyl)acrylate in an amine- or thiol-free buffer to avoid potential side reactions, resulting in efficient conversion of the peptide to the methacrylate (i.e., the modified peptide) within 1–2 h at room temperature (Figure 2C).

[0085] Peptides were incubated at 200 μM with 2 μM 14-3-3σ overnight at 4 °C, and binding was monitored using intact protein LC / MS (Figure 3A). Peptide sequence ID12, which contains a chloroacetamide warhead group 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 peptide sequences ID3 (51%), ID8 (35%), and ID11 (11%), all of which were predicted to bind Lys122. Peptide sequence ID1 also showed low levels of labeling (approximately 10%) at the expected adduct mass, as well as an unidentified smaller adduct (less than 139 Da). At this point, it was unclear whether the peptides that did not label 14-3-3σ failed to label due to reduced noncovalent binding affinity or suboptimal electrophile placement within the noncovalent complex. Therefore, taking advantage of the fact that covalent bond formation is slow and occurs over a timescale of several hours, we performed fluorescence polarization binding experiments using a BDP-labeled peptide derived from YAP-1, which binds 14-3-3σ with approximately 100 nM affinity. 14-3-3σ was added at a concentration of 0.25 μM to a premix of fluorescent (5 nM) and electrophilic (5 μM or 200 μM) peptides, and fluorescence polarization was measured at 27 °C immediately after mixing (Figure 9). Only peptide sequences ID10 and ID11 had sufficient affinity to displace the fluorescent binder at 5 μM, while all peptides except peptide sequence ID2 displaced the binder at 200 μM, the concentration at which the initial screening was performed. Thus, although many electrophilic peptides exhibit reduced noncovalent binding affinity for 14-3-3σ, the positioning of the electrophile in the noncovalent complex also appears to play an important role in covalent bond formation. These peptides were further analyzed using time course labeling experiments at lower peptide concentrations (5 μM) at 25° C. Peptide sequences ID3 and ID8 achieved 60% and 80% labeling within 5 h, respectively. Incubation at 37° C. increased the rate of labeling by approximately 4-fold (FIG. 10).Interestingly, when incubated with sequence ID11, unlabeled 14-3-3σ rapidly disappeared within 2.5 h, leaving less than 5% free 14-3-3σ. However, the reaction initially yielded a mixture of fully peptide-labeled protein (+1275 Da) and protein modified only with methacrylate groups (+112 Da). The methacrylate-labeled protein was gradually converted to fully peptide-labeled protein (Figure 3B).

[0086] The intrinsic reactivity and stability of the active peptides were compared with their labeling rates. Their stability was tested in buffer and in the presence of lysine and cysteine ​​at room temperature. The peptides showed good stability in buffer—over a 6-hour timescale in buffer, all peptides remained >90% intact. Over a timescale of several days, peptides ID8 and ID11 formed new products with the same mass as the original peptide, likely due to internal reactions within the peptide. Peptide ID3 remained unmodified even after 3 days. Incubation with lysine did not yield any products beyond those observed after long incubations in buffer, suggesting that the peptides had low intrinsic reactivity toward lysine. Reactivity toward cysteine ​​was much higher—the peptides reacted with cysteine ​​to generate both Michael adducts and substitution products, resulting in the release of thiol peptides. Peptides ID3, ID8, and ID11 reacted >50% within 2.5 hours.

[0087] method: Preparation of recombinant 14-3-3σ The pPROEXHTb expression vector encoding human 14-3-3σ with an N-terminal His6 tag was transformed into NiCo21(DE3) competent cells by heat shock. A single colony was cultured in 50 mL of LB medium (100 mg / mL ampicillin). After overnight incubation at 37°C, the culture was transferred to 2 L of TB medium (100 mg / mL ampicillin, 1 mM MgCl2) and incubated at 37°C until the OD600 nm reached 0.8-1.2. Protein expression was then induced with 0.4 mM isopropyl-β-d-thiogalactoside (IPTG), and the culture was incubated overnight at 18°C. Cells were harvested by centrifugation (8600 rpm, 20 min, 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 tablet (1 tablet / 100 mL of lysate) and benzonase (1 μL / 100 mL). After lysis using a C3 Emulsiflex-C3 homogenizer (Avestin), the cell lysate was clarified by centrifugation (20 000 rpm, 30 min, 4°C) and purified by NiCl. +2 Purification was performed using affinity chromatography (Ni-NTA superflow cartridges, Qiagen). Typically, two 5 mL columns (flow rate 5 mL / min) were used for a 2 L culture. The lysate was loaded onto the column, washed with 10 CV of 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 βME). Fractions containing 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 approximately 60 mg / ml, analyzed for purity by SDS-PAGE and Q-Tof LC / MS, and aliquots were flash-frozen for storage at -80°C.

[0088] Peptide synthesis 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 swollen in dichloromethane (DCM) for 30 min and then washed with dimethylformamide (DMF). Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 × 3 min), and coupling was carried out as follows: 4 equivalents of amino acid were mixed with 4 equivalents of HATU (azabenzotriazole tetramethyluronium hexafluorophosphate) / HOAT (1-hydroxy-7-azabenzotriazole) and 8 equivalents of DIPEA (diisopropylethylamine) in DMF, added to the resin, and mixed for 30 min. For phosphoserine and propargylglycine, 2 equivalents were used, and the reaction time was extended to 2 h. After final Fmoc deprotection, the peptide was acetylated at the N-terminus using acetic anhydride (10 equiv.) and DIPEA (20 equiv.) in DMF for 30 min. Finally, the resin was washed with DCM, dried in a desiccator, and cleaved using 94% TFA / 1% DODT (3,6-dioxa-1,8-octanedithiol) / 2.5% TIPS (triisopropylsilane) / 2.5% water with inversion for 2 h. The cleaved peptide was precipitated in cold diethyl ether:hexane, washed once with ether, dried, dissolved in 50% acetonitrile, and lyophilized.

[0089] Electrophiles were directly introduced into the crude peptide as follows: the crude peptide was 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. The reaction was monitored using LCMS and was typically complete within 1-2 hours at room temperature. The reacted peptide was then purified using reverse-phase HPLC.

[0090] To prepare fluorescently labeled peptides, a propargylglycine residue was attached to the peptide at the N-terminus prior to N-terminal acetylation, cleavage, and reaction with ethyl 2-(bromomethyl)acrylate. The pure peptides were then labeled using copper-catalyzed azide-alkyne cycloaddition (CuAAC) as follows: 5 μL of 20 mM peptide was mixed with 15 μL of BDP-TMR azide (150 nmol). Water was added to 100 μL, and approximately 50 μL of tBuOH was added to dissolve the dye. At this point, 1 μL of 100 mM CuSO4:THPTA and 200 mM sodium ascorbate (200 nmol, freshly dissolved) were added, and the reaction was continued for 1 hour. The products were purified using HPLC. The purity of all peptides was confirmed using LCMS.

[0091] LC / MS equipment and experiments LC / MS experiments for 14-3-3σ were performed on a Waters ACQUITY UPLC class H instrument in positive ion mode with electrospray ionization. A C4-BEH column (300 Å, 1.7 μm, 21 mm × 100 mm) was used for the UPLC separation. The column was maintained at 40 °C, and the autosampler was maintained at 10 °C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The experimental flow rate was 0.4 mL / min. The gradient used was 1% B in 2 min, linearly increased to 80% B in 2.5 min, held at 80% B for 0.5 min, changed to 20% B in 0.2 min, and held at 1% B for 0.8 min. MS data were collected on a Waters SQD2 detector with an m / z range of 2–3071.98 in the range of 900–1900 m / z. The desolvation temperature was 500 °C, and the flow rate was 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 28,000:34,000 Da and a resolution of 1 Da / channel.

[0092] LS / MS experiments for peptides were performed using the same instrument equipped with a C18-CSH column (300 Å, 1.7 μm, 21 mm × 100 mm) using a gradient starting at 1% B for 1 min, ramping to 95% B in 4.5 min, holding at 95% B for 0.75 min, then ramping to 1% B in 0.75 min, and holding at 1% B for 1 min. MS data were collected in the m / z range of 80–2500 using the same conditions for ionization as for proteins.

[0093] Example 2 The modified peptides can target both cysteines and lysines, controlling 14-3-3 isoform selectivity. To clarify the binding site of the peptides on 14-3-3σ, we performed trypsin digestion followed by LC / MS / MS. (The peptides and 14-3-3σ were prepared as described in Example 1.) Direct identification and quantification of modified peptides proved difficult due to the long peptide chain length, fragmentation from multiple directions, and weak relative signals. To characterize the peptide ligation site, we switched strategies and measured the relative change in the signal of unmodified peptides on 14-3-3σ compared to DMSO-treated controls. Specifically, peptides containing or immediately following residues Cys38, Lys49, and Lys122 were examined (Figure 8D). Peptide sequence ID11 specifically reduced the signal of Cys38-containing peptides and had little effect on the signal of other peptides, suggesting specific Cys38 binding. In contrast, peptides containing or followed by Lys122 were significantly, but not uniformly, depleted by peptide sequences ID3 and ID8, while the signal of the Lys49-containing peptide increased slightly. This result pointed to Lys122 as a likely binding site for peptide sequences ID3 and ID8. The uneven decrease in the signal of the Lys122-containing peptide may be due to incomplete 14-3-3σ labeling by sequences ID3 and ID8. The possibility that lysine adducts are unstable in the presence of reducing agents used prior to trypsin digestion was considered. To test this, peptides were incubated with excess TCEP (triscarboxyethylphosphine) or DTT (dithiothreitol), which rapidly released the peptide from the methacrylate within 2 hours (Figure 8A). However, under the same conditions, peptide-protein adducts were stable (Figure 8B). Proteins were incubated with fluorescently labeled methacrylate peptides, subsequently denatured in DTT-containing sample buffer, and subjected to SDS-PAGE. Again, reduction did not affect the intensity of the fluorescent band, suggesting that the protein-peptide adduct was stable after aza-Michael addition (Figure 8C).

[0094] To further confirm that lysine 122 is the target residue for peptides ID3 and ID8, 14-3-3σ was cocrystallized with both peptides. The crystal structures (Figures 4A and 4B) clearly showed a covalent bond formed between the amine of Lys122 and the methacrylate, while Lys49 remained unmodified. Comparison of the crystal structure with predictions from CovPepDock suggested that the model accurately predicted the binding pose around the phosphate and in the N-terminal portion of the peptide, but less so for the C-terminal region. More specifically, compared to the predictions, Lys122 adopted a looser conformation, the C-terminal residue was less tightly packed in the binding groove, and in peptide SEQ ID NO: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 NO:8, the C-terminal portion of the peptide shifted outward due to space occupied by the methacrylate ester moiety. The structure of the peptide sequence ID3 complex was much less affected by covalent binding due to the shorter C-terminal portion of the peptide. In contrast, both peptides showed only a minor effect of covalent binding on the structure of the N-terminal region. These results suggest that noncovalent interactions with the C-terminal portion of the peptide do not play a significant role in binding.

[0095] Because Lys122 is highly conserved in all 14-3-3 isoforms (as opposed to cysteine ​​38, which is unique to 14-3-3 sigma), peptides ID3 and ID8 were incubated with the other isoforms, along with the Cys38-targeting peptide ID12. Peptide ID12 labeled only the sigma isoform, whereas peptides ID3 and ID8 labeled all isoforms with similar efficiency. Taken together, these results conclusively verify that peptides ID3 and ID8 specifically bind to Lys122 via aza-Michael addition.

[0096] method: Binding experiment to 14-3-3σ 100X stock solutions of peptides were prepared by dissolving in DMSO + 5 mM acetic acid and stored at -80°C. Peptide conjugation to 14-3-3σ was performed in 25 mM HEPES, pH = 7.5, 100 mM NaCl, 10 mM MgCl2. Protein was diluted to 2 μM in assay buffer, and the diluted protein was added to the peptide stock solution at a 100:1 ratio and incubated under various conditions. For analysis, 24 μl of sample was mixed with 6 μl of 2.4% formic acid in water, and then 10 μl was injected into the intact protein LCMS.

[0097] Fluorescence polarization experiments: Fluorescence polarization experiments were performed in triplicate in 50 μl volumes in a 384-well plate using a Tecan plate reader in the dark. The buffer was 25 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.05% IGEPAL. To each sample, 0.5 μl of a 100X stock solution of competitor peptide (in DMSO + 5 mM acetic acid) was added, followed by 25 μl of 10 nM BDP-labeled noncovalent peptide probe. Finally, 25 μl of 0.5 μM 14-3-3σ was added to the plate, and the plate was mixed. Polarization was measured at 27°C.

[0098] LC / MS / MS characterization of labeling sites on methacrylate peptides in 14-3-3σ 14-3-3σ was diluted to 2 μM in 25 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and incubated with 5 μM peptide in a 50 μl sample. The sample was incubated at room temperature for 48 hours, resulting in approximately 75% labeling with peptide sequence ID3, 90% labeling with peptide SEQ ID NO:8, and 100% labeling with peptide sequence ID11. At this point, 50 μl of 10% SDS in 25 mM HEPES 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 at room temperature for 40 minutes in the dark. The sample was then processed using an S-trap (Protify) according to the manufacturer's instructions and subsequently desalted using an Oasis plate (Waters).

[0099] Each sample was dissolved in 50 μl of 3% acetonitrile + 0.1% formic acid, and 0.5 μl was injected onto the column. Samples were analyzed using an EASY-nLC1200 nanoflow UPLC system using a PepMap RSLC C18 column (2 μm particle size, 100 Å pore size, 75 μm diameter × 50 cm length) attached to an Exploris240 mass spectrometer using an EASY-Spray source. uLC / MS-grade solvents were used for all chromatographic steps at 300 nL / min. The mobile phases were (A) HO + 0.1% formic acid and (B) 80% acetonitrile + 0.1% formic acid. Peptides were eluted from the column into the mass spectrometer using the following gradient: 1% to 40% B in 60 min, 40% to 100% B in 5 min, held at 100% B for 20 min, 100% to 1% B in 10 min, and finally 1% B for 5 min. Ionization was achieved using a spray voltage of 1900 V at an ion transfer tube temperature of 275 °C. Data were initially acquired in data-dependent acquisition (DDA) mode. MS1 resolution was set to 120,000 (at 200 m / z), the mass range was 375–1650 m / z, the normalized AGC was set to 300%, and the maximum injection time was set to 20 ms. MS2 resolution was set to 15,000, quadrupole separation was set to 1.4 m / z, the normalized AGC was set to 100%, the maximum injection time was set to 22 ms, and the HCD collision energy was set to 30%. Three injections of 0.5 μl were performed for each sample. DDA data were analyzed using MaxQuant 1.6.3.4. The database contained the sequences of the 14-3-3σ constructs used in the study and included contaminants.

[0100] Methionine oxidation and N-terminal acetylation were variable modifications, while carbamidomethyl was a fixed modification in the analysis, with a maximum of four modifications per peptide. Digestion was defined as trypsin / P with a maximum of two missed cleavages. The PSM (peptide spectrum match) FDR (false discovery rate) was defined as 1, and the protein FDR / site decoy ratio was defined as 0.01. A second peptide was enabled, allowing for inter-experiment matches with a match time window of 0.7 min. Data were imported into Skyline, and precursors from nine peptides containing or following residues Cys38, Lys49, and Lys122 were selected for parallel reaction monitoring (PRM). For each acquisition cycle, one complete MS spectrum was acquired in the 350–1000 Da range, with a 300% AGC (automatic gain control) target, a maximum injection time of 20 ms, and a resolution of 120,000. Data for each precursor were collected during a 4-5 minute window centered around the retention time measured in the DDA experiment, with a Q1 resolution of 2 Da, an Orbitrap resolution of 15,000, an AGC target of 300%, and a maximum injection time of 160 ms. The acquired data were then analyzed in Skyline using the spectral library generated from the DDA experiment. The three most intense product ions were used for quantification relative to the DMSO control. The data were deposited to the ProteomeXchange Consortium via the PRIDE Partner Repository under dataset identifiers PXD044257 and 10.6019 / PXD044257.

[0101] Crystallization of 14-3-3σ-peptide complex: 14-3-3s was C-terminally truncated (DC = delta C) after T231 (threonine 231 in the protein) to facilitate crystallization. 14-3-3 and the sequence ID3 / sequence ID8 peptides were dissolved in complexation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl) and mixed at a molar stoichiometry of 1:2.5 or 1:5 (protein:peptide) at final protein concentrations of 10 mg / mL, 11 mg / mL, 12 mg / mL, and 12.5 mg / mL. The complex was incubated overnight at 4°C in a custom crystallization solution (0.095 M HEPES (pH 7.1, 7.3, 7.5, 7.7), 0.19 M CaCl, 24-29% (v / v) PEG400, and 5% (v / v) glycerol) and then set up for sitting drop crystallization. Crystals grew within 5-10 days at 4°C.

[0102] The crystals were removed and flash cooled in liquid nitrogen. X-ray diffraction (xRD) data were collected at the Deutsche Elektronen Synchrotron (DESY) PETRA III beamline P11, Hamburg, Germany.

[0103] Initial processing of the dataset was performed using CCP4i from the CCP4 suite. First, XIA2 / DIALS was run for data indexing and integration, and AIMLESS was run for scaling. The structure was layered by molecular replacement using Protein Data Bank (PDB) entry 5N75 as a template in MOLREP. REFMAC5 was used for initial structure refinement. The correct peptide sequence was modeled in the electron density of Coot. The presence of covalent interactions between the peptide and lysine 122 was confirmed by visual inspection of Fo-Fc and 2Fo-Fc electron density maps in Coot and constructed 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.

[0104] [Table 2]

[0105] Example 3 The modified peptide detects 14-3-3 proteins in lysates and extracellular media with high sensitivity. In Example 2, we demonstrated that methacrylate peptides can react with all 14-3-3 isoforms. We prepared BODIPY-labeled derivatives of peptides ID3, ID8, and ID12 to test their ability to function as pan-reactive 14-3-3 probes in cell lysates (Figures 5A-5B). Methacrylate sequences ID3 and ID8 formed two major bands, the lower of which corresponded to the shifted 14-3-3β band observed in Western blots. The lower band likely corresponds to six 14-3-3 isoforms with very similar sizes (α / β, ζ / δ, γ, σ, η, and θ, all 245–248 AA), while the upper band likely corresponds to the larger (255 AA) ε isoform. Binding of the peptides to 14-3-3 was highly selective; virtually no other proteins in the lysates were significantly labeled. Furthermore, the methacrylate peptide bands were enhanced after prolonged incubation (22 h vs. 1 h), suggesting their stability under these conditions. We then proceeded to test whether the peptides could detect 14-3-3 proteins in the extracellular medium. A549 cells were grown in serum-free medium for 24 or 48 h, and the medium was then filtered, concentrated, and buffer exchanged. The peptides detected 14-3-3 in the medium with very high selectivity and sensitivity. In contrast to the results observed in the lysate, 14-3-3σ was not detected in the medium by peptide sequence ID12. Therefore, the methacrylate peptide sequences ID3 and ID8 are powerful tools for the detection and quantification of 14-3-3 isoforms in lysates and extracellular media.

[0106] method: Measurement of binding to 14-3-3 isoforms using LC-MS qTOF The 14-3-3 isoforms were buffer-exchanged into complexation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl) and mixed with peptide (3 / 8) at a final concentration of 10 mg / mL at a 1:5 molar stoichiometry (protein:peptide). After overnight incubation at 4°C, the complexes were buffer-exchanged into MilliQ + 0.1% formic acid.

[0107] UPLC-QToF-MS analysis was performed on a Waters (Milford, MA, USA) Acquity I-Class UPLC system connected to a Waters Xevo G2 quadrupole time-of-flight (QToF) mass spectrometer. The instrument was controlled by MassLynx software (version 4.1, Waters, MA, USA). Full scan in positive electrospray ionization (ESI+) mode was used as the MS acquisition mode with an acquisition range of 200–2000 m / z. A 3 μm, 150 × 2.0 mm Polaris3 C8-A column (Agilent, Middelburg, The Netherlands) was used for chromatographic separation, placed in a column oven at 60 °C. The flow rate was set at 0.3 mL / min, and a gradient of 0.1% (v / v) formic acid in water (A) and 0.1% (v / v) formic acid in acetonitrile (B) was set as follows (all % v / v): 0.0–7.5 min (15% to 75% B), 7.5–8.0 min (75% B), 8.0–8.1 min (75% to 15% B), and 8.1–10.0 min (15% B). Mass spectrometry settings were as follows: capillary voltage: 0.80 kV, cone voltage: 40 V, source offset: 80 V, source temperature: 100 °C, desolvation temperature: 400 °C, cone gas: 10 L / h, desolvation gas: 800 L / h. The sample concentrations were 0.01–0.1 mg / mL, and the injection volume was 1 µL. Deconvolution was performed with the MaxEnt1 option of MassLynx software. Errors were calculated using the MaxEnt error option.

[0108] Binding of 14-3-3 proteins to peptides in the extracellular medium and lysates For experiments in lysates, A549 cells were grown in DMEM+FBS.

[0109] Cells were washed with PBS, scraped off the plates, and centrifuged at 200 g for 5 minutes. Cells were lysed in 20 mM HEPES, pH 7.5, 10 mM MgCl2, 100 mM NaCl supplemented with a protease inhibitor cocktail (Roche 11836170001). Cells were sonicated using a microprobe with 10 pulses of 2 seconds at 22% amplitude, followed by centrifugation at 21000 g for 10 minutes at 4°C. Protein concentration was estimated using BCA, and the lysate was diluted to 1.97 in lysis buffer.

[0110] For peptide incubations, 38 μl of either lysate or medium was mixed with 2 μl of a 20X stock solution of peptide (peptide SEQ ID NO:8 and peptide sequence ID3: 20 μM in 20% DMSO / lysis buffer; peptide sequence ID12: 5 μM in 20% DMSO / lysis buffer; no peptide: 20% DMSO / lysis buffer) and incubated at 25°C in the dark. Then, 13.3 μl of 4X LDS (lithium dodecyl sulfate) sample buffer with 20 mM DTT was added, and the samples were heated at 70°C for 10 min. The samples were loaded onto a Bis-Tris gradient gel (4-20%, GenScript) and run at 55 mA / 200 V using Tris-MOPS buffer. The gel was transferred to a nitrocellulose membrane, and the membrane was blocked with 5% BSA / TBST (bovine serum albumin / Tris-buffered saline + Tween 20) for 1 h at RT. The membrane was incubated overnight at 4°C with anti-14-3-3β antibody (Abcam ab15260) diluted 1:500 in 5% BSA / TBST. The membrane was washed twice with TBST and incubated with anti-rabbit horseradish peroxidase (HRP) antibody (CST7074S) diluted 1:2000 in 5% BSA / TBST for 1 hour at RT. The membrane was washed three times with TBST and imaged as follows: fluorescence using 546 nm excitation was measured using a ChemiDoc (Bio-Rad) with a 9-second exposure, and chemiluminescence was measured with a 20-second exposure. Images were processed and generated in ImageLab.

[0111] For experiments in culture, cells were grown and then transferred to FBS-free DMEM followed by incubation for 24 or 48 hours. After incubation, 8.5 ml of culture medium was filtered through a 0.2 μm filter, concentrated to approximately 200 μl using a centrifugal concentrator (Vivaspin, cutoff 8000-10000 Da), and diluted to 8 ml using 50 mM HEPES, pH 7.5, 150 mM NaCl. This was followed by two additional dilutions and concentration to approximately 200 μl. Samples were then diluted to 300 μl with 1% IGEPAL and protease inhibitors and PhosStop. Samples from 38 μl of culture medium were then incubated with peptides and analyzed as performed for lysates, with a 15-second exposure for fluorescence and a 50-second exposure for chemiluminescence.

[0112] In experiments where the gel was directly imaged, after electrophoresis, the gel was immersed in fixative (45% methanol, 45% water, and 10% acetic acid) for 10 minutes and washed twice with 100 mM Tris in water, pH = 8. The gel was then directly imaged on the ChemiDoc.

[0113] Pull-down proteomics experiments N-terminally biotinylated derivatives of peptide sequences ID3 and ID8 were synthesized and purified. A549 cells were harvested and lysed as described above. The lysate was diluted to 1.9 mg / ml in lysis buffer, and the peptides were diluted to 20 μM in 20% DMSO / lysis buffer. 142.5 μl of lysate was mixed with 7.5 μl of a 20 μM peptide stock solution, and the sample was incubated at 25°C for 22 hours. Proteins were precipitated by adding 450 μl of water, 600 μl of HPLC-grade methanol, and 150 μl of HPLC-grade chloroform, followed by vortexing and centrifugation at 21,000 × g for 10 minutes at 4°C. The upper layer was aspirated, 600 μl of methanol was added, and the sample was vortexed and centrifuged again, followed by aspirating the supernatant. The pellet was air-dried and stored at -80°C. The pellet was dissolved in 200 μl of 2.5% SDS in PBS by heating to 60° C. and shaking at 1150 rpm for 30 min. After lysis, the sample was diluted 20-fold with PBS and incubated with 10 μl of streptavidin agarose beads (Thermo) for 3 h at room temperature with end-over-end rotation.

[0114] The beads were then filtered through a spin column in a vacuum manifold. The beads were washed twice with 300 μl of 1% SDS / PBS, dispersed in 300 μl of 1% SDS / PBS, and 3 μl of 1 M DTT was added and incubated at room temperature for 30 minutes. 15 μl of freshly dissolved 0.8 M iodoacetamide was then added, followed by incubation at room temperature for 30 minutes in the dark. The solution was then removed, and the beads were washed with 350 μl of freshly dissolved 6 M urea in PBS for 3 hours, 400 μl of 20% methanol in PBS three times, PBS once, and water twice. The beads were then transferred to a test tube using 100 μl of 50 mM triethylammonium bicarbonate, and the bound protein was digested with 0.5 μg of trypsin (Promega) at 37°C with shaking at 1200 rpm for 6 hours.

[0115] The beads were centrifuged, and the supernatant was mixed 1:1 with 0.2% TFA in water. The peptides were desalted using an Oasis desalting column (Waters) and dried under vacuum. The dried peptides were dissolved in 3% acetonitrile + 0.1% formic acid (25 μl), and 2 μl was injected. Samples were analyzed using an EASY-nLC1200 nanoflow UPLC system using a PepMap RSLC C18 column (2 μm particle size, 100 Å pore size, 75 μm diameter × 50 cm length) attached to an Exploris240 mass spectrometer using an EASY-Spray source. uLC / MS-grade solvents were used for all chromatographic steps at 300 nL / min. The mobile phases were (A) HO + 0.1% formic acid and (B) 80% acetonitrile + 0.1% formic acid. Peptides were eluted from the column into the mass spectrometer using the following gradient: 1% to 40% B in 160 min, 40% to 100% B in 5 min, held at 100% B for 20 min, 100% to 1% B in 10 min, and finally 1% B for 5 min. Ionization was achieved using a spray voltage of 2100 V at an ion transfer tube temperature of 275 °C. Data were initially acquired in data-dependent acquisition (DDA) mode. MS1 resolution was 120,000 (at 200 m / z), the mass range was 375–1650 m / z, the normalized AGC was set to 300%, and the maximum injection time was set to 20 ms. MS2 resolution was 15,000, quadrupole separation was 1.4 m / z, the normalized AGC was 50%, the maximum injection time was set to auto, and the HCD collision energy was 30%. Four samples were analyzed per condition.

[0116] Data analysis was performed using Fragpipe (version 19.1) with the Msfragger search engine (version 3.8), IonQuant 1.8.10, and Philosopher 4.8.1. Analysis was performed using the Human Proteome Database (Uniprot) as of December 2022, with streptavidin added manually as a contaminant. Msfragger analysis was performed using trypsin as the enzyme cleaving after Arg and Lys, with a maximum of two missed cleavages, peptide lengths of 7–50, and removal of the N-terminal methionine. N-terminal acetylation and methionine oxidation were defined as variable modifications, and carbamidomethyl was defined as a fixed modification. A false discovery rate of 0.01 was used at both the peptide and protein levels. Label-free quantification was performed using IonQuant with a 1-minute tolerance, enabling inter-experiment agreement. After analysis, the combined protein files were analyzed using Perseus. Intensities were transformed to Log2 values, quadruplicates of each type were grouped, and all proteins with at least three valid values ​​in one of the groups were retained in the analysis. Missing values ​​were replaced by imputation from a normal distribution (downshift 1.8, width 0.3), and differences and p-values ​​were calculated using Student's t-test. Data have been deposited to the ProteomeXchange Consortium via the PRIDE Partner Repository under dataset identifier PXD044294.

[0117] Example 4 Modified Protein (Im9) Covalent Binder Recombinant proteins were modified into covalent binders using 2-(bromomethyl)acrylate. The bacterial colicin E9 toxin / antitoxin system was chosen as a model system. This system consists of a highly toxic nuclease (E9) bound intracellularly by an inhibitory partner called the immunity protein (Im9). This complex can be excreted and internalized by target cells, displacing Im9, causing E9-induced toxicity. The affinity of the Im9 / E9 complex is very high and structurally well characterized.

[0118] A computational pipeline using the Rosetta Relax application (Tyka, MD et al., Alternate States of Proteins Revealed by Detailed Energy Landscape Mapping. J. Mol. Biol. 2011, 405 (2), 607-618; Leman, JK; et al., Macromolecular Modeling and Design in Rosetta: Recent Methods and Frameworks. Nat. Methods 2020, 17 (7), 665-680) was performed, which applied covalent constraints between the methacrylate side chain and the target lysine to enforce the covalent bond between them while performing all-atom refinements using relatively small movements that sampled local conformational space. Based on the noncovalent complex of colicin E9 and Im9 (PDB: 1EMV), we mutated position 20 of Im9, which is within a Cα-Cα distance of <14 Å from the target Lys97, to our methacrylate side chain and found five mutations that yielded sub-Å models with particularly favorable interface and constraint scores (interface backbone RMSD <1 Å). From these designs, we selected an Im9 mutant (C23A / E41C) onto which a methacrylate "warhead group" was introduced to react with Lys97 of the E9 nuclease (Figure 7A). E9 and its Im9 mutants were expressed and purified. Preparation of the methacrylate-modified Im9 mutant under native buffer conditions was impractical because the cysteine ​​modification was slow and competed with modifications at other sites, as observed by the appearance of multiply labeled species before the complete formation of the single-labeled protein, possibly suggesting that the cysteine ​​was not fully exposed. Preparation under denaturing conditions (50% acetonitrile) was much more efficient, resulting in rapid and selective modification on timescales ranging from minutes to an hour, and, combined with HPLC purification, yielded >95% singly labeled protein (Figure 7D).

[0119] To evaluate the ligation of methacrylate-modified Im9 to Lys97 of E9, the modified Im9 mutants and E9 were incubated, and the crosslinked complex was monitored by intact protein LCMS. These results show approximately 50% conversion to the covalent complex within 5 h and nearly quantitative conversion within 16 h (Figure 7B). To verify Lys97 ligation, point mutation experiments were performed by individually mutating lysines 55, 81, 89, 97, and 125 of E9 to arginine. Mutants K81R and K125R dramatically inhibited bacterial growth, suggesting that a decrease in binding affinity likely caused E9-mediated toxicity. Binding of K55R, K89R, and K97R to the methacrylate-modified Im9 mutants was also tested (Figure 7C). The K55R and K89R mutations had no effect on ligation efficiency, whereas mutation of Lys97 almost completely abolished the formation of the covalent complex, suggesting Lys97 as the target binding site, consistent with the model.

[0120] The effect of covalent binding was investigated by measuring the stability of the mutations, which influence the structure and complex. To this end, pure Im protein and its complex with E9 were analyzed using SEC-MALS. The estimated Mw of the protein from the elution volume was consistent with the MALS measurements, suggesting that the Im protein is monomeric and that the C23A / E41C mutations in the Im protein cause the protein to adopt a somewhat compact structure, which may explain the difficulty in modifying the protein in native buffer. The methacrylate-modified variant behaved more similarly to the WT, and a similar trend was observed for the complex with E9. These results suggest that the covalent complex adopted a structure similar to that of the native complex. To estimate the effect of covalent binding on the stability of the complex, differential scanning fluorimetry (DSF) was used to monitor the thermal stability of the complex (Figure 9). Im9 protein in its free and methacrylate-modified forms unfolded at approximately 50 °C, whereas E9 protein showed no discernible transition. The non-covalent complex shows minimal differences compared to free Im9, whereas the covalent complex is quite stable and unfolds at 72° C. (FIG. 9).

[0121] Provided herein is the single labeling of Im9 with a methacrylate electrophile (Figures 7A-7D). To generate a covalent protein reagent, the protein is produced by standard recombinant expression, allowing any number of cysteines to be mutated, supporting the single introduction of an electrophile. As exemplified herein, electrophilic Im9 irreversibly binds E9 (Figure 7B). However, binding was abrogated by mutation of the target lysine to arginine (Figure 7C). Such irreversible protein-protein binding could have profound effects, such as exceptionally strong thermal stabilization or improved in vivo efficacy, as shown by the irreversible Im9 / E9 complex (Figures 11A-11B). By analogy with peptides, this approach would likely work even better for targeting cysteines on proteins.

[0122] method: Cloning of Im9 and E9 mutants The pET21d plasmid encoding either E41+wild-type Im9 or Im9 was used. For mutation of E41 to cysteine, PCR was performed using this plasmid as a template with the following primers: ImFor: GAAATGACTGAGCACCCTAGT (sequence ID 13) ImRev:ACAAAAGTGTGTAACCAATTTAACCAGTTC (Sequence ID 14)

[0123] The PCR product was purified and 1 μg was phosphorylated using 10 units of T4 PNK (NEB) in 20 μl of T4 ligase buffer (NEB) for 1 hour at room temperature. This was followed by the addition of 400 units of T4 ligase (NEB) for 2 hours at room temperature. The product was transformed into DH5α and plated on ampicillin plates. After colony selection and identification of the correct sequence, this step was repeated with the following primers to introduce a second mutation (C23A): Im2For:GCTAATGCGGACACTTCCAGTG (sequence ID 15) Im2Rev:AATTGTTGTTACAAGCTGTAAAAATTCAG (Sequence ID 16) For the E9 mutation, the same procedure was used with the following primer set: Lys55 for:CGGGCTGTATGGGAAGAGGTGTC (Sequence ID 17) Lys55rev:CCGAAAATCGTCGAAGCTTTTAAATTC (Sequence ID 18) Lys81for:CGAGGTTATTCTCCGTTTACTCCAAAG (Sequence ID 19) Lys81rev:TGAAACACTAGACTTATTGCTTGGG (Sequence ID 20) Lys89for:CGGAATCAACAGGTCGGAGGG (sequence ID 21) Lys89rev:TGGAGTAAACGGAGAATAACCTTTTG (Sequence ID 22) Lys97for:CGAGTCTATGAACTTCATCATGACAAG (sequence ID 23) Lys97rev:TCTCCCTCCGACCTGTTG (Sequence ID 24) Lys125for:CGGCGACATATCGATATTCACCG (sequence ID 25) Lys125rev:AGGTGTAGTCACTCGGATATTATC (Sequence ID 26)

[0124] Expression and purification of E9 and Im9 mutants The plasmid was transformed into BL21(DE3) bacteria, which were grown in 2YT+NPS+1 mM MgSO4 at 37°C to an OD=0.6, rapidly cooled to 16°C on ice, and induced with 1 mM IPTG for 16 hours.

[0125] For purification of Im9, cells were dispersed in 30 ml of lysis buffer (25 mM Tris, pH 7.5, 50 mM NaCl, 10 mM imidazole) plus protease inhibitors and sonicated (55%, 1 min, 5-second pulse). After this, MgCl2 was added to 1 mM, and 5 μl of Benzonase Nuclease (Fisher) was added. The lysates were centrifuged (20,000 rpm for 20 min), and the lysates were filtered through a 0.45 μm filter. Each lysate was then loaded onto a 5 ml Ni-NTA column pre-equilibrated with lysis buffer, and the column was washed with 4 CV of lysis buffer. Im9 was eluted with 25 mM Tris, pH=7.5, 50 mM NaCl, 500 mM imidazole, extensively dialyzed (three times) against 20 mM NaPi, pH=7.2, 100 mM NaCl, 0.2 μm filtered, and flash-frozen at −80°C.

[0126] Purification of E9 was carried out using the same procedure, except that elution was carried out using 6 M GuHCl. Some precipitation was observed during dialysis.

[0127] Methacrylate labeling of Im9 mutants Labeling was performed in protein storage buffer (20 mM NaPi, pH = 7.2, 100 mM NaCl). Im9(C23A / E41C) was doubled to a concentration of 1.88 mM, resulting in some precipitation. At this point, 1.1 equivalents of ethyl-(3-bromomethacrylate) (pre-dissolved in acetonitrile) were added. After 1 hour at room temperature, 70% labeling was observed, and an additional 0.7 equivalents were added. After 1 hour, the sample was diluted with 0.1% TFA in water, filtered, and purified using HPLC.

[0128] Reaction between Im9 methacrylate and E9 The purified protein was diluted to 20 μM in 20 mM NaPi, pH 7.2, 50 mM NaCl. The Im methacrylate solution was then mixed with the E9 solution in a 1:1 ratio to give a 10 μM conjugate. The reaction was incubated at room temperature for 4.5 hours, then stopped by diluting the conjugate 5-fold with 0.1% TFA / water, followed by LC-MS analysis.

[0129] Sec-MALS characterization of the Im-E9 complex Samples containing 200 μM of isolated Im constructs or Im-E9 complexes were prepared in 25 mM NaPi, pH 7.2, 100 mM NaCl. A miniDAWN TREOS multi-angle light scattering detector (Wyatt Technology, Santa Barbara, CA) equipped with three detector angles (43.6°, 90°, and 136.4°) and a 658.9 nm laser beam, a Wyatt QELS dynamic light scattering module for hydrodynamic radius determination, and an Optilab T-rEX refractometer (Wyatt Technology) were used in series with a Superdex 75 Increase 10 / 300 GL column (Cytiva) for size exclusion chromatography analysis. 420–770 μg of each sample was injected onto the column in 150–200 μL. Experiments were performed using an AKTA Pure system equipped with a UV-900 detector (Cytiva) at a flow rate of 0.8 ml / min and using PBS, pH 7.4, as the running buffer. All experiments were performed at room temperature (25°C). Data collection and SEC-MALS analysis were performed with ASTRA 6.1 software (Wyatt Technology). The refractive index of the solvent was defined as 1.331, and the viscosity was defined as 0.8945 cP (common parameters for PBS buffer at 658.9 nm). The dn / dc (refractive index increment) value for all samples was defined as 0.185 mL / g (standard value for proteins).

[0130] Differential scanning fluorescence measurements on Im-E9 complexes A 50 μl sample of E9:Im complex at a concentration of 50 μM was prepared and incubated overnight at room temperature in 20 mM NaPi, pH 7.2, 50 mM NaCl. SYPRO Orange (X5000 stock solution) was diluted 200-fold in buffer, and 13 μl of this stock solution was added to each sample, diluting the protein to 40 μM. Each sample was divided into three technical replicates and heated to 95°C in a thermal cycler for 1.5 hours while measuring fluorescence.

[0131] Introducing new residues into Rosetta Our methacrylate side chains were introduced into Rosetta using the protocol described by 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). Because the reaction between the methacrylate warhead group and the lysine amine forms two different stereoisomers, we implemented these as separate residues. We used the GaussView interface to draw each stereoisomer, and then optimized the structures using Gaussian software with the following options: HF / 6-31G(d)scf=tight test. Each optimized structure was converted to a mol file using the OpenBabel toolbox (http: / / openbabel.org), which was then converted to a Rosetta residue "parameter file" using the molfile_to_params_polymer.py script provided with Rosetta. To allow residues to form covalent bonds to other residues, CONNECT records were added to each stereoisomer parameter file to identify which atoms participate in inter-residue covalent bonds, as described by Drew et al. (Drew et al. Adding Diverse Noncanonical Backbones to Rosetta: Enabling Peptidomimetic Design. PLoS One 2013, 8(7), e67051) for oligooxopiperazines. Virtual atoms were added to each parameter file, and their internal coordinates were defined according to the optimal positions of the lysine NZ atoms as predicted by Gaussian optimization. These virtual atoms were used during the modeling process to prioritize accurate covalent bond geometries. Rotamer libraries were generated using the Rosetta MakeRotLib application.

[0132] Appropriate covalently linked variants of lysine were implemented through the residue patch system, utilizing existing definitions and a rotamer library optimized for use in Rosetta. The reacted lysine was modeled as described above, and a patch file was created that removed the lysine 3HZ atom and added a virtual atom with internal coordinates consistent with the CONNECT record and the Gaussian-optimized structure. A PROTON_CHI record was added to allow for sampling of new rotamers around the coupled CE-NZ bond.

[0133] Design of 14-3-3σ peptide binders Using PDB ID: 3MHR as the template structure, we designed Lys49- and Lys122-linked peptides for 14-3-3σ. Using the Rosetta fixed backbone design application (fixbb), we mutated each lysine to a covalently linked variant, and relevant peptide positions (Cα-Cα distance to the target lysine <14 Å) were mutated to each of our methacrylate side chain stereoisomers; these included positions 126-131 of Lys49 and positions 126-133 of Lys122. CovPepDock was applied to generate 200 models of each of these mutant complexes (100 for each stereoisomer). To prioritize covalent bond formation in the correct geometry, AtomPair constraints were applied between each covalently bonded atom and its virtual alternative within the associated residue, as described in our previous work. The HARMONIC score function was used, centered at 0 and with a standard deviation of 0.3. The 10 top interface-scoring models for each complex were manually inspected, focusing on near-native models with constraint scores <2, and the top four peptides were selected.

[0134] For the second set of peptides, we searched the PDB for X-ray crystal structures of 14-3-3σ in complex with peptides 3–15 amino acids in length. The results were filtered for structures in which the peptide binds near Lys122 (Cα-Cα distance < 14 Å) but not near Cys38 (Cα-Cα distance > 12 Å). This resulted in the PDB IDs for 3IQU, 3P1N, 4IEA, 4QLI, and 7NWF. Similarly, Lys122-binding peptides were designed based on each of these structures by mutating positions 257–260 of 14-3-3σ 3IQU, 372–374 of 3P1N, 620–625 of 4IEA, 175–180 of 4QLI, and 592–595 of 7NFW. The native Cys180 of the 4QLI peptide was mutated to a serine to avoid possible cyclization or side reactions that may occur due to the addition of a second cysteine ​​that introduces a methacrylate warhead group.

[0135] Design of colicin E9 protein binders PDB ID: 1EMV was used as the template structure. Similar to the peptide design protocol, we used the Rosetta fixed backbone design application (fixbb) to mutate Lys97 of colicin E9 to our covalently linked variant, and positions 30-41 and 48-55 of Im9 to each stereoisomer of our methacrylate side chain. Using the RosettaScripts interface and FastRelax Mover, we then generated 200 models of each complex (100 for each stereoisomer), applying constraints similar to those described in the peptide design methodology section. To select constructs for synthesis and testing, the 10 top interface-scoring models were manually inspected for each mutant complex, focusing on near-native models with constraint scores <2.

[0136] Example 5 Selectivity and off-target characterization using chemical proteomics To further characterize the selectivity and off-target activity of the methacrylate peptides, we synthesized biotin-labeled derivatives of peptide sequences ID3 and ID8, incubated them with A549 lysate, enriched biotinylated proteins using streptavidin beads and trypsin digestion, and subsequently characterized the bound proteins by LC-MS / MS. All 14-3-3 isoforms bound efficiently, demonstrating the best targets with few off-targets, confirming that peptide sequences ID3 and ID8 were selective, pan-14-3-3-reactive probes (Figure 6). Several off-targets were identified, many of which were NAD / NADP-dependent enzymes, such as aldo-keto reductases, aldolases, and dehydrogenases. These contain defined binding pockets for phosphate-containing cofactors with nearby lysine residues. Enzymes with phosphate-containing substrates, including several glycolytic enzymes, were also prominent off-targets (Figure 10). We speculate that phosphorylated peptides compete for these binding sites and form covalent adducts with these proteins. Nevertheless, the fluorescence imaging results suggested that the 14-3-3 proteins were highly selectively targeted and that only a small fraction of off-targets were modified due to the lack of more specific sequence recognition.

[0137] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. 1. A modified protein or peptide, including a recombinant protein or synthetic peptide modified with an electrophile, wherein the electrophile is covalently attached to a thiol group of a cysteine ​​within the protein or peptide, the electrophile having a structure represented by Formula I; 【Chemistry 1】 (In the formula, R １ includes substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle; X is O, NR ３ or substituted or unsubstituted alkylene; and R ３ is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle, aryl, heteroaryl or heterocycle; is represented by the structure A modified protein or peptide capable of covalently binding a target protein.

2. 2. The modified protein or peptide of claim 1, wherein the electrophile has non-covalent recognition with the target protein and is in proximity to a target residue of the target protein prior to the covalent binding to the target protein.

3. The target residue in the target protein is a cysteine ​​residue (SH) or a lysine residue (NH ２ 3. The modified protein or peptide of claim 2, wherein

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

5. The electrophile is represented by Formula II: 【Chemistry 2】 5. The modified protein or peptide according to any one of claims 1 to 4, which is represented by the structure:

6. 6. The modified protein or peptide of claim 1, wherein the electrophile is ethyl methacrylate.

7. The following peptide: Ac-RSApSmCPSL-NH ２ (Sequence ID 3), Ac-RAHpSmCPASLQ-NH ２ (SEQ ID NO: 8) or Ac-RAHpSSPASmCQ-NH ２ 7. The modified peptide of any one of claims 1 to 6, comprising: (SEQ ID 11) wherein pS is phosphoserine and mC is methacrylate-modified cysteine.

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

9. 9. The modified peptide of claim 8, wherein the peptide covalently binds Cys38, Lys122, or Lys49 of a 14-3-3σ target protein.

10. 7. The modified protein of any one of claims 1 to 6, comprising a modified immunity (Im9) protein (SEQ ID 27).

11. 11. The modified protein of claim 10, wherein the Im9 protein (SEQ ID NO: 27) binds the E9 target protein.

12. 12. A method for preparing a modified protein or peptide according to any one of claims 1 to 11, comprising: a. identifying the target protein; b. Designing candidate covalent binders of a peptide or protein based on previously characterized non-covalent binders of said target protein; c) synthesizing a modified peptide or protein that allows recognition of the target protein at the recognition site, wherein the modified protein or peptide includes a cysteine ​​that reacts with the electrophile of Formula I. A method comprising:

13. 13. The method of claim 12, wherein the design of the peptide is based on computational modeling including RosettaCovPepDock.

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

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

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

17. 17. The protein-protein covalent conjugate of claim 16, wherein the modified protein is Im9 protein and the target protein is E9.

18. 18. The protein-protein covalent conjugate of claim 17, which exhibits greater thermal stability than the non-covalent complex.

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

20. The modified peptide is Ac-RSApSmCPSL-NH ２ (Sequence ID 3), Ac-RAHpSmCPASLQ-NH ２ (SEQ ID NO: 8) or Ac-RAHpSSPASmCQ-NH ２ 20. The peptide-protein covalent conjugate of claim 19, wherein pS is phosphoserine and mC is methacrylate-modified cysteine; and the target protein is 14-3-3.

21. A modified protein or modified peptide according to any one of claims 1 to 11 for use in selective labelling, fluorescent labelling, inhibition, drug conjugation or conjugation of a target protein.

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