Covalent polypeptide inhibitors, conjugates, kits and uses targeting creatine kinase b

Abstract

The application discloses a covalent polypeptide inhibitor targeting creatine kinase B, a conjugate, a kit and application. The general formula of the covalent polypeptide inhibitor is Z1-X1-X2-X3-X4-X5-X6-X7-Z2, wherein X5 is a non-natural amino acid with a covalent reaction group, and can be covalently crosslinked with a Cys283 residue on creatine kinase B. The covalent polypeptide inhibitor has strong covalent binding capacity for creatine kinase B, can realize specific recognition and labeling at the whole proteome level, effectively inhibit the enzyme activity, and can be used as a chemical probe for enrichment, detection and function research of active creatine kinase B, and has a wide application prospect in biological research, drug discovery and disease diagnosis related to creatine kinase B.

Description

Technical Field

[0001] This application relates to the fields of chemical biology and pharmaceutical technology, specifically to covalent peptide inhibitors, conjugates, kits, and applications targeting creatine kinase B. Background Technology

[0002] Creatine kinase B (CKB) is an important cytoplasmic isoenzyme in the creatine kinase (CK) family. Widely distributed in vertebrates, CKB primarily catalyzes the reversible phosphate transfer reaction between creatine and ATP, producing phosphocreatine and ADP. As a "high-energy phosphate bond reserve" within cells, CKB plays a central role in maintaining local energy homeostasis in cells with high energy demands.

[0003] As research into the creatine kinase family deepens, its crucial role in the development and progression of certain diseases is gradually being revealed. Existing studies have shown that CK severely impacts cellular processes dependent on high ATP demand, such as cancer cell proliferation, particularly in aggressive acute myeloid leukemia, liver cancer, breast cancer metastasis, and pancreatic cancer growth and metastasis. Inhibition of CK can significantly weaken the metabolic adaptation of tumor cells, with minimal impact on basal energy metabolism in somatic cells and normal tissues. This makes CK family proteins highly promising new targets for anti-tumor therapy. Furthermore, CKB has been found to possess non-classical biological functions such as metabolic and signal regulation. For example, CKB can be localized in the mitochondria of brown adipocytes, regulating the ineffective creatine cycle (FCC), thereby affecting adaptive thermogenesis and obesity. Recent research has made a breakthrough by discovering that CKB also possesses "part-time" protein kinase activity, interacting with and directly phosphorylating the key S104 site of glutathione peroxidase 4 (GPX4), thereby blocking the binding of GPX4 to HSC70 and effectively alleviating the GPX4-induced ferroptosis.

[0004] Despite the diverse and important biological functions of CKB, the discovery of specific inhibitors targeting CKB has not been widely reported. Creatine analogues such as β-guanidinium propionate (3-GPA) and cyclic creatine can competitively inhibit CKB enzymatic activity; however, these competitive substrate analogues often have low affinity and require high concentrations under physiological conditions to exert effective inhibitory effects. I2 imidazoline receptor ligand inhibitors, represented by BU99006, are thought to covalently bind to the Cys283 site of CKB, but lack specificity. Recent studies have used ultra-deep chemical proteomics to screen for inhibitors CTi that can covalently bind to cysteine ​​residues near the active site of CK; however, different subtypes of the CK family have similar three-dimensional structures, and pan-subtype inhibitors CTi show similar affinity for the mitochondrial subtype CKMT and the muscle tissue subtype CKM of creatine kinase. Furthermore, small molecule inhibitors can inhibit the enzymatic activity of CKB as a metabolic enzyme, but it remains unclear whether they can effectively block the protein-protein interaction interface formed between CKB and other proteins.

[0005] Given the aforementioned significant limitations of existing CKB inhibitors, there is an urgent need in this field to develop a novel inhibitor that can potently and selectively inhibit CKB enzyme activity in order to meet the pressing need for in-depth research into the biological functions of CKB and the development of targeted anti-tumor drug molecules. Summary of the Invention

[0006] Therefore, it is necessary to provide covalent peptide inhibitors, conjugates, kits, and applications targeting creatine kinase B.

[0007] The first aspect of this application provides a covalent polypeptide inhibitor targeting creatine kinase B, wherein the general formula of the covalent polypeptide inhibitor is Z1-X1-X2-X3-X4-X5-X6-X7-Z2, wherein X1-X2-X3-X4-X5-X6-X7 is an amino acid sequence, and X1 is selected from methionine or its conserved alternative amino acids.

[0008] X2 is selected from small-sized amino acid residues or negatively charged amino acid residues;

[0009] X3 is selected from small-sized amino acid residues or hydrophobic amino acid residues;

[0010] X4 is selected from small-sized amino acid residues or charged amino acid residues;

[0011] The molecular weight of the small-sized amino acid residues is less than 100 Da;

[0012] X5 is a non-natural amino acid with a covalently reactive group, which can covalently crosslink with the Cys283 residue on creatine kinase B.

[0013] X6 is selected from negatively charged amino acid residues;

[0014] X7 is selected from negatively charged amino acid residues or hydrophobic amino acid residues.

[0015] Z1 is selected from hydrogen, acyl, fluorescent group, biotin, isotope tag or bioorthogonal reactive group;

[0016] Z2 is selected from hydroxyl, amino, cell-penetrating peptide or stabilizing modification groups.

[0017] In some embodiments, the covalent reactive group includes any one of chloroacetamide, acrylamide, fluorosulfate, vinyl sulfone, and sulfonyl fluoride.

[0018] Optionally, X5 is selected from any one of 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, and ornithine, whose side chains are modified with chloroacetamide or acrylamide groups.

[0019] In some embodiments, X2, X3 and X4 are each independently selected from any one of serine, glycine, alanine and glutamic acid.

[0020] In some embodiments, X6 is selected from either glutamic acid or aspartic acid.

[0021] In some embodiments, X7 is selected from any one of aspartic acid, leucine, and methionine.

[0022] In some embodiments, the amino acid sequence is selected from any of the following:

[0023] (1) Met-Glu-Ser-Ala-X5-Glu-Asp;

[0024] (2) Met-Ser-Ser-Gly-X5-Asp-Leu;

[0025] (3) Met-Pro-Leu-Asp-X5-Asp-Leu;

[0026] (4) Met-Ala-Ala-Arg-X5-Glu-Met;

[0027] X5 is 2,3-diaminopropionic acid with a chloroacetamide group modified on the side chain.

[0028] In some embodiments, Z1 is 5-hexyneylyl and Z2 is amino;

[0029] Optionally, the sequence of the covalent peptide inhibitor is 5-hexynyl-Met-Glu-Ser-Ala-Dap(ClAc)-Glu-Asp-NH2, wherein Dap(ClAc) is 2,3-diaminopropionic acid with a chloroacetamide group modified on the side chain.

[0030] Optionally, the structural formula of the covalent peptide inhibitor is shown below:

[0031] .

[0032] A second aspect of this application provides a conjugate comprising the covalent peptide inhibitor described in the first aspect of this application and a carrier and / or reporter tag attached to the covalent peptide inhibitor;

[0033] Optionally, the report label includes one or more of rhodamine-based fluorescent dyes, cyanine-based fluorescent dyes, and biotin and its derivatives; the carrier includes one or more of cell-penetrating peptides and liposomes.

[0034] A third aspect of this application provides a kit comprising the covalent peptide inhibitor described in the first aspect of this application or the conjugate described in the second aspect of this application;

[0035] Optionally, the kit may also include a protease inhibitor.

[0036] A fourth aspect of this application provides a pharmaceutical composition comprising the covalent polypeptide inhibitor described in the first aspect of this application or the conjugate described in the second aspect of this application;

[0037] Optionally, pharmaceutically acceptable excipients may also be included.

[0038] The fifth aspect of this application provides the use of the covalent peptide inhibitor described in the first aspect of this application or the conjugate described in the second aspect of this application in the preparation of products for the prevention and / or treatment of creatine kinase B-mediated related diseases;

[0039] Optionally, the creatine kinase B-mediated diseases are energy metabolism disorders or ferroptosis-related diseases;

[0040] Further optionally, the creatine kinase B-mediated diseases include one or more of the following: aggressive acute myeloid leukemia, liver cancer, breast cancer, pancreatic cancer, and adaptive thermogenic metabolic syndrome.

[0041] The sixth aspect of this application provides the use of the covalent peptide inhibitor described in the first aspect of this application or the conjugate described in the second aspect of this application in any of the following aspects:

[0042] (1) Application in the preparation of in vitro biochemical reagents or tool drugs for specifically binding to and inhibiting the activity of creatine kinase B;

[0043] (2) Application in the preparation of chemical proteomics probes for the specific labeling, enrichment and quantification of active creatine kinase B protein in complex whole proteome samples;

[0044] (3) Application in the preparation of regulators for blocking protein-protein interactions between creatine kinase B and glutathione peroxidase 4.

[0045] The aforementioned covalent peptide inhibitors targeting creatine kinase B exhibit strong covalent binding to creatine kinase B, enabling specific recognition and labeling of creatine kinase B at the whole-proteome level. They can not only effectively inhibit the enzymatic activity of creatine kinase B, but also serve as chemical probes for the enrichment, detection, and functional study of active creatine kinase B in complex biological samples, showing broad application prospects in creatine kinase B-related biological research, drug discovery, and disease diagnosis. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0047] Figure 1 This is a schematic diagram of the covalent peptide design structure targeting CKB in one embodiment of this application; where a represents ADP, creatine, and Mg in the CKB catalytic pocket. 2+ a) Schematic diagram of the binding mode and the position of key residues; b) Structural model of the binding of non-covalently designed peptide MESALED with CKB; c) Modeled structure of covalent peptide CKB-5 with CKB after introducing non-natural amino acid LC4 containing covalent reactive groups.

[0048] Figure 2 The chemical structural formulas of a series of covalent peptide inhibitors / probes (CKB-1 to CKB-5) targeting CKB designed and synthesized in one embodiment of this application are shown below.

[0049] Figure 3This document presents the specific labeling and binding site characterization results of covalent peptides on recombinant CKB protein in one embodiment of this application; where a) are the gel fluorescence imaging and Coomassie brilliant blue staining results of different peptide probes labeling recombinant CKB protein; b) are the comparison results of activity-dependent labeling of peptide CKB-5 on wild-type CKB (WT), C283S active mutant, and SDS-denatured wild-type CKB; c) are the complete proteomic spectrum (Intact MS) analysis results of the 1:1 covalent adduct formed by peptide CKB-5 and recombinant CKB; and d) are the LC-MS / MS secondary mass spectrometry analysis results for identifying the covalent modification site (Cys283) of peptide CKB-5.

[0050] Figure 4 This is an embodiment of the present application showing the specific labeling results of covalent peptides on active CKB in mouse brain tissue lysates; wherein a is the gel fluorescence imaging results of a series of designed and synthesized covalent peptides labeled in mouse brain tissue lysates and the corresponding Coomassie brilliant blue staining results; b is the gel fluorescence imaging results of CKB-5 labeled in mouse brain tissue lysates at concentration gradients and the immunoblotting results of the target protein CKB.

[0051] Figure 5 This is a volcano plot showing the labeling selectivity of peptide CKB-5 at the whole proteome level of mouse brain lysate, analyzed by chemical proteomics and label-free quantitative methods in one embodiment of this application.

[0052] Figure 6 This is the kinetic measurement result of the inhibitory effect of the covalent peptide CKB-5 on CKB kinase activity in one embodiment of this application; where a is the half-maximal inhibitory concentration (IC50). 50 b is the concentration-activity fitting curve; b is the determination of the second-order inactivation rate constant (k inact / K I The concentration-apparent rate constant (k) obs Fitted curve. Detailed Implementation

[0053] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0055] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0056] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0057] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0058] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0059] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0060] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0061] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0062] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0063] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0064] In this application, the terms "room temperature" or "normal temperature" generally refer to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0065] Traditional creatine kinase B inhibitors have the following limitations: First, competitive inhibitors, represented by creatine analogs, have low affinity, requiring high concentrations under physiological conditions to effectively inhibit enzyme activity; second, covalent inhibitors, represented by BU99006, while binding to the Cys283 site, lack specific recognition ability for creatine kinase B; third, pan-subtype small molecule inhibitors CKi have similar affinity for different subtypes of the creatine kinase family (such as mitochondrial subtype CKMT and muscle subtype CKM), resulting in poor selectivity; in addition, traditional small molecule inhibitors are small in size, making it difficult to effectively block the protein-protein interaction interface between creatine kinase B and downstream proteins (such as GPX4), thus failing to intervene in its non-classical biological functions.

[0066] Based on this, the embodiments of this application at least provide a covalent peptide inhibitor targeting creatine kinase B, a conjugate, a kit, and its application. The embodiments of this application break through the dependence on natural ligands, and for the first time obtain an artificial peptide backbone that fits the CKB substrate binding groove using a deep learning algorithm, and precisely introduce covalently reactive amino acids using a geometric hash matching algorithm to target the reactive Cys283 site in the CKB substrate binding groove. This covalent peptide exhibits good specificity for CKB at the whole proteome level, especially among CK family members.

[0067] In this application, the term "conservatively substituted amino acid" refers to an amino acid that has been replaced with another amino acid that has similar physicochemical properties (such as charge, hydrophobicity, size, and side chain structure). For example, the conservedly substituted amino acids for methionine include isoleucine, leucine, and valine.

[0068] In a first aspect of this application, a covalent peptide inhibitor targeting creatine kinase B is provided, having the general formula Z1-X1-X2-X3-X4-X5-X6-X7-Z2, wherein X1-X2-X3-X4-X5-X6-X7 is an amino acid sequence, and X1 is selected from methionine or its conserved alternative amino acids.

[0069] X2 is selected from small-sized amino acid residues or negatively charged amino acid residues.

[0070] X3 is selected from small-sized amino acid residues or hydrophobic amino acid residues.

[0071] X4 is selected from small-sized amino acid residues or charged amino acid residues.

[0072] Small-sized amino acid residues have a molecular weight of less than 100 Da;

[0073] X5 is a non-natural amino acid with a covalently reactive group, which can covalently cross-link with the Cys283 residue on creatine kinase B.

[0074] X6 is selected from negatively charged amino acid residues;

[0075] X7 is selected from negatively charged amino acid residues or hydrophobic amino acid residues.

[0076] Z1 is selected from hydrogen, acyl, fluorescent group, biotin, isotope tag, or bioorthogonal reactive group;

[0077] Z2 is selected from hydroxyl, amino, cell-penetrating peptides, or stabilizing modification groups.

[0078] It should be noted that the predicted structure of the amino acid sequence exhibits a relatively extended linear conformation, which can occupy the open conformational pocket of creatine kinase B when it is not bound to a natural substrate; its main chain carbonyl oxygen and the side chains of negatively charged amino acid residues can form hydrogen bonds or electrostatic interactions with at least one residue of Arg130, Arg132, Arg236, Arg292 or Arg320 on creatine kinase B.

[0079] In some embodiments, the small-sized amino acid residues can be glycine, alanine, serine, or proline.

[0080] In some embodiments, the bioorthogonal reactive group can be, but is not limited to, alkynyl or azide.

[0081] In some embodiments, the covalent reactive group in the non-natural amino acid includes any one of chloroacetamide, acrylamide, fluorosulfate, vinyl sulfone, and sulfonyl fluoride. Exemplarily, the covalent reactive group is selected from chloroacetamide or acrylamide.

[0082] In some embodiments, X5 is selected from any one of 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, and ornithine, whose side chains are modified with chloroacetamide or acrylamide groups.

[0083] In some embodiments, X2, X3 and X4 are each independently selected from any one of serine, glycine, alanine and glutamic acid.

[0084] In some embodiments, X6 is selected from either glutamic acid or aspartic acid.

[0085] In some embodiments, X7 is selected from any one of aspartic acid, leucine, and methionine.

[0086] In some embodiments, the amino acid sequence of the covalent peptide inhibitor is selected from any of the following:

[0087] (1) Met-Glu-Ser-Ala-X5-Glu-Asp (SEQ ID NO: 1);

[0088] (2) Met-Ser-Ser-Gly-X5-Asp-Leu (SEQ ID NO: 2);

[0089] (3) Met-Pro-Leu-Asp-X5-Asp-Leu (SEQ ID NO: 3);

[0090] (4) Met-Ala-Ala-Arg-X5-Glu-Met (SEQ ID NO: 4);

[0091] X5 is 2,3-diaminopropionic acid with a chloroacetamide group modified on the side chain.

[0092] In some embodiments, Z1 is selected from 5-hexynyl and Z2 is selected from amino.

[0093] In some embodiments, the sequence of the covalent peptide inhibitor is 5-hexynoyl-Met-Glu-Ser-Ala-Dap(ClAc)-Glu-Asp-NH2, wherein Dap(ClAc) is 2,3-diaminopropionic acid with a chloroacetamide group modified on the side chain; further, the structural formula of the covalent peptide inhibitor is shown below:

[0094]

[0095] In some embodiments, the above-mentioned covalent peptide inhibitors can be prepared using conventional techniques in the art. For example, the above-mentioned covalent peptide inhibitors can be prepared using the Fmoc solid-phase synthesis method.

[0096] In a second aspect of this application, a conjugate is provided, comprising the aforementioned covalent peptide inhibitor and a carrier and / or reporter tag attached to the covalent peptide inhibitor. It should be noted that the conjugate may be formed by coupling the covalent peptide inhibitor to the carrier or reporter tag via its terminal alkynyl group or side chain modification group (e.g., the alkynyl group via click chemistry).

[0097] In this application, the term "report label" refers to a functional group covalently linked to the aforementioned covalent polypeptide inhibitor for the purpose of detecting, visualizing, enriching, or identifying creatine kinase B protein.

[0098] In some implementations, the report label includes, but is not limited to, one or more of rhodamine-based fluorescent dyes, cyanine-based fluorescent dyes, and biotin and its derivatives, for fluorescent imaging or enrichment identification of creatine kinase B protein in complex proteomics.

[0099] In some implementations, biotin derivatives include, but are not limited to, desulfurized biotin (DBIA), DADPS-biotin, etc.

[0100] In some embodiments, the carrier includes, but is not limited to, one or more of cell-penetrating peptides, polymer materials, nanomaterials, and liposomes.

[0101] In some embodiments, the polymeric material includes, but is not limited to, one or more of polyester, polyanhydride, polyethylene glycol, and chitosan.

[0102] In a third aspect of this application, a kit is provided that includes the covalent peptide inhibitor or the conjugate described above.

[0103] In some implementations, the kit may be a companion diagnostic kit or a molecular probe kit.

[0104] In some implementations, the kit also includes protease inhibitors. It is understood that protease inhibitors are used to protect the integrity of proteins in the sample and prevent degradation of CKB by endogenous or exogenous proteases.

[0105] In a fourth aspect of this application, a pharmaceutical composition is provided comprising the above-described covalent peptide inhibitor or the above-described conjugate.

[0106] In some embodiments, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0107] In some embodiments, pharmaceutically acceptable excipients include, but are not limited to, one or more of carriers, solvents, diluents and excipients, which can be prepared into different dosage forms as needed.

[0108] In a fifth aspect of this application, the use of the aforementioned covalent peptide inhibitor or the aforementioned conjugate in the preparation of products for the prevention and / or treatment of creatine kinase B-mediated related diseases is provided.

[0109] In some implementations, the product includes a drug.

[0110] In this application, "drug" includes any agent, compound, composition, or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. There is no particular limitation on the scope of the physiological and / or pharmacological effects produced by a "drug" in vivo; it may have systemic effects or only local effects. There is no particular limitation on the activity of a "drug"; it may be an active substance that can interact with other substances or an inert substance that does not interact with other substances.

[0111] In some implementations, the diseases mediated by creatine kinase B are energy metabolism disorders or ferroptosis-related diseases.

[0112] In some implementations, creatine kinase B-mediated diseases include one or more of the following: aggressive acute myeloid leukemia, liver cancer, breast cancer, pancreatic cancer, and adaptive thermogenic metabolic syndrome.

[0113] In a sixth aspect of this application, the above-described covalent peptide inhibitor or the above-described conjugate is provided for use in any of the following aspects:

[0114] (1) Application in the preparation of in vitro biochemical reagents or tool drugs for specifically binding to and inhibiting the activity of creatine kinase B;

[0115] (2) Application in the preparation of chemical proteomics probes for the specific labeling, enrichment and quantification of active creatine kinase B protein in complex whole proteome samples;

[0116] (3) Application in the preparation of regulators for blocking protein-protein interactions between creatine kinase B and glutathione peroxidase 4.

[0117] In a seventh aspect of this application, a method for preventing and / or treating creatine kinase B-mediated diseases is provided, comprising administering an effective amount of the aforementioned covalent peptide inhibitor to a subject.

[0118] In this application, "effective amount" means the amount required to elicit a biological or medical response in an individual, such as the amount of reagent that brings about a positive physiological and / or pharmacological effect on an individual, including but not limited to improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease.

[0119] In some implementations, the subject is preferably a mammal, and more preferably a human.

[0120] The following are some examples.

[0121] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0122] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0123] Example 1: Computational Design and Synthesis of CKB-Targeting Covalent Peptides

[0124] Computational design of CKB covalent peptides:

[0125] This embodiment breaks away from the traditional design approach based on natural ligands, employing a strategy combining deep learning generative models and geometric hashing algorithms to design, from scratch, an artificial covalent peptide that specifically targets the CKB substrate binding pocket. First, using the unbound open conformation of CKB (reference PDB ID: 3B6R) as a template, Cys283, with potentially high activity, was selected as a hotspot residue. Using a deep learning diffusion model and sequence design tools, non-covalent peptide backbones (such as the preferred sequence Met-Glu-Ser-Ala-Leu-Glu-Asp) that can form multiple electrostatic and hydrogen bond interactions with conserved arginine residue clusters (such as R130, R132, R236) within the CKB catalytic pocket were designed and screened.

[0126] Subsequently, this embodiment utilizes a self-developed covalent peptide design algorithm. This method employs an efficient geometric hash matching strategy to quickly search for appropriate introduction positions of a series of covalent reactive groups at the peptide-protein interface, in order to satisfy the interaction between the non-covalent peptide template and the target protein as much as possible. Using this algorithm, by replacing the 5th leucine residue in the sequence with a non-natural amino acid containing a chloroacetamide group (such as chloroacetyl-2,3-diaminopropionic acid, LC4), the algorithm predicts that the reactive group will be well-aligned near the CKB Cys283 residue. The modeling results are as follows: Figure 1 As shown. The final preferred covalent peptide was named CKB-5, with the following amino acid sequence: 5-hexynoyl-Met-Glu-Ser-Ala-X-Glu-Asp-NH2 (where X is a 2,3-diaminopropionic acid residue with a chloroacetamide group modified on the side chain, and the N-terminal hexynoyl group is used for subsequent click chemical coupling). The other covalent peptides tested in the examples of this application were named CKB-1, CKB-2, CKB-3, and CKB-4, respectively, and their detailed structures are shown below. Figure 2 As shown.

[0127] Synthesis of CKB-targeting covalent peptides:

[0128] Add 2 g (0.15 mmol / g) of RinkAmide MBHA resin coupled with starting amino acid residues to a 150 mL peptide synthesis tube, swell with 50 mL DCM (dichloromethane) for 20 min, then wash three times with DMF (N,N-dimethylformamide). Add 20 mL piperidine / DMF (1:4, v:v) and shake for 2 × 10 min to remove the Fmoc protecting group (9-fluorenylmethoxycarbonyl), then wash three times with DMF and drain. Amino acids (1 mmol), 1-hydroxy-benzotriazole (HOBT, 1 mmol, 135 mg), O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU, 1 mmol, 380 mg), N,N-Diisopropylethylamine (DIEA, 2 mmol, 350 μL), and DMF (20 mL) were briefly mixed and activated before being added to a peptide synthesis tube and shaken at 30 °C for 1 h. After the reaction, the sample was washed three times with DMF. Subsequently, Fmoc was removed and the subsequent amino acids were ligated as described above.

[0129] A covalent warhead was introduced after N-terminal hexynic acid capping, starting from the Dap side chain protected by 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl, Dde). The resin carrying the protected peptide was treated with 2% N2H4·H2O / DMF for 2 × 5 min, followed by washing three times with DMF and draining. Chloroacetyl chloride (1 mmol), DIEA (2 mmol, 350 μL), and DMF (20 mL) were briefly mixed, activated, and added to the peptide synthesis tube, and shaken at 30 °C for 1 h. After the reaction, the sample was washed three times with DMF.

[0130] The resin was washed three times with DMF, rinsed with methanol, and drained. Subsequently, it was cleaved with a cleavage buffer (TFA / triisopropylsilane / H₂O, 95:2.5:2.5, v:v:v). The resulting solution was concentrated under reduced pressure and washed with cold diethyl ether. The crude product was purified using semi-preparative HPLC (Newstyle, Hanbon). The molecular weight of the synthesized peptide was confirmed by ESI-MS (LCQ Fleet, Thermo), and the purity (>95%) was analyzed by HPLC (Prominence LC-20A, Shimadzu).

[0131] Example 2: Recombinant CKB protein labeled with covalent peptides

[0132] Preparation of recombinant CKB protein and its active mutants:

[0133] The cDNA encoding CKB WT (with GST- and His-tags) was cloned into the pET-28a vector, and the sequence was verified by sequencing. The resulting plasmid was transformed in E. coli DH5α strain, and single clones were selected, amplified, and extracted. The point mutation plasmid encoding CKB C283S was prepared by circular PCR, with the following primers: forward: 5'-CTGGGCTACATCCTCACAAGCCCATCCAACCTGGGCAC-3' (SEQ ID NO: 5); reverse: 5'-GTGCCCAGGTTGGATGGGCTTGTGAGGATGTAGCCCAG-3' (SEQ ID NO: 6). The plasmid was then transformed into E. coli BL21(DE3) and induced for expression with 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at 30°C for 16 h. Bacteria were lysed by autoclaving in PBS, and the resulting lysates were purified using a Ni-NTA column (HisSep, Yeasen) on an AKTA starter (Cytiva) protein purification system. The elution fractions were analyzed by SDS-PAGE, and the target fractions were combined and concentrated to 2 mg / mL by ultrafiltration (Amicon Ultra, 10 MWCO, Merck). The buffer was then replaced with 10% glycerol / PBS. The resulting recombinant protein was aliquoted and stored at -80°C for later use.

[0134] Recombinant proteins were labeled using covalent peptides and coupled with fluorescent groups.

[0135] Add each covalent peptide to 100 μL of 1 μM recombinant protein to a working concentration of up to 100 μM (PBS as a control), then incubate at 25°C and 1000 rpm for 4 h. After the reaction, add 10 μL of click reaction premix (14.4 mg / mL TCEP : 50 mM CuSO4 : 1.7 mM TBTA = 1:1:3, v:v:v) and 1 μL of 20 mM TAMRA-N3 (Thermo), mix thoroughly, and incubate at 29°C and 1000 rpm for 1 h.

[0136] In-gel ABPP detection probe labeling:

[0137] Add loading buffer to a final concentration of 1×, incubate at 95°C for 10 min, and separate by SDS-PAGE (10% Tris-Gly gel). The gel was imaged using the rhodamine method in a multi-functional imaging system (ChemiDoc MP, Biorad).

[0138] Intact MS characterizes the formation of covalent adducts:

[0139] Add 5 eq. of covalent peptide or an equal volume of PBS to 100 μL of 25 μM recombinant protein solution, followed by ultrafiltration (Vivacon, 10 kDa MWCO, Sartorius) to remove uncovalently bound peptides, and then replace the solvent with 100 μL of pure water. Analyze on UPLC-MS: LC: Acquity UPLC (Waters); column: Aeris 3.6 μm WIDEPORE XB-C8 (Phenomenex); MS: SQ Detector 2 (Waters). The obtained MS spectra were deconvolved using the MaxEnt algorithm.

[0140] LC-MS / MS analysis of the labeling sites of covalent peptides for recombinant CKB:

[0141] Sample pretreatment: Add 5 eq. of covalent peptide or an equal volume of PBS to 100 μL of 25 μM recombinant protein solution, followed by ultrafiltration (Vivacon, 10 kDa MWCO, Sartorius) to remove uncovalently bound peptides, and replace the solvent with 100 μL of 6 M Urea. Add DTT to a final concentration of 10 mM and react at 37 ℃ and 1150 rpm for 30 min. Then, add IAA (iodoacetamide) to a final concentration of 20 mM and react at 37 ℃ and 1150 rpm for 30 min. Finally, dilute with 500 μL of PBS, add 5 μg of trypsin, and digest at 37 ℃ and 1150 rpm for 16 h. After digestion, collect the supernatant and desalt using a C18 column: wash with H2O containing 0.1% FA (formic acid), and elute with 50% ACN (acetonitrile) / H2O (containing 0.1% FA). Combine the eluents and concentrate them by vacuum centrifugation.

[0142] LC-MS / MS method: Dried peptides were dissolved in H2O containing 0.1% FA (mobile phase A) and loaded onto the surface. High-performance liquid chromatography (HPLC) (Ultimate 3000, Thermo) was used for injection, separation in a capillary direct-jet column (15 cm long, 75 μm inner diameter, 360 μm outer diameter, C18 packing material, 3 μm particle size), and detection was performed by high-resolution mass spectrometry (QExactive Plus, Thermo). The effective gradient for LC separation was 60 min, with a gradient of 5–45% mobile phase B (ACN containing 0.1% FA) at a flow rate of 300 nL / min. Mass spectrometry data acquisition used data-dependent (DDA) mode, with the primary ion scan range of 350–1800 m / z, resolution = 70000, AGC = 3E6, and MIT = 50 ms. The secondary spectrum was fragmented using collision-induced dissociation (HCD) mode, with resolution = 17500, TopN = 20, NCE = 28, AGC = 1E5, MIT = 45 ms, isolation window = 1.6 m / z, and dynamic exclusion = 25.0 s. Those skilled in the art can make adaptive adjustments based on the instrument model.

[0143] Data processing: The obtained mass spectrometry data were searched in MaxQuant software (v 2.1.3.0) based on the corresponding species' whole protein database (containing 50% bait sequences) in Uniprot, using the software's default parameters. Since the preferred covalent peptides do not have trypsin recognition sites (K / R), the molecular weight of the covalent peptides was used as a fixed modification on cysteine ​​residues for parameter settings during data processing. The obtained MS / MS spectra were visualized using pLabel (v.2.4.3.0).

[0144] This embodiment evaluated the labeling ability of the designed covalent peptide CKB-5 for recombinant CKB and verified its specificity for the Cys283 site. The test results are as follows: Figure 3 .

[0145] The results show that among the designed and screened artificial covalent peptides, all probes CKB-1 to CKB-5 with chloroacetyl groups as covalent warheads can effectively label recombinant CKB protein. CKB-5, designed using the optimal sequence of this application, produced a strong and dose-dependent labeling effect on recombinant CKB, reaching near saturation at a concentration of approximately 50 μM. This labeling dependence on the Cys283 active site was confirmed by the CKB-C283S mutant and SDS denaturation assays. Furthermore, intimate MS results showed that the preferred compound CKB-5 forms a 1:1 covalent adduct with recombinant CKB protein, and LC-MS / MS results confirmed that the labeling of recombinant CKB protein by CKB-5 strictly occurs at the Cys283 site designed and expected in this application.

[0146] Example 3: Labeling of CKB in mouse brain lysate using covalent peptides

[0147] Preparation of mouse tissue lysates:

[0148] Untreated adult male C57BL6 / J mice were euthanized by cervical dislocation and rapidly harvested on ice. Brain tissue samples were immediately added to ice-cold PBS buffer containing a 1× protease inhibitor mixture (COPLETE Tablet, Roche) and homogenized in an automated homogenizer (TissueLyser II, Qiagen) (2 Hz, 90 s, 5 cycles). The resulting homogenate was centrifuged at 20000×g for 10 min, and the supernatant was collected. Protein concentration was determined by the BCA method and adjusted to 2 mg / mL. The homogenate was then aliquoted and stored at -80°C for later use.

[0149] Labeling and fluorescent conjugation of CKB in mouse brain tissue lysates:

[0150] To 100 μL (2 mg / mL) of the above-mentioned mouse brain lysate, the covalently involved peptide designed in this application was added to a final concentration of 200 μM (PBS was used as a blank control), and the mixture was incubated at 25°C and 1000 rpm for 4 h with shaking. After the reaction was complete, the protein was precipitated using methanol-chloroform precipitation to thoroughly wash away unbound free peptides and small molecules. After washing with cold methanol, the protein was reconstituted in 100 μL of 0.4% SDS / PBS buffer. Subsequently, 10 μL of click chemistry premix (composition: 14.4 mg / mL TCEP (tris(2-carboxyethyl)phosphine): 50 mM CuSO4: 1.7 mM TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) = 1:1:3, v:v:v) and 1 μL of 20 mM TAMRA-N3 fluorescent dye were added. After thorough mixing, the mixture was incubated at 29°C and 1000 rpm for 1 h. After the reaction, excess fluorescent dye was removed again by methanol-chloroform precipitation, and the solution was reconstituted in 100 μL of 1.2% SDS / PBS for later use.

[0151] In-gel ABPP detection and immunoblotting verification:

[0152] Add loading buffer to the reconstituted sample to a final concentration of 1×, boil at 95°C for 10 min for denaturation, and separate by SDS-PAGE (10% Tris-Gly gel). The gel is placed in a multi-functional imager (ChemiDoc MP, Biorad) and the rhodamine channel is used for imaging to acquire the fluorescent signal of the probe label. After imaging, or after separation by SDS-PAGE of parallel prepared gels, the gels are electroblotted onto a 0.45 μm PVDF membrane (Millipore). Block with 5% BSA for 30 min, followed by overnight incubation at 4°C with a specific primary antibody targeting CKB (abcam ab92452). After washing, incubate with HRP-conjugated secondary antibody (CST #7074) at room temperature for 30 min. After thorough washing, add chemiluminescent developing solution (Clarity, Biorad), and perform chemiluminescent imaging in the imager. Precisely align the molecular weight and intensity of the fluorescent bands with the immunoblot bands.

[0153] This embodiment evaluated the labeling strength and specificity of each designed covalent peptide for endogenous CKB in mouse brain lysate. The test results are as follows: Figure 4 .

[0154] The results show that among the designed covalent peptides, peptides CKB-1, 2, 4, and 5, with chloroacetyl groups as covalent warheads, can specifically label proteins at approximately 50 kDa in mouse brain lysates in a concentration-dependent manner. This result is consistent with the molecular weight of CKB. CKB-5 showed the highest labeling intensity, approaching saturation at 6.25–12.5 μM. Immunoblot experiments further confirmed the specific labeling of endogenous CKB by CKB-5. This indicates that CKB-5 provided in this application can not only serve as an effective binding agent for CKB but also as a specific chemical probe to characterize the enrichment and distribution of CKB proteins in complex biological samples.

[0155] Example 4: Confirmation of the selectivity of covalent peptides in the whole proteome using chemical proteomics and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0156] Labeling and biotin conjugation of CKB in complex tissue lysates:

[0157] A covalently substituted peptide was added to 500 μL of 2 mg / mL mouse brain lysate to a final concentration of 10 μM (using an equal volume of DMSO as a control), followed by incubation at 25°C and 1000 rpm for 4 h. After the reaction, small molecules were removed by methanol-chloroform precipitation, followed by washing with cold methanol and reconstitution in 500 μL of 0.4% SDS / PBS. 50 μL of click reaction premix (14.4 mg / mL TCEP : 50 mM CuSO4 : 1.7 mM TBTA = 1:1:3, v:v:v) and 5 μL of 20 mM Biotin-N3 (Thermo) were added to the reconstituted mixture, and after thorough mixing, incubation was performed at 29°C and 1000 rpm for 1 h. After the reaction, small molecules were again removed by methanol-chloroform precipitation, followed by washing with cold methanol and reconstitution in 100 μL of 1.2% SDS / PBS.

[0158] Enrichment of biotinylated protein targets:

[0159] The reconstituted mixture was heated at 90°C for 5 min, centrifuged at 20000×g for 10 min to remove copper, and the supernatant was collected for later use. Separately, 50 μL of wet-volume streptavidin agarose beads (Thermo) were washed according to the manufacturer's instructions (1700×g, 3 min). The washed agarose beads were resuspended in 2.5 mL of PBS, followed by the addition of the supernatant, and incubated at 30°C for 4 h. After complete binding, the agarose beads were washed once, three times, and three times with 0.2% SDS / PBS, PBS, and water, respectively, and residual washings were aspirated.

[0160] On-bead digestion and sample pretreatment for chemical proteomics:

[0161] Resuspend the washed agarose beads in 6M urea / PBS, add DTT to a final concentration of 10 mM, and react at 37 °C and 1150 rpm for 30 min. Then, add IAA to a final concentration of 20 mM and react at 37 °C and 1150 rpm for 30 min. Wash three times with PBS and aspirate the wash buffer. Resuspend the agarose beads in 1 M urea / PBS, then add 2 μg trypsin and digest at 37 °C and 1150 rpm for 16 h. After digestion, collect the supernatant and desalt using a C18 microcentrifuge column: wash with H2O containing 0.1% FA, and elute with 50% ACN / H2O (containing 0.1% FA). Combine the eluents and concentrate by vacuum centrifugation for later use.

[0162] LC-MS / MS mass spectrometry data acquisition:

[0163] The dried peptide fragments were dissolved in H2O containing 0.1% FA (mobile phase A) and loaded onto the surface. High-performance liquid chromatography (HPLC) was used for injection, separation in a capillary direct-jet column (15 cm long, 75 μm inner diameter, 360 μm outer diameter, C18 packing material, 3 μm particle size), and detection was performed by high-resolution mass spectrometry (QExactive Plus, Thermo). The effective gradient for HPLC separation was 60 min, with a gradient of 5–45% mobile phase B (ACN containing 0.1% FA) at a flow rate of 300 nL / min. Mass spectrometry data acquisition was performed in data-dependent (DDA) mode, with the primary ion scan range of 350–1800 m / z, resolution = 70000, AGC = 3E6, and MIT = 50 ms. The secondary spectra were fragmented using collision-induced dissociation (HCD) mode, with resolution = 17500, TopN = 20, NCE = 28, AGC = 1E5, MIT = 45 ms, isolation window = 1.6 m / z, and dynamic exclusion = 25.0 s. Those skilled in the art can routinely adjust the above chromatographic and mass spectrometric parameters according to actual analytical needs and equipment model.

[0164] Mass spectrometry data processing and label-free quantitative analysis:

[0165] The obtained mass spectrometry data were searched in MaxQuant software (v 2.1.3.0) based on the Uniprot mouse whole protein database (containing 50% bait sequences) and quantified using the label-free quantification (LFQ) method with the software's default parameters. The quantification results were processed for missing values ​​and visualized using the R (v 4.4.1) package DEP (v 3.19), with the MinDet interpolation algorithm. The screening threshold for differentially expressed proteins was |FC| > 2 and p < 0.05.

[0166] This embodiment evaluated the selectivity of the preferred compound CKB-5 in the whole proteome derived from mouse brain lysate, and the test results are as follows: Figure 5 .

[0167] The results show that, with the DMSO-treated group as the control, CKB-5 exhibits excellent selectivity for labeling CKB in the whole proteome of mouse brain tissue lysate, and can most significantly enrich CKB protein, while no off-target enrichment of other CK family subtypes (such as mitochondrial subtype CKMT1 / 2 or muscle subtype CKM) was observed.

[0168] Example 5: Using covalent peptides as inhibitors to achieve CKB activity inhibition.

[0169] Enzyme activity test method:

[0170] This embodiment uses the ADP-Glo ​​(Promega) method to test the activity of CKB in catalyzing the phosphorylation of its substrate creatine. After reacting the covalently bound peptide with 1 μM recombinant protein, uncovalently bound peptides were removed by ultrafiltration (Vivacon, 10 kDa MWCO, Sartorius), and then diluted to 13.3 nM in test buffer as the sample (with a CKB-free test buffer as a blank control). Subsequently, following the manufacturer's instructions, 2 μL of 1 mM creatine and 5 μL of 500 μM ATP were added to 20 μL of the above sample, and the reaction was allowed to proceed for 20 min. After the reaction, ATP was quenched with ADP-Glo, and ADP luminescence was catalyzed using KDR. The luminescence intensity was read using a multi-functional microplate reader (SynergyH1, BioTek), and the obtained data were fitted using GraphPad software (v8.0).

[0171] Determination of the half-maximal inhibitory concentration (IC50):

[0172] A preferred covalent peptide CKB-5 concentration gradient (e.g., 0 to 200 μM) was set up in this embodiment of the application and co-incubated with recombinant CKB protein (final concentration approximately 13.3 nM) at 37 °C for 4 h. Three biological replicates were set up for each concentration gradient. After incubation, the residual enzyme activity of CKB was read using the enzyme activity assay method described above, and a covalent peptide concentration-activity response curve was plotted. The half-maximal inhibitory concentration (IC50) of the covalent peptide was calculated by nonlinear regression fitting.

[0173] Second-order rate constant k of covalent reaction inact / K I Measurement:

[0174] A reaction time gradient from 0 min to 120 min was set; for each time point, a covalent peptide concentration gradient from 0 μM to 100 μM was set, with three biological replicates per group. After incubation, the residual enzyme activity of CKB was detected using the aforementioned enzyme activity assay method. For each covalent peptide concentration, a scatter plot of time versus the natural logarithm of residual activity ln(activity%) was plotted and linearly fitted; the absolute value of the slope of the resulting line was the inactivation rate constant k. obs Subsequently, the concentration-k of the covalent peptide was plotted. obs The curve is obtained by fitting k using the Michaelis-Menten equation. inact K I and their ratios.

[0175] This embodiment evaluated the inhibitory effect of the preferred covalent peptide CKB-5 as a covalent inhibitor on CKB activity. The test results are as follows: Figure 6 .

[0176] Enzyme activity and covalent reaction kinetics assays showed that CKB-5 exhibited strong inhibitory activity against recombinant CKB, with an IC50 of 2.307 ± 1.030 μM, while the second-order rate constant of the covalent reaction, k... inact / K I It can reach 53.242 ± 3.707 M. -1 s -1 This indicates that CKB-5, as an artificial heptapeptide designed entirely from scratch through computation, can achieve potent, rapid, and irreversible covalent inhibition of CKB kinase. It not only effectively blocks the classic metabolic enzyme activity of CKB, but the steric hindrance generated by its substrate binding groove also provides a solid foundation for blocking the interaction between CKB and other downstream proteins (such as GPX4), and has great potential for development into a long-acting targeted inhibitor.

[0177] In summary, this application, based on a combination of deep learning and computer-aided geometric hashing algorithms, overcomes the dependence on natural peptide ligands and successfully develops a series of novel covalent peptide inhibitors targeting CKB. Recombinant protein characterization, kinetic measurements, and mass spectrometry analysis of the whole proteome of complex mouse tissues confirm that the novel peptide inhibitors provided in this application overcome the inherent defects of existing pancreatine kinase small molecule inhibitors (such as CTi), which are prone to intrafamily subtype cross-reactivity and off-target effects. While achieving highly selective binding to the CKB substrate binding groove, it also achieves precise and irreversible covalent modification of the highly reactive site Cys283 near the substrate binding groove. Furthermore, this application not only achieves potent and sustained inhibition of CKB enzyme activity, but the steric hindrance occupied by its binding also provides a new tool for blocking protein-protein interactions between CKB and downstream substrates (such as GPX4). Simultaneously, it can also serve as a specific chemical probe to characterize the enrichment and distribution of CKB proteins in complex biological systems, possessing broad application prospects and translational value.

[0178] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0179] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A covalent peptide inhibitor targeting creatine kinase B, characterized in that, The general formula of the covalent peptide inhibitor is Z1-X1-X2-X3-X4-X5-X6-X7-Z2, wherein X1-X2-X3-X4-X5-X6-X7 is an amino acid sequence, and X1 is selected from methionine or its conserved alternative amino acids. X2 is selected from small-sized amino acid residues or negatively charged amino acid residues; X3 is selected from small-sized amino acid residues or hydrophobic amino acid residues; X4 is selected from small-sized amino acid residues or charged amino acid residues; The molecular weight of the small-sized amino acid residues is less than 100 Da; X5 is a non-natural amino acid with a covalently reactive group, which can covalently crosslink with the Cys283 residue on creatine kinase B. X6 is selected from negatively charged amino acid residues; X7 is selected from negatively charged amino acid residues or hydrophobic amino acid residues. Z1 is selected from hydrogen, acyl, fluorescent group, biotin, isotope tag or bioorthogonal reactive group; Z2 is selected from hydroxyl, amino, cell-penetrating peptide or stabilizing modification groups.

2. The covalent peptide inhibitor targeting creatine kinase B as described in claim 1, characterized in that, The covalent reactive group includes any one of chloroacetamide, acrylamide, fluorosulfate, vinyl sulfone, and sulfonyl fluoride. Optionally, X5 is selected from any one of 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, and ornithine, whose side chains are modified with chloroacetamide or acrylamide groups.

3. The covalent peptide inhibitor targeting creatine kinase B as described in claim 1, characterized in that, One or more of the following conditions must be met: (i) X2, X3 and X4 are each independently selected from any one of serine, glycine, alanine and glutamic acid; (ii) The X6 is selected from either glutamic acid or aspartic acid; and, (iii) X7 is selected from any one of aspartic acid, leucine and methionine.

4. The covalent polypeptide inhibitor targeting creatine kinase B as described in any one of claims 1 to 3, characterized in that, The amino acid sequence is selected from any of the following: (1) Met-Glu-Ser-Ala-X5-Glu-Asp; (2) Met-Ser-Ser-Gly-X5-Asp-Leu; (3) Met-Pro-Leu-Asp-X5-Asp-Leu; (4) Met-Ala-Ala-Arg-X5-Glu-Met; X5 is 2,3-diaminopropionic acid with a chloroacetamide group modified on the side chain.

5. The covalent peptide inhibitor targeting creatine kinase B as described in claim 4, characterized in that, Z1 is 5-hexyneylyl, and Z2 is amino. Optionally, the sequence of the covalent peptide inhibitor is 5-hexynyl-Met-Glu-Ser-Ala-Dap(ClAc)-Glu-Asp-NH2, wherein Dap(ClAc) is 2,3-diaminopropionic acid with a chloroacetamide group modified on the side chain. Optionally, the structural formula of the covalent peptide inhibitor is shown below: 。 6. A coupling agent, characterized in that, The conjugate includes the covalent peptide inhibitor according to any one of claims 1 to 5, and a carrier and / or reporter tag attached to the covalent peptide inhibitor; Optionally, the report label includes one or more of rhodamine-based fluorescent dyes, cyanine-based fluorescent dyes, and biotin and its derivatives; the carrier includes one or more of cell-penetrating peptides and liposomes.

7. A reagent kit, characterized in that, The kit comprises the covalent polypeptide inhibitor according to any one of claims 1 to 5 or the conjugate according to claim 6; Optionally, the kit may also include a protease inhibitor.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the covalent polypeptide inhibitor according to any one of claims 1 to 5 or the conjugate according to claim 6; Optionally, pharmaceutically acceptable excipients may also be included.

9. The use of the covalent peptide inhibitor according to any one of claims 1 to 5 or the conjugate according to claim 6 in the preparation of products for the prevention and / or treatment of creatine kinase B-mediated diseases; Optionally, the creatine kinase B-mediated diseases are energy metabolism disorders or ferroptosis-related diseases; Further optionally, the creatine kinase B-mediated diseases include one or more of the following: aggressive acute myeloid leukemia, liver cancer, breast cancer, pancreatic cancer, and adaptive thermogenic metabolic syndrome.

10. The use of the covalent peptide inhibitor according to any one of claims 1 to 5 or the conjugate according to claim 6 in any of the following aspects: (1) Application in the preparation of in vitro biochemical reagents or tool drugs for specifically binding to and inhibiting the activity of creatine kinase B; (2) Application in the preparation of chemical proteomics probes for the specific labeling, enrichment and quantification of active creatine kinase B protein in complex whole proteome samples; (3) Application in the preparation of regulators for blocking protein-protein interactions between creatine kinase B and glutathione peroxidase 4.

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