Kit, polypeptide targeting P53 and its application in the preparation of drugs for treating cancer

Abstract

The present invention provides a kit, a polypeptide targeting P53, and its application in the preparation of a drug for treating cancer. The polypeptide specifically binds to the P53 mutant, and the polypeptide is selected from any one or more of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NOs: 17 to 22. By means of AI-assisted design and screening, it is verified that the above polypeptide has the ability to specifically target and bind to the P53 mutant and has significant anti-breast cancer cell activity.

Description

Technical Field

[0001] The present invention relates to the field of anti-cancer drug development, and in particular, to a kit, a polypeptide targeting p53, and their applications in the preparation of drugs for treating cancer. Background Art

[0002] p53 is a collective term for a series of homologous isomeric proteins known as tumor suppressor proteins (also known as p53 protein or p53 tumor protein). p53 is a tumor suppressor protein and transcription factor that can respond to cellular stress or DNA damage and activate a variety of transcriptional targets. p53 can coordinate a variety of responses, including cell cycle arrest, DNA repair, metabolic changes, antioxidant effects, anti-angiogenic effects, autophagy, senescence, and apoptosis. The p53 protein can maintain the stability of the genome, avoiding or reducing the occurrence of mutations, and is therefore known as the guardian of the genome.

[0003] The p53 protein plays an important role in the mechanism of preventing cancer. For example, apoptosis, cell senescence, genetic stability, and inhibition of angiogenesis. Mutations in TP53 (the gene encoding p53) are the most common gene mutations in cancer, and partial or complete loss of function occurs in more than 50% of tumors. p53 mutations provide a selective advantage for tumor cells, enabling them to bypass cell cycle checkpoints, avoid apoptosis and senescence, and proliferate under conditions where normal cells cannot proliferate. The mutated p53 protein may lose the ability to effectively bind to DNA, resulting in the inability to form the p21 protein to send a signal to stop cell division. Therefore, damaged cells will divide uncontrollably and eventually form tumors, such as breast cancer.

[0004] Breast cancer is one of the most common malignant tumors in women. According to the latest global cancer burden data released by the International Agency for Research on Cancer (IARC) of the World Health Organization in 2020, the number of newly diagnosed breast cancers worldwide reached 2.26 million in 2020, exceeding lung cancer (2.21 million cases) for the first time to become the world's largest cancer. China is a major country in breast cancer. In 2020, there were approximately 420,000 newly diagnosed breast cancers, resulting in nearly 120,000 deaths, usually occurring in the breast glandular epithelial tissue. Breast cancer is more common in women, and male breast cancer accounts for 0.5% to 1% of all breast cancer patients. The treatment methods for breast cancer include surgical resection, radiotherapy, chemotherapy, and hormone therapy, etc. In recent years, molecular targeted therapy, as a new means of breast cancer treatment, has shown certain efficacy in breast cancer treatment and has been increasingly valued by the academic community.

[0005] Molecular targeted therapy uses specific gene fragments in tumor cells as therapeutic sites and aims to treat diseases by regulating or blocking the functions of these gene fragments. Breast cancer molecular targeted therapy refers to the treatment targeting the signal pathways related to the occurrence and development of breast cancer and their oncogene-related expression products. Molecular targeted drugs control the changes in cell gene expression by blocking the signal transduction of tumor cells or related cells, thereby inhibiting or killing tumor cells. Targeted therapy has strong specificity, significant effects, and basically does not damage normal tissues. Therefore, tumor targeted therapy is the most promising approach in tumor treatment.

[0006] A polypeptide is a molecular compound composed of three or more amino acid molecules linked by peptide bonds. As one of the important substances of life, peptide substances widely exist in living organisms to regulate the functional activities of various systems, organs, and cells in the body. In recent years, polypeptide drugs have attracted much attention in the development of anti-tumor drugs due to their advantages such as high targeting, low immunogenicity, high tissue permeability, and safety. In 2019, the global market valuation of anti-tumor polypeptide therapy was 8.6 billion US dollars and is expected to maintain a stable growth trend in the next decade. As a small molecule compound, polypeptides can effectively bind to p53 mutant proteins, thereby restoring the normal function of p53 proteins and inhibiting the occurrence of cancer. Therefore, developing effective polypeptide drugs that can restore the activity of P53 mutant proteins not only promotes the research on tumor growth but also has very important clinical value.

[0007] Therefore, how to develop a batch of polypeptide drug molecules with potential medicinal value targeting P53 as an anti-cancer target has also become a hot issue in the development of anti-tumor drugs. Summary of the Invention

[0008] The main object of the present invention is to provide a kit, a polypeptide targeting P53, and its application in the preparation of drugs for treating cancer, so as to provide more polypeptide drug molecules with potential medicinal value for the development of anti-cancer drugs.

[0009] To achieve the above object, according to one aspect of the present invention, there is provided a polypeptide targeting P53, which specifically binds to P53 mutants, and the polypeptide is selected from any one or more of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0010] Further, the P53 mutant is a mutant selected from at least one of the following site mutations: S121F, V122G, C135V, C141V, W146Y, C182S, V203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268D, E294M or S315R; preferably, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification; preferably, the chemical group modification is any one or more of PEG modification, acetylation modification or amidation modification; preferably, the PEG modification is a linear PEG modification, a PEG modification with a single functional group or a PEG modification with a double functional group; preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain and Cys side chain of the polypeptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40,000; preferably, the N-terminus and C-terminus of the polypeptide have acetylation modification and amidation modification respectively; preferably, the N-terminus of the polypeptide has a fatty acid modification, more preferably a Myr modification; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine modification; preferably, the hydrophilic amino acid modification is adding 1-4 hydrophilic amino acids at the N-terminus, C-terminus or both NC termini of the polypeptide, more preferably, the hydrophilic amino acids are Glu, Lys, Ser or Gly; further preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys or Ser-Gly-Ser; preferably, the cysteine modification is adding cysteine at any one of the following positions of the polypeptide: N-terminus, C-terminus, both NC termini or the middle of the peptide chain; more preferably, adding cysteine in the middle of the peptide chain includes inserting one or more cysteines in the middle of the peptide chain, or one or more cysteines are connected to the middle of the peptide chain in a branched form; preferably, the polypeptide exists in the form of a linear polypeptide with a cell-penetrating peptide or a cyclic polypeptide without a cell-penetrating peptide; preferably, the linear polypeptide with a cell-penetrating peptide is selected from any one or more of the following: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0011] To achieve the above object, according to the second aspect of the present invention, there is provided a polypeptide drug, which includes any one of the above polypeptides and a pharmaceutically optional excipient.

[0012] Further, the polypeptide drug is an anticancer drug, preferably an anti-breast cancer drug; preferably, the concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM.

[0013] According to the third aspect of the present invention, there is provided an application of the above-mentioned polypeptide in the preparation of a medicament for treating cancer.

[0014] Further, the cancer includes any one or more of the following: breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterine cancer, ovarian cancer, liposarcoma, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymoma, and central nervous system cancer; preferably, the central nervous system cancer is brain cancer.

[0015] In order to achieve the above object, according to one aspect of the present invention, there is provided a kit for detecting P53 mutants, which kit includes the above-mentioned polypeptide.

[0016] Further, the polypeptide exists in the form of a polypeptide-protein conjugate; preferably, the protein in the polypeptide-protein conjugate is selected from any one of the following: bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, or casein; more preferably, the polypeptide is conjugated to the protein through a linking sequence to form a polypeptide-protein conjugate, and further preferably, the linking sequence is CGSG.

[0017] Further, the polypeptide is coated on a solid-phase carrier; preferably, the solid-phase carrier includes an enzyme-linked immunosorbent assay (ELISA) plate, a membrane carrier, or microspheres; preferably, the membrane carrier includes a nitrocellulose membrane, a glass fiber membrane, or a nylon membrane; preferably, a positive control is also coated on the membrane carrier, and the polypeptide and the positive control are sequentially arranged on the membrane carrier according to the detection sequence; preferably, the kit further includes at least one of the following: (1) an enzyme-labeled secondary antibody, and more preferably the enzyme-labeled secondary antibody is an HRP-labeled secondary antibody; (2) a colloidal gold conjugate pad, on which a specific binding substance of colloidal gold-labeled antigen and the positive control is coated; (3) a labeled pad, on which fluorescently labeled microspheres are coated, and the microspheres are loaded with a specific binding substance of the positive control; preferably, the positive control is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin, or rabbit immunoglobulin, and correspondingly, the specific binding substance of the positive control is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin, or anti-rabbit immunoglobulin.

[0018] Further, the kit includes a chip, on which a polypeptide array composed of polypeptides is pre-set.

[0019] Applying the technical solution of the present invention, by using the P53 mutant protein as the target protein of the anti-cancer drug and combining the means of AI-assisted design and screening, a number of novel polypeptide compounds with anti-cancer activity have been found. These polypeptides have the advantages of specific targeting, low toxicity and low side effects as drugs, and thus have potential application value for developing into anti-cancer drugs. In particular, the polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, when used in the form of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16 with a cell-penetrating peptide for cell experiments, were found to have the highest anti-cancer activity. For example, their activity against breast cancer is higher than that of other polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0021] Figure 1 Shows a box plot of the binding affinity between the candidate polypeptides screened in the embodiments of the present application and the P53 mutant;

[0022] Figures 2a to 2g Respectively show the HPLC chromatograms of the synthesis of the polypeptides shown in SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16 in the embodiments of the present application;

[0023] Figures 3a to 3g Respectively show the MTT result graphs of the proliferation effects of each candidate anti-cancer polypeptide on MDA-MB-231 cells in the embodiments of the present application;

[0024] Figures 4a to 4g Respectively show the effect graphs of the inhibitory effects of the polypeptides shown in SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16 on the proliferation of MDA-MB-231 cells in the embodiments of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0026] Term Explanation:

[0027] Polypeptide: In the present application, it refers to any peptide segment that can specifically bind to the target P53 mutant, which is predicted or screened.

[0028] Polypeptide-carrier protein conjugate: In the present application, it refers to a conjugate formed by coupling a polypeptide with a carrier protein. Among them, one carrier protein can be coupled with one or more polypeptides. When multiple polypeptides are coupled, the multiple polypeptides have the same amino acid sequence. According to the differences in the physicochemical properties of the specifically coupled polypeptide sequences, the types of specific carrier proteins, and the different coupling methods, the number of polypeptides coupled to each carrier protein varies. In the present application, it is preferably 2 - 50, more preferably 3 - 45, 5 - 40, 5 - 35, 5 - 30, 8 - 30, 10 - 30, 12 - 30, 15 - 30; or, more preferably, any one of 6 - 36, 8 - 32, 10 - 28, 10 - 26, 10 - 24, 10 - 22, 10 - 20, 10 - 18, 10 - 16, and 10 - 15.

[0029] The polypeptide chip technology is a detection technology based on the polypeptide chip. It utilizes the contact between the various polypeptides on the polypeptide chip and the sample, and then uses image acquisition technology to collect each characteristic signal on the polypeptide chip (specifically, it can be manifested as a fluorescence image carrying each characteristic signal), and then outputs the signal intensity of each characteristic in the chip, that is, the polypeptide chip detection result data. Based on the sample detection signal output from the polypeptide chip detection result data, the analysis of the analyte in the sample that binds to the polypeptides on the polypeptide chip, the analysis of the sample, etc. can be realized.

[0030] Motif: Motif. In biology, it is a mathematical statistical model based on data. Typically, it is a segment of sequence (Sequence), or it can also be a structure. It is the sequence prediction of a specific group (group). For example, a DNA sequence can be defined as a transcription factor binding site, that is, the sequence tends to be bound by transcription factors. For proteins, the sequence motif can be defined as the protein sequence belonging to a given protein family. A simple motif can be, for example, a pattern, and this pattern is shared by all members of this group (group).

[0031] As mentioned in the background art, there is an urgent need in the prior art to develop anti-cancer polypeptide drugs targeting different targets. In the present invention, P53 is selected as the target protein of the anti-cancer drug, and through the combination of AI computational analysis and functional verification, anti-cancer active polypeptide drugs are rapidly screened out. The iCXOncP2a polypeptide screened out in the present invention shows a significant effect of inhibiting the growth of breast cancer cells, which can provide ideas for the discovery of new active compounds in subsequent drug research and development. At the same time, the novel anti-cancer polypeptide sequence provided by us can be used as a lead compound for subsequent drug research and development.

[0032] Based on the above research results, the applicant has proposed a series of technical solutions for this application. In a typical embodiment, a polypeptide targeting P53 is provided, which specifically binds to the P53 mutant, and the polypeptide is selected from any one or more of the following: RRRLIHNRHY (SEQ ID NO: 1), RRHNTPHPRL (SEQ ID NO: 2), RRRPILTIITLE (SEQ ID NO: 3), RRVFIRFWHN (SEQ ID NO: 17), RRAFLRLNHNKLS (SEQ ID NO: 18), RRHSIPHPEYR (SEQ ID NO: 19), RRAFIRLNYNK (SEQ ID NO: 20), RRLYVRLSHNS (SEQ ID NO: 21), RRHNRHSPEPK (SEQ ID NO: 22).

[0033] As described above, in this application, by using the P53 mutant protein as the target protein of the anti-cancer drug and combining AI-aided design and screening methods, the above novel polypeptide compounds with anti-cancer activity are found. These polypeptides have the advantages of specific targeting, low toxicity and low side effects as drugs. To a certain extent, it makes up for the deficiencies of current treatment methods. At the same time, it has an important enlightenment effect on the research of drugs targeting the p53 mutant protein as an anti-cancer target. Among the above polypeptides, the compounds shown in SEQ ID NO: 1 and SEQ ID NOs: 17 to 22 are preferred, and these compounds have the highest anti-cancer activity. For example, their anti-breast cancer activity is higher than that of other polypeptides.

[0034] The above P53 mutant is any mutant that can cause the loss of its tumor suppressor function. In this application, it includes, but is not limited to, mutants with at least one site mutation as follows: S121F, V122G, C135V, C141V, W146Y, C182S, V203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268D, E294M or S315R.

[0035] Polypeptides have a significant structural advantage. That is, without affecting the original functional polypeptide fragments, new functional chemical modifications can be introduced at one or both ends of the polypeptide through solid-phase synthesis or biosynthesis to obtain multifunctionality. The mechanism by which this multifunctional chemical modification enables pH-sensitive liposomes with receptor / ligand binding ability to enter cells will induce the active endocytosis of liposomes by tumor tissue cells and then release drugs. Common polypeptide modifiers can be classified into four categories according to the modification sites: C-terminal modification (amidation, sulfation, etc.), N-terminal modification (acetylation, fatty acylation, etc.), middle residue modification (glycosylation modification combined with Ser-, Tyr-, Asn-, Thr-; phosphorylation modification combined with Ser-, Tyr-, Thr-, etc.), and cyclization modification.

[0036] Therefore, according to the requirements of drug development, the above polypeptides can also be variously modified as needed during development. In a preferred embodiment, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification.

[0037] For example, to further improve the affinity of some peptide segments, in a preferred embodiment, the above chemical group modification is PEG modification; preferably, the PEG modification is linear PEG modification, PEG modification with a single functional group, or PEG modification with a double functional group; preferably, the site of PEG modification is selected from any one or more of the N-terminus, C-terminus, Lys side chain, and Cys side chain of the polypeptide; preferably, the PEG modification has a molecular weight of 500 - 40,000; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine modification; preferably, the hydrophilic amino acid modification is adding 1 - 4 hydrophilic amino acids at the N-terminus, C-terminus, or both NC termini of the polypeptide, and more preferably, the hydrophilic amino acids are Glu, Lys, Ser, or Gly; further preferably, the 1 - 4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys, or Ser-Gly-Ser.

[0038] Since chemically synthesized polypeptides often carry free amino groups and free carboxyl groups, and the polypeptide sequence often represents the sequence of its parent protein, in order to make the synthesized protein closer to the parent protein in terms of sequence and activity, the ends of the polypeptide are usually blocked, generally by acetylating the N-terminus and amidating the C-terminus. Such modification will reduce the total charge of the polypeptide and lower its solubility, and thus can also enable the polypeptide to mimic the original state of the α-amino group and carboxyl group in its parent protein. Therefore, in some other embodiments, the above chemical group modification is double-end modification of the polypeptide, and more preferably, the N-terminus and C-terminus of the polypeptide are respectively acetylated and amidated.

[0039] In some embodiments, in order to better achieve the directional coupling of peptide segments, it is preferably possible to perform cysteine modification on the polypeptide. Specifically, this includes but is not limited to adding cysteine at the N-terminus, C-terminus, or both the N- and C-termini of the peptide segment, or adding cysteine in the middle of the peptide chain of the polypeptide. When adding cysteine in the middle of the peptide chain of the peptide segment, one or more cysteines can be inserted into the middle of the peptide chain (i.e., inserted between two amino acid residues), or one or more cysteines can be connected to the middle of the peptide chain in a branched form (i.e., as the side chain of a certain amino acid in the middle of the peptide chain).

[0040] It should be noted that the above methods for modifying polypeptides can use the most mature and widely used chemical modification methods currently, including liquid phase methods and solid phase methods.

[0041] In some embodiments, in order to more effectively utilize the above polypeptide molecule as a drug to penetrate the cell membrane and enter the body to target and bind to the P53 mutant, preferably, the polypeptide exists in the form of a linear polypeptide with a cell-penetrating peptide or a cyclic polypeptide without a cell-penetrating peptide. The cell-penetrating peptide is usually set at the C-terminus of the polypeptide or one end of the drug molecule, and can be used to bring the polypeptide molecule or drug molecule into a specific type of cell, and then the molecule undergoes biochemical reactions within the cell. The cell-penetrating peptide is usually rich in basic amino acids such as arginine (Arg) or lysine (Lys). Examples of relatively commonly used cell-penetrating peptides are as follows: (1) H7R8: HHHHHHHHRRRRRRRR (SEQ ID NO: 7); (2) YGRKKRRQRRR (SEQ ID NO: 8); or (3) GRRRRRRRRRPPQ (SEQ ID NO: 9).

[0042] Preferably in this application, the amino acid sequences of the linear polypeptides with cell-penetrating peptides are myr-RRRLIHNRHY (SEQ ID NO: 4), myr-RRHNTPHPRL (SEQ ID NO: 5), myr-RRRPILTIITLE (SEQ ID NO: 6), myr-RRVFIRFWHN (SEQ ID NO: 11), myr-RRAFLRLNHNKLS (SEQ ID NO: 12), myr-RRHSIPHPEYR (SEQ ID NO: 13), myr-RRAFIRLNYNK (SEQ ID NO: 14), myr-RRLYVRLSHNS (SEQ ID NO: 15), or myr-RRHNRHSPEPK (SEQ ID NO: 16).

[0043] In the second typical embodiment of the present application, a polypeptide drug is provided, and the polypeptide drug includes any one of the above polypeptides and pharmaceutically optional excipients. The drug containing the above polypeptide molecule has the advantages of high ability to target and bind to the P53 mutant, high anti-cancer activity, low toxicity and low side effects.

[0044] The above pharmaceutically optional excipients may vary according to the dosage form and / or administration method and administration route of the drug to be prepared, and can be reasonably selected from existing pharmaceutical excipients. They include, but are not limited to, pharmaceutically acceptable carriers, excipients or adjuvants and other excipients. Moreover, the polypeptide drug can be made into different dosage forms to adapt to diverse administration routes. For example, the administration routes include oral administration, injection administration or transdermal administration, etc. And the dosage forms of the drug include capsules, tablets, oral liquids, injections or transdermal absorbents, etc.

[0045] Specifically, the drugs of the present application can be made into capsules, tablets, oral liquids, injections or transdermal absorbents, etc. according to methods known in the pharmaceutical industry. Preferably, the injection is an intravenous injection. When preparing capsules, tablets and oral liquids suitable for oral administration, sucrose, lactose, galactose, corn starch, gelatin, microcrystalline cellulose, carboxymethyl cellulose, etc. can be used as carriers or excipients. In addition, the drugs of the present application can also be made into solutions and suspensions suitable for oral administration using methods and auxiliary components known in the pharmaceutical industry. If preparing solutions and suspensions suitable for parenteral administration, distilled water, water for injection, isotonic sodium chloride or glucose solution, or low-concentration (such as 1-100 mM) phosphate buffer solution (PBS) can be used as carriers or diluents. One or more other auxiliary components or additives can be added to these parenteral preparations. For example, ascorbic acid can be used as an antioxidant, and sodium benzoate, etc. can be used as preservatives. Other solubilizers, disintegrants, colorants, dispersants or surfactants can also be contained in the preparations of these dosage forms.

[0046] Since the above polypeptide drug has anti-cancer activity, it can be used as an anti-cancer drug, preferably an anti-breast cancer drug. In some embodiments, the polypeptide exists in the form of a linear polypeptide with a cell-penetrating peptide to further improve the transmembrane performance of the polypeptide drug. Preferably, it is the polypeptide shown in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11 to SEQ ID NO:16 above.

[0047] In the above polypeptide drug, the concentration of the polypeptide varies according to different dosage forms or administration methods. In a preferred embodiment, the concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM, and it has anti-cancer activity within this concentration range. More preferably, the concentration is 1 μM to 100 μM, and further preferably 10 μM to 100 μM. Specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 μM.

[0048] In the third typical embodiment of the present application, there is provided the use of any one of the above polypeptides in the preparation of a drug for treating cancer. Since P53 is a tumor suppressor protein and plays an important role in inhibiting the cancer occurrence mechanism. Therefore, the polypeptides screened in the present application that can specifically bind to the P53 mutant all have potential medicinal development value for treating cancer to a certain extent. Especially the medicinal value for anti-breast cancer.

[0049] The above cancers include but are not limited to any one or more of the following: breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterine cancer, ovarian cancer, liposarcoma, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymic cancer, and central nervous system cancer; preferably, the central nervous system cancer is brain cancer. Preferably, the above cancer is breast cancer.

[0050] It should be noted that the above treatment includes effects such as inhibiting the proliferation of cancer cells to different degrees. The specific inhibition degrees include inhibiting the proliferation of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or even 100% of cancer cells.

[0051] In the fourth typical embodiment of the present application, a kit for detecting P53 mutants is provided, and the kit includes any one of the above polypeptides. Since the above polypeptides have been verified to have the ability to specifically target and bind to P53 mutants through a polypeptide chip combined with AI-assisted screening and experimental verification, and thus have anti-cancer activity by binding to P53 mutants, especially the anti-breast cancer cell activity of the polypeptides shown in any one of SEQ ID NO: 1 or SEQ ID NO: 4, SEQ ID NO: 17-22 or any one of SEQ ID NO: 11-16. Therefore, the kit containing the above polypeptides can specifically and accurately detect the presence or absence and the expression level of P53 mutants.

[0052] Since the polypeptide molecules are relatively small, to improve the detection ability, the above polypeptides can be prepared in the form of polypeptide-carrier protein conjugates for detection. According to the requirements for preparing the polypeptide-carrier protein conjugates, a specific and suitable carrier protein can be selected to form the polypeptide-carrier protein conjugates. In some preferred embodiments, the polypeptide exists in the form of a polypeptide-protein conjugate. The carrier proteins in the present application include but are not limited to BSA (bovine serum albumin), OVA (ovalbumin), KLH (keyhole limpet hemocyanin) or CS (casein). According to the amino acid sequence composition of different polypeptides, in order to facilitate conjugation with the carrier protein, it is necessary to conjugate with the carrier protein through a linking sequence (also called a linker or linker). In the present application, the linking sequence is preferably CGSG.

[0053] It should be noted that the above polypeptide-carrier protein conjugate is a recombinantly expressed protein. The recombinantly expressed polypeptide-carrier protein conjugate can be used to detect not only P53 mutant proteins, but also, based on protein homology, to a large extent, detect P53 protein homologous isoforms or proteins with different mutation sites.

[0054] According to the physicochemical properties of the polypeptide amino acids, the different carrier proteins used, and the different conjugation methods, the number of polypeptides that can be conjugated to each carrier protein is also different. Considering the conjugation efficiency and the antibody recognition and binding ability, it is preferred that each carrier protein is conjugated with 2 to 50 polypeptides, more preferably 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, 15 to 30; or, more preferably, any one of 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16 and 10 to 15.

[0055] The above-mentioned kit can be prepared into various different types of detection kits according to specific needs. However, considering the convenience of detection and the judgment of detection results, the polypeptide in the kit can be set in the form of pre-coated on a solid-phase carrier. The specific pre-coated solid-phase carrier is reasonably designed according to needs. More preferably, the solid-phase carrier includes an enzyme-linked immunosorbent assay (ELISA) plate (mostly made of polystyrene material), a membrane carrier or microspheres; further preferably, the membrane carrier includes a nitrocellulose membrane (the most widely used), a glass fiber membrane or a nylon membrane. Even more preferably, a positive control is also coated on the membrane carrier, and the polypeptide-carrier protein conjugate and the positive control are sequentially arranged on the nitrocellulose membrane according to the detection sequence.

[0056] According to the different specific detection methods of the kit, the specific supporting reagents in the kit are also correspondingly different, but all can be combined with the supporting reagents according to the preparation method of the known kit. Preferably, the above-mentioned kit further includes at least one of the following: (1) an enzyme-labeled secondary antibody, and more preferably the enzyme-labeled secondary antibody is an HRP-labeled secondary antibody (corresponding to an ELISA detection kit); (2) a colloidal gold conjugate pad, on which a specific conjugate of a colloidal gold-labeled polypeptide-carrier protein conjugate and a positive control is coated (corresponding to an immunochromatographic assay kit); (3) a labeled pad, on which fluorescently labeled microspheres are coated, and the microspheres are loaded with a specific conjugate of a positive control (corresponding to an immunofluorescence detection kit).

[0057] The above-mentioned immunochromatographic assay kit and immunofluorescence detection kit are relatively more convenient to detect, and only the C line of the positive control and the T line of the test sample need to be established. The positive control pre-coated at the C line of the positive control only needs to be a specific conjugate with a detection label that can be carried over during the serum chromatography process of the test sample, and there are no special limitations on the specific antigen or antibody of the specific positive control. Preferably, the above-mentioned positive control is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin. Correspondingly, the specific conjugate of the positive control is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

[0058] The above-mentioned anti-mouse immunoglobulin can be a sheep anti-mouse immunoglobulin or a rabbit anti-mouse immunoglobulin according to the different immunized objects, or other immunizable animal anti-mouse immunoglobulins. Similarly, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin can also be anti-immunoglobulins from different species sources according to the different immunized animals. The above-mentioned immunoglobulin can be any one of IgM, IgG, IgA, IgD or IgE. These anti-immunoglobulin antibodies can be monoclonal antibodies or polyclonal antibodies.

[0059] In the above-mentioned kit, according to the number of samples to be detected, the specifications of the enzyme-linked immunosorbent assay (ELISA) plates used are also different, and a reasonable selection can be made from 12- to 384-well ELISA plates. In the pre-coated ELISA plates, according to the different detection targets in different polypeptide-carrier protein conjugates, the coating amounts of the polypeptide-carrier protein conjugates in each well also vary. Similarly, the coating amounts of the polypeptide-carrier protein conjugates on the membrane carrier (such as nitrocellulose membrane) are also different. For example, it can be 0.8-8 μg / cm, more preferably 0.8-7 μg / cm, 0.8-6 μg / cm, 0.8-5 μg / cm, 0.8-4 μg / cm, 0.8-3 μg / cm, 0.8-2 μg / cm, 0.8-1.8 μg / cm, 0.8-1.7 μg / cm, 0.8-1.6 μg / cm, 0.8-1.5 μg / cm, 0.8-1.4 μg / cm or 0.8-1.2 μg / cm.

[0060] In some preferred embodiments, the kit includes a chip, on which polypeptides are pre-set, and the polypeptides form a polypeptide array. Detecting by setting the above-mentioned polypeptides on the polypeptide chip can not only improve the specificity of detection by using the target-binding ability of the polypeptides to the P53 mutants, but also improve the detection throughput by using the array form, so it is suitable for large-scale screening of cancers.

[0061] The beneficial effects of the present application will be further described below in conjunction with specific embodiments. It should be noted that the embodiments of the present application mainly include the following parts:

[0062] A. Data analysis: According to the peptide library contained in the existing polypeptide chip, screen the binding peptide segments that are significantly different between the two groups of target target proteins and positive control polypeptides;

[0063] B. AI calculation: Randomly shear the characteristics of specific polypeptides, calculate the binding force with the P53 mutant protein, screen the polypeptides with the top-ranked binding forces and compare them with the existing knowledge base;

[0064] C. Experimental verification: Synthesize potential polypeptides for cell experiments on polypeptide anti-cancer, and verify to screen active polypeptides.

[0065] Example 1: Polypeptide library screening and AI analysis

[0066] 1. Utilize the polypeptide database in the polypeptide chip. By using the crystal structure of the P53 mutant protein (see the following PDB file numbers and website, specifically covering mutants with any one or more mutations among the following 17 mutation sites: S121F, V122G, C135V, C141V, W146Y, C182S, V203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268D, E294M or S315R, and using the protein sequence numbered P04637D as the wild-type P53 protein sequence for site alignment), compare the binding energy of the polypeptides in the peptide library that bind to the P53 mutant protein with that of the positive polypeptides, and preliminarily screen out the binding peptide segments with significant differences as the first candidate polypeptide set. For the first candidate polypeptide set, use the AutoDock CrankPep software to calculate the binding force between each candidate polypeptide and the P53 mutant protein based on the Docking strategy, and then screen out the second candidate polypeptide set with higher binding force.

[0067] 1) Search for the three-dimensional structure of the P53 mutant protein

[0068] Download the three-dimensional conformation file of the P53 mutant protein from the PDB database. [RCSB PDB - 4MZR: Crystal structure of a polypeptide p53 mutant bound to DNA](https: / / www.rcsb.org / structure / 4MZR).

[0069] 2) Prepare the receptor protein

[0070] Extract the receptor protein coordinates: The 4MZR PDB file contains the protein, ligand, and water molecules; first, extract the coordinates of the protein.

[0071] Add hydrogen: The coordinates of hydrogen atoms are usually missing in the crystal structure (because hydrogen atoms have few electrons and the proton nucleus has a weak attraction for electrons, so it is difficult to locate). However, in the docking process, hydrogen atoms, especially polar hydrogen atoms, are necessary for calculating electrostatic interactions. Therefore, hydrogen atoms need to be added to the protein.

[0072] Define the 3D search space for ligand binding: If the binding site is unknown, theoretically, a rectangular box can be defined to contain the entire protein or any specific region.

[0073] 3) Prepare the reference ligand

[0074] Extract the atomic positions of the reference ligand from the 4MZR PDB structure.

[0075] Similar to the protein structure, the ligand structure also lacks hydrogen atoms. Therefore, it is necessary to add hydrogen atoms and define which bonds are rotatable for flexible docking.

[0076] 4) Prepare the docking configuration file

[0077] The docking configuration file contains information on the input receptor (protein), ligand (compound), and default search parameters.

[0078] 5) Docking D and visualize the docking results using PyMol.

[0079] The results are shown in Figure 1 . Figure 1 Shown is a box plot of the binding affinity between the polypeptides in the second candidate polypeptide set obtained by screening and the target protein P53 mutant (the abscissa represents the binding energy, and the larger the absolute value, the stronger the binding ability).

[0080] 2. According to Figure 1 the binding strength of each candidate polypeptide, considering the length of the polypeptide, the degree of binding strength fluctuation, and the magnitude of the average binding strength, and combining with the relevant knowledge of the known target protein, potential functional peptide segments are selected to verify their anti-cancer activity. In this application, 3 polypeptides (see Table 1) are selected to further verify the activity of the peptide segments. And according to the comparison with the currently publicly available polypeptide library data (NCBI polypeptide group), it is found that all the polypeptides in this application are novel sequences.

[0081] Table 1:

[0082] Serial number Polypeptide sequence Binding energy kcal / mol 1 RRRLIHNRHY(SEQ ID NO: 1) -187.71 2 RRHNTPHPRL(SEQ ID NO: 2) -183.72 3 RRRPILTIITLE(SEQ ID NO: 3) -184.72 4 RRVFIRFWHN(SEQ ID NO: 17) -191.21 5 RRAFLRLNHNKLS(SEQ ID NO: 18) -179.99 6 RRHSIPHPEYR(SEQ ID NO: 19) -176.46 7 RRAFIRLNYNK(SEQ ID NO: 20) -174.63 8 RRLYVRLSHNS(SEQ ID NO: 21) -174.62 9 RRHNRHSPEPK(SEQ ID NO: 22) -164.87

[0083] Note: The larger the absolute value of the binding energy in the table, the higher the possible binding strength of the polypeptide.

[0084] It should be noted that the above sequences are based on the original polypeptide sequences screened by the polypeptide chip, with R amino acids added at the N-terminus, making the polypeptide carry a positive charge at the N-terminus, which is beneficial for the polypeptide to cross the cell membrane and enter the cell. Therefore, the sequences in the above table are the sequences with two additional R amino acids at the N-terminus.

[0085] Example 2: Polypeptide synthesis

[0086] 1) Entrust GeneScript to synthesize the following candidate peptide segments (myr modification at the N-terminus) and positive control polypeptides:

[0087] Table 2:

[0088] Serial number Name Polypeptide sequence 1 iCXOncP2a myr - RRRLIHNRHY(SEQ ID NO: 4) 2 iCXOncP1a myr - RRVFIRFWHN(SEQ ID NO: 11) 3 iCXOncP3a myr - RRAFLRLNHNKLS(SEQ ID NO: 12) 4 iCXOncP4a myr - RRHSIPHPEYR(SEQ ID NO: 13) 5 iCXOncP5a myr - RRAFIRLNYNK(SEQ ID NO: 14) 6 iCXOncP6a myr - RRLYVRLSHNS(SEQ ID NO: 15) 7 iCXOncP10a myr - RRHNRHSPEPK(SEQ ID NO: 16) 8 iCXOncP8a myr - RRHNTPHPRL(SEQ ID NO: 5) 9 iCXOncP9a myr - RRRPILTIITLE(SEQ ID NO: 6) 10 Positive control RRRRRRRRRRPILTRITE(SEQ ID NO: 10)

[0089] Among them, the HPLC chromatograms of the synthesized polypeptides shown in SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 are as Figures 2a to 2g shown.

[0090] Example 3: Verification Test of Peptide Segment Biological Function

[0091] I. Chemicals and Reagents:

[0092] Table 3:

[0093] Reagent Source Dulbecco's Modified Eagle Medium(DMEM) Invitrogen(CA, USA) Fetal bovine serum(FBS) HyClone Laboratories, Inc(USA) Dimethyl sulfoxide(DMSO) Sigma MTT kit Sangon Biotech(E606334 - 0500)

[0094] II. MTT Experiment Steps:

[0095] 1. All tubes need to be centrifuged before opening.

[0096] 2. Preparation of MTT solution: Dissolve MTT Reagent (Component A) with MTT Solvent (Component B) to prepare a 5 mg / ml MTT solution. It can be used immediately after preparation, or stored at -20°C in the dark. It can also be appropriately aliquoted and stored at -20°C in the dark according to needs.

[0097] 3. Add 10 μL of MTT solution to each well, so that the final concentration of MTT in each well is 0.5 mg / ml.

[0098] 4. After gently mixing, incubate in a 5% CO2, 37°C incubator for 4 hours.

[0099] 5. Carefully aspirate the culture medium in each well to prevent rupture of the cell monolayer.

[0100] 6. Add 100 μL of Formazan Solubilization Solution (Component C) to each well.

[0101] 7. Place the 96-well plate on an oscillator and gently shake for 10 minutes until it is found that all formazan has dissolved under an ordinary optical microscope.

[0102] 8. Measure the absorbance at 570 nm with an enzyme-linked immunosorbent detector.

[0103] III. Inhibition of MDA-MB-231 (ATCC) Cell Activity

[0104] (1) Cell culture: Maintain MDA-MB-231 (ATCC) cells in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and place them in an environment of 37 °C, humid, and containing 5% carbon dioxide.

[0105] (2) In vitro detection of the biological activity of polypeptides: Use the MTT (USE) assay to detect cell proliferation. First, seed MDA-MB-231 (ATCC) cells in a 96-well plate, with 5×10 3 cells in each well. Add 100 μL of DMEM containing 10% FBS to each well, and leave 8 wells empty as blank controls. Incubate overnight (37 °C, 5% CO2) to allow the cells to attach to the wells. Add designed polypeptides dissolved precisely in DMSO at different concentrations (0.1 μM, 1 μM, 10 μM, 100 μM), as well as positive controls. Add DMEM containing 2% FBS to each well. Incubate (37 °C, 5% CO2) for 48 hours to allow the polypeptides to take effect.

[0106] Add 0.05% MTT solution to each well. Incubate (37 °C, 5% CO2) for 3 hours to allow MTT metabolism. Discard the medium, resuspend formazan (the metabolite of MTT) in 100 μL of DMSO, and mix the formazan evenly in the solvent. Read the optical density at 570 nm and record the results. Plot a growth curve with the concentration on the x-axis and the cell survival rate on the y-axis. The results are as Figures 3a to 3g shown.

[0107] Figures 3a to 3g The results show that the polypeptides all showed anti-cancer activity to varying degrees. Among them, the anti-cancer activities of iCXOncP2a, iCXOncP1a, iCXOncP3a, iCXOncP4a, iCXOncP5a, iCXOncP6a, and iCXOncP10a were particularly significant, and they could effectively inhibit the growth of cancer cells at a concentration of 10 - 100 μM.

[0108] Based on the experimental data, the IC 50 of iCXOncP1a was calculated to be 10.1 μM, the IC 50 of iCXOncP2a was 37.3 μM, the IC 50 of iCXOncP3a was 38.5 μM, the IC 50 of iCXOncP4a was 37.5 μM, the IC 50 of iCXOncP5a was 36.9 μM, the IC 50 of iCXOncP6a was 31.7 μM, and the IC 50 of iCXOncP10a was 37.5 μM.

[0109] The IC of the positive control polypeptide ReACp53 50 = 58.5 uM (Positive control reference: Soragni A, Janzen DM, Johnson LM, Lindgren AG, Thai-Quynh Nguyen A, Tiourin E, Soriaga AB, Lu J, Jiang L, Faull KF, Pellegrini M, Memarzadeh S, Eisenberg DS. A Designed Inhibitor of p53 Aggregation Rescues p53 Tumor Suppression in Ovarian Carcinomas. Cancer Cell. 2016 Jan 11;29(1):90-103.).

[0110] The above IC 50 Value calculation method: Based on the inhibition rate of the polypeptide on tumor cells at different concentrations, plot a regression curve with the abscissa - polypeptide concentration and the ordinate - the inhibition rate of tumor cells, obtain the regression equation and R value, and use 50% inhibition rate as the y value to calculate the IC 50 value.

[0111] Thus, it can be seen that the anti-cancer effects of the polypeptides iCXOncP2a, iCXOncP1a, iCXOncP3a, iCXOncP4a, iCXOncP5a, iCXOncP6a and iCXOncP10a of the present application are better than those of the positive control. The druggability of the polypeptide compounds can be further confirmed through the research on these polypeptides in the future.

[0112] Example 4: Microscopic observation of cell proliferation

[0113] By treating cells with polypeptides of different concentrations (1 μM, 10 μM, 100 μM) of iCXOncP2a, iCXOncP1a, iCXOncP3a, iCXOncP4a, iCXOncP5a, iCXOncP6a and iCXOncP10a, the results are as Figures 4a to 4g shown. The cancer cells grow slowly after the polypeptide treatment, and no cancer cells survive under the treatment of 100 μM.

[0114] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: Through the polypeptide chip combined with AI-assisted screening and experimental verification, it is verified that the above polypeptides have the ability to specifically target and bind to the P53 mutant, and thus have anti-cancer activity by binding to the P53 mutant. In particular, the polypeptides shown in any one of SEQ ID NO: 1 and SEQ ID NOs: 17-22, and the polypeptides with a transmembrane peptide shown in any one of SEQ ID NO: 4 and SEQ ID NOs: 11-16 have significant anti-breast cancer cell activity.

[0115] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Sequence Listing <110> Zhuhai Carboncloud Intelligence Technology Co., Ltd. <120> Kit, Polypeptide Targeting P53 and Its Application in the Preparation of Drugs for Treating Cancer <130> PN162519SZTY <160> 22 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> P53 Mutant Binding Peptide <400> 1 Arg Arg Arg Leu Ile His Asn Arg His Tyr 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> P53 Mutant Binding Peptide <400> 2 Arg Arg His Asn Thr Pro His Pro Arg Leu 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> P53 Mutant Binding Peptide <400> 3 Arg Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> iCXOncP2a, N-terminal Myr-modified <400> 4 Arg Arg Arg Leu Ile His Asn Arg His Tyr 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> iCXOncP8a, N-terminal Myr-modified <400> 5 Arg Arg His Asn Thr Pro His Pro Arg Leu 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> iCXOncP9a, N-terminal Myr-modified <400> 6 Arg Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu 1 5 10 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(15) <223> Cell-penetrating peptide <400> 7 His His His His His His His Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Cell-penetrating peptide <400> 8 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> Cell-penetrating peptide <400> 9 Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Gln 1 5 10 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(18) <223> Positive control <400> 10 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Ile Leu Thr Arg Ile 1 5 10 15 Thr Glu <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> iCXOncP1a, N-terminal Myr modification <400> 11 Arg Arg Val Phe Ile Arg Phe Trp His Asn 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> iCXOncP3a, N-terminal Myr modification <400> 12 Arg Arg Ala Phe Leu Arg Leu Asn His Asn Lys Leu Ser 1 5 10 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> iCXOncP4a, N-terminal Myr modification <400> 13 Arg Arg His Ser Ile Pro His Pro Glu Tyr Arg 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> iCXOncP5a, N-terminal Myr modification <400> 14 Arg Arg Ala Phe Ile Arg Leu Asn Tyr Asn Lys 1 5 10 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> iCXOncP6a, N-terminal Myr modification <400> 15 Arg Arg Leu Tyr Val Arg Leu Ser His Asn Ser 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> iCXOncP10a, N-terminal Myr modification <400> 16 Arg Arg His Asn Arg His Ser Pro Glu Pro Lys 1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Unmodified P53 mutant binding peptide <400> 17 Arg Arg Val Phe Ile Arg Phe Trp His Asn 1 5 10 <210> 18 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> Unmodified P53 Mutant-Binding Peptide <400> 18 Arg Arg Ala Phe Leu Arg Leu Asn His Asn Lys Leu Ser 1 5 10 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Unmodified P53 Mutant-Binding Peptide <400> 19 Arg Arg His Ser Ile Pro His Pro Glu Tyr Arg 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Unmodified P53 Mutant-Binding Peptide <400> 20 Arg Arg Ala Phe Ile Arg Leu Asn Tyr Asn Lys 1 5 10 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Unmodified P53 Mutant-Binding Peptide <400> 21 Arg Arg Leu Tyr Val Arg Leu Ser His Asn Ser 1 5 10 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Unmodified P53 Mutant Binding Peptide <400> 22 Arg Arg His Asn Arg His Ser Pro Glu Pro Lys 1 5 10

Claims

1. A polypeptide targeting P53 mutants, characterized in that, The polypeptide specifically binds to the P53 mutant, the N-terminus of the polypeptide has a Myr modification, and the polypeptide is SEQ ID NO: 11; The P53 mutant is a mutant selected from at least one of the following site mutations: S121F, V122G, C135V, C141V, W146Y, C182S, V203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268D, E294M or S315R.

2. A polypeptide drug, characterized in that, The polypeptide drug comprises the polypeptide as claimed in claim 1 and a pharmaceutically optional excipient.

3. The medicament according to claim 2, characterized in that, The concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM.

4. Use of the polypeptide as claimed in claim 1 in the preparation of a drug for treating breast cancer.

5. A kit for detecting P53 mutants, characterized in that, The kit comprises the polypeptide as claimed in claim 1.

6. The kit according to claim 5, wherein The polypeptide is coated on a solid-phase carrier.

7. The kit according to claim 6, characterized in that, The solid-phase carrier comprises an enzyme-linked immunosorbent assay (ELISA) plate, a membrane carrier or a microsphere.

8. The kit according to claim 7, characterized in that, The membrane carrier comprises a nitrocellulose membrane, a glass fiber membrane or a nylon membrane.

9. The kit according to claim 8, characterized in that, A positive control is further coated on the membrane carrier, and the polypeptide and the positive control are sequentially arranged on the membrane carrier in the detection order.

10. The kit according to claim 9, wherein The kit further comprises at least one of the following: (1) An enzyme-labeled secondary antibody, which is an HRP-labeled secondary antibody; (2) A colloidal gold conjugate pad, on which a colloidal gold-labeled antigen and a specific binding substance of the positive control are coated; (3) A labeled pad, on which fluorescently labeled microspheres are coated, and the microspheres are loaded with a specific binding substance of the positive control.

11. The kit according to claim 10, characterized in that, The positive control is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin. Correspondingly, the specific binding substance of the positive control is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

12. The kit according to claim 5, wherein The kit comprises a chip, on which a polypeptide array composed of the polypeptide is pre-set.

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