WO2023093303A1 - 靶向p53的多肽及其在制备用于治疗癌症的药物中的应用 - Google Patents

靶向p53的多肽及其在制备用于治疗癌症的药物中的应用 Download PDF

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WO2023093303A1
WO2023093303A1 PCT/CN2022/123235 CN2022123235W WO2023093303A1 WO 2023093303 A1 WO2023093303 A1 WO 2023093303A1 CN 2022123235 W CN2022123235 W CN 2022123235W WO 2023093303 A1 WO2023093303 A1 WO 2023093303A1
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polypeptide
seq
cancer
modification
drug
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PCT/CN2022/123235
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French (fr)
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周方行
张星艳
王博文
张燕丽
李英睿
文洁
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深圳碳云智肽药物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4748—Details p53
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a polypeptide targeting P53 and its application in the preparation of medicines for treating cancer.
  • Breast cancer is one of the most common malignant tumors in women. According to the latest global cancer burden data in 2020 released by the International Agency for Research on Cancer (IARC) of the World Health Organization, the number of new cases of breast cancer in the world will reach 2.26 million in 2020, surpassing lung cancer for the first time (221 million cases) has become the world's leading cancer. China is a big breast cancer country. In 2020, there will be about 420,000 new cases of breast cancer and nearly 120,000 deaths. Breast cancer usually occurs in the glandular epithelial tissue of the breast, most of which are women, and male breast cancer accounts for 0.5% to 1% of all breast cancer patients. Breast cancer treatments include surgical resection, radiation therapy, chemotherapy, and hormone therapy.
  • molecular targeted therapy as a new method for the treatment of breast cancer, has shown certain curative effect in the treatment of breast cancer, and has received increasing attention from the academic community.
  • Molecular targeted therapy uses specific gene fragments in tumor cells as treatment sites, and achieves the purpose of treating diseases by regulating or blocking the function of these gene fragments.
  • Molecular targeted therapy of breast cancer refers to the treatment of signaling pathways related to the occurrence and development of breast cancer and their expression products related to oncogenes.
  • 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 effect, and basically does not damage normal tissues. Therefore, targeted tumor therapy is the most promising solution in tumor treatment.
  • p53 is the collective name for a family of homologous proteins known as tumor suppressor proteins (also known as p53 proteins or p53 oncoproteins).
  • p53 is a tumor suppressor protein and transcription factor that activates multiple transcriptional targets in response to cellular stress or DNA damage.
  • p53 coordinates multiple responses, including cell cycle arrest, DNA repair, metabolic alterations, antioxidant effects, antiangiogenic effects, autophagy, senescence, and apoptosis.
  • the p53 protein can maintain the stability of the genome and avoid or reduce the occurrence of mutations, so it is called the guardian of the genome.
  • the p53 protein plays an important role in avoiding cancer mechanisms, such as apoptosis, cell senescence, genetic stability, and inhibition of angiogenesis.
  • Mutations in TP53 are the most common genetic mutations in cancer, causing partial or complete loss of function in more than 50% of tumors. Mutations in p53 confer a selective advantage to tumor cells, enabling them to circumvent 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 form an effective binding to DNA, resulting in the inability of the p21 protein to form and send out the signal to stop cell division. As a result, damaged cells divide uncontrollably and eventually form tumors.
  • Polypeptides are molecular compounds of three or more amino acids linked by peptide bonds.
  • peptide substances widely exist in living organisms to regulate the functional activities of various system organs and cells in the body.
  • peptide drugs have attracted much attention in the development of anti-tumor drugs due to their high targeting, low immunogenicity, high tissue permeability, and safety.
  • the global anti-tumor peptide therapy market was valued at US$8.6 billion, and it is expected to maintain a steady growth trend in the next decade.
  • the polypeptide can effectively bind to the p53 mutant protein, thereby restoring the normal function of the p53 protein and inhibiting the occurrence of cancer. Therefore, the development of effective polypeptide drugs that can restore the activity of P53 mutant proteins can not only promote the study of tumor growth, but also have very important clinical value.
  • the present invention aims to provide a polypeptide targeting P53 and its application in the preparation of medicines for treating cancer, so as to provide more polypeptide drug molecules with potential medicinal value for the development of anticancer medicines.
  • a polypeptide targeting P53 mutant is provided.
  • 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, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: twenty two.
  • the P53 mutant is a mutant with at least one mutation selected from the following sites: Y220C, R175H or R273H; preferably, the polypeptide is a modified peptide; preferably, the modification is a chemical group modification or an amino acid modification; preferably Preferably, the chemical group modification is any one or more of PEG modification, acetylation modification or amidation modification; preferably, the PEG modification is linear PEG modification, PEG modification with monofunctional group or PEG modification with bifunctional group ;
  • the site of PEG modification is selected from any one or more of the N-terminal, C-terminal, Lys side chain and Cys side chain of the polypeptide;
  • PEG modification is PEG modification with a molecular weight of 500-40000;
  • the N-terminal and C-terminal of the polypeptide have acetylation modification and amidation modification respectively;
  • the N-terminal of the polypeptide has fatty acid modification, more preferably
  • polypeptide drug includes any one or more of the above polypeptides and pharmaceutically optional auxiliary materials.
  • 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 ⁇ M ⁇ 100 ⁇ M.
  • the drug is a polypeptide drug or a combined drug.
  • the cancer includes any one or more of the following: breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, Leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterus cancer, ovarian cancer, fat cancer, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymus cancer and central nervous system Systemic cancer; preferably, the central nervous system cancer is brain cancer; preferably, the application includes the function of inhibiting the proliferation of human breast cancer cells through the polypeptide.
  • kits for detecting P53 mutants includes any one or more of the above polypeptides.
  • polypeptide exists in the form of a polypeptide-protein conjugate; preferably, the protein in the polypeptide-protein conjugate is selected from any of the following: bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or casein more preferably, the polypeptide is coupled to the protein through a linking sequence to form a polypeptide-protein conjugate, and further preferably, the linking sequence is CGSG.
  • the polypeptide is coated on a solid-phase carrier; preferably, the solid-phase carrier includes a microtiter plate, a membrane carrier or microspheres; preferably, the membrane carrier includes a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane; preferably Preferably, the membrane carrier is also coated with a positive control substance, and the polypeptide and the positive control substance are sequentially arranged on the membrane carrier according to the order of detection; preferably, the kit also includes at least one of the following: (1) an enzyme-labeled secondary antibody, more preferably The enzyme-labeled secondary antibody is a HRP-labeled secondary antibody; (2) colloidal gold binding pad, the colloidal gold binding pad is coated with a specific conjugate of colloidal gold-labeled antigen and positive control; (3) labeling pad, the labeling pad is coated with Fluorescently labeled microspheres loaded with specific binders for positive controls; preferably, the positive controls are selected from mouse immunoglobulins, human immunoglobulins, sheep immunoglobulin
  • the kit includes a chip on which a polypeptide array composed of polypeptides is preset.
  • the present invention uses the P53 mutant protein as the target protein of the anticancer drug, screens the potential polypeptide fragments on a large scale through the polypeptide chip, and then through the means of screening and verification in vivo and in vitro, and aims at the technical problems existing in the existing treatment methods, finds the New peptide compounds with anti-cancer activity; these peptides have the advantages of specific target, low toxicity and low side effects as drugs, so they have potential application value as anti-cancer drugs.
  • Figure 1a- Figure 1g shows the results of HPLC detection of polypeptide synthesis in the embodiment of the present invention
  • Figure 2a- Figure 2c shows the MTT result graph of the effect of the anti-cancer polypeptide on the proliferation of MDA-MB-231 cells in the embodiment of the present invention.
  • Figures 3a-3c show the effects of the polypeptides in the examples of the present invention on the inhibition of MDA-MB-231 cell proliferation.
  • Polypeptide In this application, it refers to any predicted or screened peptide that can specifically bind to the target P53 mutant.
  • Polypeptide-carrier protein conjugate refers to a conjugate formed by coupling a polypeptide with a carrier protein, wherein one carrier protein can be coupled to one or more polypeptides, and when multiple polypeptides are coupled, the multiple polypeptides have the same amino acid sequence. According to the differences in the physical and chemical properties of the specific coupled polypeptide sequences, the different types of specific carrier proteins, and the different coupling methods, the number of coupled polypeptides on each carrier protein is different.
  • 2- 50 pieces preferably 2- 50 pieces, more preferably 3-45 pieces, 5-40 pieces, 5-35 pieces, 5-30 pieces, 8-30 pieces, 10-30 pieces, 12-30 pieces, 15-30 pieces; or, more preferably 6 ⁇ 36, 8 ⁇ 32, 10 ⁇ 28, 10 ⁇ 26, 10 ⁇ 24, 10 ⁇ 22, 10 ⁇ 20, 10 ⁇ 18, 10 ⁇ 16 and 10 ⁇ 15 any of the situations.
  • Peptide chip technology is a detection technology based on a peptide chip, which utilizes the contact between a wide variety of peptides on the peptide chip and the sample, and then uses image acquisition technology to collect each characteristic signal on the peptide chip (specifically, it can be expressed as a fluorescent image carrying each characteristic signal). ), and then output the signal intensity of each feature in the chip, that is, the detection result data of the peptide chip. Based on the sample detection signal output from the detection result data of the peptide chip, the analysis of the analyte in the sample combined with the peptide on the peptide chip, the analysis of the sample, etc. can be realized.
  • Motif in biology, is a mathematical statistical model based on data, typically a sequence (Sequence), can also be a structure, 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, a sequence that tends to be bound by a transcription factor.
  • group a DNA sequence can be Defined as a transcription factor binding site, that is, a sequence that tends to be bound by a transcription factor.
  • a sequence motif can be defined as a protein sequence belonging to a given protein family.
  • a simple motif could be, for example, a pattern that is shared by all members of the group.
  • P53 mutant protein is selected as the target protein of anti-cancer drugs, and large-scale screening through polypeptide chips has potential Peptide fragments, and then through in vivo and in vitro screening and verification methods, quickly screened out peptide drugs with anticancer activity.
  • a polypeptide targeting P53 is provided, the polypeptide specifically binds to a P53 mutant, and the polypeptide is selected from any one or more of the following: RRGPARVSQVPKHL (SEQ ID NO: 1), RRWLPPFGVFS ( SEQ ID NO: 2), RRNDYVLRLNKHS (SEQ ID NO: 3), RRYSRAPWSG (SEQ ID NO: 4), RRVEHENAFG (SEQ ID NO: 5), RRPNFPLAQSSD (SEQ ID NO: 6), RRFESKKRSG (SEQ ID NO: 7 ), RRYWYKNHLYHG (SEQ ID NO: 8), RRLLDAGPSEG (SEQ ID NO: 9), RREPAKSYWSQVE (SEQ ID NO: 10), RRPALGNRLWDAQVE (SEQ ID NO: 11), RRPNYLDQFADG (SEQ ID NO: 12), RRNH
  • the present invention uses the P53 mutant protein as the target protein of anticancer drugs, screens potential polypeptide fragments on a large scale through a polypeptide chip, and then screens and verifies them in vivo and in vitro, aiming at the technical problems existing in existing treatment methods, New peptide compounds with anti-cancer activity have been found; these peptides have the advantages of specific target, low toxicity and low side effects as drugs, so they have potential application value as anti-cancer drugs.
  • the compounds shown in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 22 are preferred.
  • the activity is the highest, for example, the activity against breast cancer is higher than that of other polypeptides.
  • P53 mutants are any mutants that can lead to the loss of its tumor suppressor function, in this application, including but not limited to mutants with at least one of the following mutations: Y220C, R175H or R273H.
  • Peptides have a significant structural advantage. On the basis of not 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. . This multifunctional chemical modification of the mechanism of pH-sensitive liposomes with receptor/ligand binding ability to enter cells will induce tumor tissue cells to actively endocytose liposomes and then release drugs.
  • C-terminal modification (amidation, sulfation, etc.), N-terminal modification (acetylation, fatty acidation, etc.), middle residue modification (with Ser- , Tyr-, Asn-, Thr-binding glycosylation modification; Ser-, Tyr-, Thr-binding phosphorylation modification, etc.) and cyclization modification.
  • the above polypeptides can also be modified in various ways as needed during development.
  • the polypeptide is a modified peptide segment; preferably, the modification is chemical group modification or amino acid modification.
  • the above-mentioned chemical group modification is PEG modification; preferably, PEG modification is linear PEG modification, PEG modification with a single functional group or PEG modification with bifunctional groups; preferably, the site of PEG modification is selected from any one or more of the N-terminal, C-terminal, Lys side chain and Cys side chain of the polypeptide; preferably, the PEG modification has a molecular weight of 500- PEG modification of 40000; preferably, the amino acid modification is hydrophilic amino acid modification or cysteine modification; preferably, the hydrophilic amino acid modification is to add 1-4 hydrophilic Water-based amino acid, more preferably, the hydrophilic amino acid is Glu, Lys, Ser or Gly; further preferably, 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys or Ser- Gly-Ser.
  • the above-mentioned chemical group modification is double-terminal modification of the polypeptide, more preferably, acetylation modification and amidation modification are respectively performed on the N-terminal and C-terminal of the polypeptide.
  • the polypeptide in order to better achieve directional coupling of peptides, it is preferable to modify the polypeptide with cysteine. Specifically, including but not limited to adding at the N-terminal, C-terminal or both ends of the NC of the peptide, or adding cysteine in the middle of the peptide chain of the polypeptide.
  • cysteine When cysteine is added in the middle of the peptide chain of a peptide, one or more cysteines can be inserted in the middle of the peptide chain (that is, inserted between two amino acid residues), or one or more cysteines can be inserted
  • the acid is connected in the middle of the peptide chain in the form of a branch (that is, as a side chain of an amino acid in the middle of the peptide chain).
  • modification method of the above polypeptide can adopt the most mature and widely used chemical modification method at present, including liquid phase method and solid phase method.
  • a polypeptide drug is provided, and the polypeptide drug includes any one of the above-mentioned polypeptides and pharmaceutically optional excipients.
  • the medicine containing the above-mentioned polypeptide molecule has the advantages of high target binding ability with P53 mutant, high anticancer activity, low toxicity and low side effects.
  • the above-mentioned optional pharmaceutical excipients can be different 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. Including but not limited to pharmaceutically acceptable carriers, excipients or adjuvants and other auxiliary materials.
  • the polypeptide drug can be made into different dosage forms to adapt to various administration routes, specifically, the administration routes include oral administration, injection administration or transdermal administration and the like.
  • the dosage forms of medicines include capsules, tablets, oral liquids, injections or transdermal absorbers, etc.
  • the drug of the present application can be made into tablets, capsules, tablets, oral liquids, injections or transdermal absorbers according to known methods in the pharmaceutical industry.
  • the injection is an intravenous injection.
  • sucrose, lactose, galactose, corn starch, gelatin, microcrystalline cellulose, carboxymethyl cellulose, etc. can be used as carriers or excipients.
  • the drugs of the present application can also be prepared into solutions and suspensions suitable for oral administration by using methods and auxiliary ingredients known in the pharmaceutical industry.
  • distilled water, water for injection, isotonic sodium chloride or glucose solution, or low concentration (such as 1-100mm) phosphate buffer saline (PBS) can be used as carrier or diluent.
  • PBS phosphate buffer saline
  • One or more other auxiliary ingredients or additives may be added to these preparations for parenteral administration, for example, ascorbic acid may be used as an antioxidant, and sodium benzoate may be used as a preservative.
  • the formulations of these dosage forms may also contain other solubilizers, disintegrants, colorants, dispersants or surfactants.
  • the above-mentioned polypeptide drugs can be used as anticancer drugs due to their anticancer activity, preferably anticancer drugs.
  • the concentration of the polypeptide varies according to different dosage forms or administration methods.
  • the concentration of the polypeptide in the polypeptide drug is 0.1 ⁇ M-100 ⁇ M, and all have anticancer activity within this concentration range. More preferably, the concentration is 1 ⁇ M to 100 ⁇ M, further preferably 10 ⁇ M to 100 ⁇ M.
  • 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 ⁇ .
  • the use of any one of the above-mentioned polypeptides in the preparation of a medicament for treating cancer is provided.
  • P53 is a tumor suppressor protein, it plays an important role in inhibiting the mechanism of cancer occurrence. Therefore, the polypeptides screened in this application that can specifically bind to P53 mutants all have potential pharmaceutical development value for treating cancer to a certain extent. Especially the medicinal value of anti-breast cancer.
  • the anti-breast cancer drug is used to inhibit the function of proliferation of human breast cancer cells, so as to achieve the purpose of anti-breast cancer.
  • the above-mentioned 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, fat cancer, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymus cancer and central nervous system Cancer; preferably, the central nervous system cancer is brain cancer.
  • the aforementioned cancer is breast cancer.
  • the above-mentioned treatment includes different degrees of inhibition of cancer cell proliferation and other effects, and the specific degree of inhibition includes one of more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, and more than 35%. More than %, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% , more than 96%, more than 97%, more than 98%, more than 99%, or even 100% of cancer cell proliferation.
  • a kit for detecting P53 mutants including any one of the above-mentioned polypeptides. Since the above-mentioned polypeptide has the ability to specifically target and bind to P53 mutants through polypeptide chips combined with AI-assisted screening and experiments, it has anti-cancer activity by binding to P53 mutants, especially SEQ ID NO: 1-SEQ ID NO: 22 or the polypeptide shown in any one of the modified sequences thereof has anti-breast cancer cell activity. Therefore, the presence or absence of the P53 mutant and the level of expression can be specifically and accurately detected using the kit comprising the above polypeptide.
  • the above polypeptide can be prepared in the form of a polypeptide-carrier protein conjugate for detection.
  • a specific and suitable carrier protein can be selected to form the polypeptide-carrier protein conjugate.
  • the polypeptide is in the form of a polypeptide-protein conjugate.
  • Carrier proteins in this application include, but are not limited to, BSA (bovine serum albumin), OVA (ovalbumin), KLH (keyhole limpet hemocyanin) or CS (casein).
  • the linker sequence is preferably CGSG.
  • polypeptide-carrier protein conjugate is a protein expressed through recombinant synthesis.
  • the above-mentioned recombinantly expressed polypeptide-carrier protein conjugates can also be used to detect homologous isomeric proteins or different mutation sites with P53 proteins to a large extent based on protein homology. protein.
  • the number of polypeptides that can be coupled to each carrier protein is also different.
  • it is preferable to couple 2 to 50 polypeptides per carrier protein 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 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, Any one of 10-22, 10-20, 10-18, 10-16 and 10-15.
  • the polypeptide in the kit can be pre-coated on a solid-phase carrier.
  • the specific pre-coated solid phase carrier is reasonably designed according to the needs.
  • the solid phase support includes microtiter plates (mostly polystyrene materials), membrane supports or microspheres; further preferably, the membrane supports include nitrocellulose membranes (the most widely used), glass cellulose membranes or nylon membranes
  • the membrane carrier is also coated with a positive control substance, and the polypeptide-carrier protein conjugate and the positive control substance are sequentially arranged on the nitrocellulose membrane according to the order of detection.
  • the specific supporting reagents in the kits are also different, but all of them can be combined according to the preparation methods of the known kits.
  • the above kit also includes at least one of the following: (1) enzyme-labeled secondary antibody, more preferably enzyme-labeled secondary antibody is HRP-labeled secondary antibody (corresponding to ELISA detection kit); (2) colloidal gold binding pad , the colloidal gold binding pad is coated with colloidal gold-labeled polypeptide-carrier protein conjugates and the specific binder of the positive control (corresponding to the immunocolloidal gold detection kit); (3) the marker pad, the marker pad is coated with Fluorescently labeled microspheres loaded with specific binders for positive controls (corresponding to the immunofluorescence detection kit).
  • the above-mentioned immunocolloidal gold detection kit and immunofluorescence detection kit are relatively more convenient to detect, and only need to establish the C line of the positive control and the T line of the test sample.
  • the pre-coated positive control substance at the C line of the positive control as long as it can be combined with a specific binder with a detection label that can be carried along with the serum chromatography process of the sample to be tested, the specific positive control substance
  • the specific antigen or antibody is not particularly limited.
  • the above-mentioned positive control is selected from mouse immunoglobulin, human immunoglobulin, goat immunoglobulin or rabbit immunoglobulin, and correspondingly, the specific binder of the positive control is selected from anti-mouse immunoglobulin, anti-human immunoglobulin Immunoglobulin, anti-goat immunoglobulin or anti-rabbit immunoglobulin.
  • anti-mouse immunoglobulin can be goat anti-mouse immunoglobulin or rabbit anti-mouse immunoglobulin, or anti-mouse immunoglobulin of other immunizable animals according to the different immunized objects.
  • anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin can also be anti-immunoglobulins of different species origin depending on the animal to be immunized.
  • the aforementioned immunoglobulin may be any one of IgM, IgG, IgA, IgD or IgE. These anti-immunoglobulin antibodies may be monoclonal or polyclonal.
  • the specifications of the enzyme plate used are also different, and a reasonable choice can be made from the 12-384-well enzyme plate.
  • the coating amount of polypeptide-carrier protein conjugates in each well is also different.
  • the amount of coating of the polypeptide-carrier protein conjugate on the membrane support is also different.
  • it can be 0.8-8 ⁇ g/cm, more preferably 0.8-7 ⁇ g/cm, 0.8-6 ⁇ g/cm, 0.8-5 ⁇ g/cm, 0.8-4 ⁇ g/cm, 0.8-3 ⁇ g/cm, 0.8-2 ⁇ g/cm, 0.8 -1.8 ⁇ g/cm, 0.8-1.7 g, 0.8-1.6 ⁇ g/cm, 0.8-1.5 ⁇ g/cm, 0.8-1.4 ⁇ g/cm, or 0.8-1.2 ⁇ g/cm.
  • the kit includes a chip on which polypeptides are pre-prepared, wherein the polypeptides are composed of the above-mentioned polypeptides to form a polypeptide array.
  • the present invention determines the inhibitory activity IC50 value of the candidate anticancer polypeptides on MDA-MB-231 cells (human breast cancer cells) in vitro at different concentrations (0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M), and determines the activity of 22 kinds of polypeptides.
  • Anticancer activity that is, the anti-breast cancer iCXOncP11a, iCXOncP12a, iCXOncP13a, iCXOncP15a, iCXOncP17a, iCXOncP18a, iCXOncP19a, iCXOncP22a, iCXOncP31a, iCXOncP32a, iCXOncP33a, iC XOncP34a, iCXOncP35a, iCXOncP36a, iCXOncP16a, iCXOncP20a, iCXOncP21a, iCXOncP23a, iCXOncP24a, iCXOncP25a , iCXOncP27a and iCXOncP29a polypeptides. And through verification, we found that all the peptides
  • Fluorescently labeled samples were diluted according to seven concentration gradients of 1:500, 1:1000, 1:5000, 1:10000, 1:50000, 1:500000 and 1:5000000, and the median of the final dilution concentration was 1 : 10000.
  • the chips in the assay cassette were disassembled, cleaned and dried, then assembled into the imaging cassette, and put into the ImageXpress micro 4 imager of Molecular Device for scanning and imaging. Finally, a TIFF image file is obtained for each detection sample, which is the original data.
  • the above-mentioned P53 mutant protein was used to oversaturate and incubate the IST chip to make the mutant protein bind to the peptides in the chip.
  • Antibody PAB1620 (Merck product number: MABE339) will bind to P53 with normal structure. Detection of the binding of PAB1620 can explain the recovery of P53 protein structure. Therefore, use PAB1620 for high-throughput short peptide array chip detection, and screen out those that can restore P53 Constructively active peptides.
  • the 4MZR PDB file contains proteins, ligands and water molecules; first extract the coordinates of the protein.
  • the atomic positions of the reference ligands were extracted from the 4MZR PDB structure.
  • the structure of the ligand also lacks hydrogen atoms, so it is necessary to add hydrogen atoms and define which bonds are rotatable for flexible docking.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal bovine serum
  • DMSO Dimethyl sulfoxide
  • MTT solution Dissolve MTT Reagent (component A) with MTT Solvent (component B) and prepare a 5 mg/ml MTT solution. It can be used immediately after preparation, or directly stored at -20°C in the dark, or can be properly divided and stored at -20°C in the dark.
  • MDA-MB-231 source: ATCC company
  • human breast cancer cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin ), and cultivated in an environment with a temperature of 37°C and a carbon dioxide (CO 2 ) content of 5%.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • CO 2 carbon dioxide
  • MTT (USE) assay was used to detect cell proliferation.
  • MDA-MB-231 cells were planted in a 96-well plate, each well contained 5 ⁇ 103 cells, each well contained 100 ⁇ L DMEM of 10% FBS, and left blank 8 wells were used as blank control. Incubate overnight (37°C, 5% CO 2 ) to allow the cells to attach. Different concentrations (0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M) of the designed polypeptide accurately dissolved in DMSO were added, and DMEM containing 2% FBS was added to each well. Incubate (37°C, 5% CO 2 ) for 48 hours to allow the polypeptide to work.
  • IC 50 value based on the inhibitory rate of the polypeptide on tumor cells at different concentrations, draw a regression curve in which the abscissa is the concentration of the polypeptide and the ordinate is the inhibitory rate of the tumor cell, and the regression equation and R value are obtained, and the 50 The % inhibition rate is y value, and the value of IC 50 is calculated.
  • breast cancer cells were treated with 22 kinds of polypeptides at different concentrations (1 ⁇ M, 10 ⁇ M, 100 ⁇ M). As shown in Figure 3a, Figure 3b and Figure 3c, the cancer cells after polypeptide treatment grew slowly, Under the treatment of 100 ⁇ M, the cancer cells did not survive.
  • the IC 50 is much smaller than the LD 50 , which is higher than other peptides Pharmaceutical development value.
  • polypeptide IC50 ( ⁇ M) LD50 ( ⁇ M) Positive control 52.96 69.47 iCXOncP11a 37.64 33.16 iCXOncP12a 12.98 32.71 iCXOncP13a 41.23 237.8 iCXOncP15a 40.95 19.94 iCXOncP17a 44.36 53.62 iCXOncP18a 48.52 419.4 iCXOncP19a 31.3 23.78 iCXOncP22a 6.739 24.56 iCXOncP31a 20.82 25.67 iCXOncP32a 14.27 20.18 iCXOncP33a 47.64 165.5 iCXOncP34a 42.47 192.2 iCXOncP35a 72.62 155.5 iCXOncP36a 33.57 193.1
  • the present invention selects the P53 mutant as the target protein of the anticancer drug, and screens potential polypeptide fragments on a large scale through the polypeptide chip. Then, through in vivo and in vitro screening and verification methods, peptide drugs with anticancer activity were quickly screened out.

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Abstract

本发明公开了一种靶向P53的多肽及其在制备用于治疗癌症的药物中的应用。其中，该多肽特异性结合P53突变体，且多肽选自如下任意一条或多条：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10等。本发明通过多肽芯片大规模筛选有潜力的多肽片段，再通过体内外筛选验证的手段，寻找到了具有抗癌活性的全新多肽化合物；这些多肽作为药物具有靶标专一、低毒及低副作用的优势，因而具有开发为抗癌药物的潜在应用价值。

Description

靶向P53的多肽及其在制备用于治疗癌症的药物中的应用 技术领域
本发明涉及生物医药技术领域，具体而言，涉及一种靶向P53的多肽及其在制备用于治疗癌症的药物中的应用。
背景技术
乳腺癌是女性最常见的恶性肿瘤之一，根据世界卫生组织国际癌症研究机构(IARC)发布的2020年全球最新癌症负担数据，2020年全球新发乳腺癌达到226万例，首次超过肺癌(221万例)成为全球第一大癌症。而中国是乳腺癌大国，2020年新发乳腺癌约42万例，并导致近12万人死亡。乳腺癌通常发生在乳房腺上皮组织，发病中以女性居多，男性乳癌占全部乳癌患者的0.5％到1％。乳腺癌的治疗方法包括外科手术切除、放射治疗、化学治疗和激素治疗等。近年来，分子靶向治疗作为乳腺癌治疗的一种新手段，在乳腺癌治疗中显示出一定的疗效，日益受到学术界的重视。分子靶向治疗是以肿瘤细胞中特有的基因片段为治疗位点，通过调节或阻断这些基因片段功能达到治疗疾病的目的。乳腺癌分子靶向治疗是指针对乳腺癌发生、发展有关的信号通路及其癌基因相关表达产物进行治疗。分子靶向药物通过阻断肿瘤细胞或相关细胞的信号转导，来控制细胞基因表达的改变，从而抑制或杀死肿瘤细胞。靶向治疗特异性强，效果显著，基本上不损伤正常组织，因此肿瘤靶向治疗是肿瘤治疗中最有前景的方案。
p53是一系列被称为肿瘤抑制蛋白(也称为p53蛋白或p53肿瘤蛋白)的同源异构蛋白的统称。p53是一种肿瘤抑制蛋白和转录因子，它可以对细胞应激或DNA损伤作出响应，激活多种转录靶标。p53可协调多种反应，包括细胞周期阻滞、DNA修复、代谢改变、抗氧化作用、抗血管生成作用、自噬、衰老和凋亡。p53蛋白能保持基因组的稳定性，避免或减少突变的发生，因此被称为基因组守护者。p53蛋白在避免癌症发生机制上扮演重要的角色，例如，细胞凋亡(apoptosis)、细胞衰老(cell senescence)、基因组稳定性(genetic stability)、抑制血管新生(angiogenesis)。TP53(编码p53的基因)突变是癌症中最常见的基因突变，在超过50％的肿瘤中丧失部分或全部功能。p53突变为肿瘤细胞提供了选择性优势，使其能够避开细胞周期检查点，避免凋亡和衰老，并在正常细胞无法增值的条件下增殖。突变后的p53蛋白可能丧失与DNA形成有效结合的能力，造成p21蛋白无法形成，无法发出停止细胞分裂的信号。因此，受损细胞将不受控制的进行细胞分裂，最终形成肿瘤。近年来，越来越多的研究将p53突变蛋白作为一个中药的抗癌靶点分子，进行药物开发；但仍旧缺乏通过对靶点突变的p53蛋白的功能进行恢复，从而达到抑制癌细胞生长的相关的药物报道。
多肽是由肽键连接的三个或三个以上氨基酸分子化合物。作为重要的生命物质之一，肽类物质广泛存在于生命体中调节体内各个系统器官和细胞的功能活动。近年来多肽类药物凭借其高靶向性、低免疫原性、高组织渗透性，及安全性等优点在抗肿瘤药物的研制中备受关 注。2019年，全世界抗肿瘤多肽治疗的市场估值为86亿美元，并预期会在未来近十年保持稳定增长的趋势。多肽作为小分子化合物，可以有效的与p53突变蛋白结合，从而恢复p53蛋白的正常功能，抑制癌症的发生。因此，开发有效的、使P53突变蛋白恢复活性多肽药物，不仅能够促进肿瘤生长的研究，还具有非常重要的临床价值。
因此，开发以P53为抗癌靶点的具有潜在药用价值的多肽药物分子是抗肿瘤药物研制的一大热点问题。
发明内容
本发明旨在提供一种靶向P53的多肽及其在制备用于治疗癌症的药物中的应用，，以便为抗癌药物研制提供更多具有潜在药用价值的多肽药物分子。
为了实现上述目的，根据本发明的一个方面，提供了一种靶向P53突变体的多肽。该多肽特异性结合P53突变体，且多肽选自如下任意一条或多条：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21及SEQ ID NO：22。
进一步地，P53突变体为选自如下至少一个位点突变的突变体：Y220C、R175H或R273H；优选地，多肽为修饰后的肽段；优选地，修饰为化学基团修饰或氨基酸修饰；优选地，化学基团修饰为PEG修饰、乙酰化修饰或酰胺化修饰中的任意一种或多种；优选地，PEG修饰为直链PEG修饰、具有单官能团的PEG修饰或者具有双官能团的PEG修饰；优选地，PEG修饰的位点选自多肽的N端、C端、Lys侧链和Cys侧链中的任意一个或多个；优选地，PEG修饰为分子量为500～40000的PEG修饰；优选地，多肽的N端和C端分别具有乙酰化修饰和酰胺化修饰；优选地，多肽的N端具有脂肪酸修饰，更优选为Myr修饰；优选地，氨基酸修饰为亲水性氨基酸修饰或半胱氨酸修饰；优选地，亲水性氨基酸修饰为在多肽的N端、C端或者NC两端添加1-4个亲水性氨基酸，更优选地，亲水性氨基酸为Glu、Lys、Ser或Gly；进一步优选地，1-4个亲水性氨基酸选自如下任意一种：Glu-Glu、Lys-Lys或Ser-Gly-Ser；优选地，半胱氨酸修饰为在多肽的如下任一位置添加半胱氨酸：N端、C端、NC两端或肽链中间；更优选地，在肽链中间添加半胱氨酸包括在肽链中间插入一个或多个半胱氨酸，或者一个或多个半胱氨酸以支链形式连接于肽链的中间。
根据本发明的另一个方面，提供一种多肽药物。该多肽药物包括上述任一种或多种多肽以及药学上可选的辅料。
进一步地，多肽药物为抗癌药物，优选为抗乳腺癌药物；优选地，多肽药物中多肽的浓度为0.1μM～100μM。
根据本发明的再一个方面，提供一种上述多肽在制备用于治疗个体肿瘤或癌症的药物中的应用，可选的，药物为多肽药物或联合药物。
进一步地，癌症包括如下任意一种或多种：乳腺癌、肺癌、鼻咽癌、喉癌、胃癌、肝癌、食道癌、肠癌、胰腺癌、胆囊癌、肾癌、膀胱癌、前列腺癌、白血病、淋巴癌、血管瘤、骨癌、宫颈癌、子官癌、卵巢癌、脂肪癌、脑瘤、鳞癌、皮肤癌、甲状腺癌、唇癌、黑色素癌、舌癌、胸腺癌以及中枢神经系统癌症；优选地，中枢神经系统癌症为脑癌；优选的，应用包括通过多肽抑制人乳腺癌细胞增殖的功能。
根据本发明的又一方面，提供了一种检测P53突变体的试剂盒。该试剂盒包括上述任一种或多种多肽。
进一步地，多肽以多肽-蛋白偶联物的形式存在；优选地，多肽-蛋白偶联物中的蛋白选自如下任意一种：牛血清蛋白、卵清白蛋白、钥孔血蓝蛋白或酪蛋白；更优选，多肽通过连接序列与蛋白偶联形成多肽-蛋白偶联物，进一步优选地，连接序列为CGSG。
进一步地，多肽被包被于固相载体上；优选地，固相载体包括酶标板、膜载体或微球；优选地，膜载体包括硝酸纤维素膜、玻璃纤维素膜或尼龙膜；优选地，膜载体上还包被有阳性对照物，多肽和阳性对照物按检测顺序在膜载体上依次设置；优选地，试剂盒还包括如下至少之一：(1)酶标二抗，更优选酶标二抗为HRP标记的二抗；(2)胶体金结合垫，胶体金结合垫上包被有胶体金标记的抗原和阳性对照物的特异性结合物；(3)标记垫，标记垫上包被有荧光标记的微球，微球上负载有阳性对照物的特异性结合物；优选地，阳性对照物选自鼠免疫球蛋白、人免疫球蛋白、羊免疫球蛋白或兔免疫球蛋白，相应地，阳性对照物的特异性结合物选自抗鼠免疫球蛋白、抗人免疫球蛋白、抗羊免疫球蛋白或抗兔免疫球蛋白。
进一步地，试剂盒包括芯片，芯片上预置有多肽组成的多肽阵列。
本发明以P53突变蛋白作为抗癌药物的靶点蛋白，通过多肽芯片大规模筛选有潜力的多肽片段，再通过体内外筛选验证的手段，针对现有治疗手段中存在的技术问题，寻找到了具有抗癌活性的全新多肽化合物；这些多肽作为药物具有靶标专一、低毒及低副作用的优势，因而具有开发为抗癌药物的潜在应用价值。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1a-图1g示出了本发明实施例中多肽合成HPLC检测结果图；
图2a-图2c示出了本发明实施例中抗癌多肽对MDA-MB-231细胞增殖效果MTT结果图；以及
图3a-图3c示出了本发明实施例中多肽对MDA-MB-231细胞增殖抑制作用效果图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
术语解释：
多肽：本申请中指预测的或者筛选的能够与靶点P53突变体特异性结合的任意一条肽段。
多肽-载体蛋白偶联物：本申请中指一条多肽与载体蛋白偶联形成的偶联物，其中，一个载体蛋白可以偶联一条或多条多肽，多条多肽偶联时，多条多肽具有相同的氨基酸序列。根据具体偶联的多肽序列的理化性质的差异、具体载体蛋白的种类的不同以及偶联方法的不同，每个载体蛋白上所偶联的多肽的条数有所差异，本申请中优选2～50条，更优选为3～45条、5～40条、5～35条、5～30条、8～30条、10～30条、12～30条、15～30条；或者，更优选为6～36条、8～32条、10～28条、10～26条、10～24条、10～22条、10～20条、10～18条、10～16条及10～15条中的任意一种情况。
多肽芯片技术是基于多肽芯片的检测技术，其利用多肽芯片上的种类繁多的多肽与样本的接触，然后利用图像采集技术采集多肽芯片上各个特征信号(具体可表现为携带各个特征信号的荧光图像)，进而输出芯片中每个特征的信号强度，即多肽芯片检测结果数据。基于多肽芯片检测结果数据输出的样本检测信号，可实现对与多肽芯片上的多肽结合的样本中的待测物的分析，样本的分析等。
Motif：基序，在生物学中是一个基于数据的数学统计模型，典型的是一段序列(Sequence)，也可以是一个结构，是特定的组(group)的序列预测，例如，一个DNA序列可以定义为转录因子结合位点，也就是序列倾向于被转录因子结合。对蛋白质来说，序列基序(Sequence motif)可以被定义为蛋白质序列属于一个给定的蛋白质家族。一个简单的motif可以是，比如一个模式(pattern)，而这个模式被这个组(group)中的所有成员共享。
如背景技术所提到的，现有技术中急需开发针对不同靶点的抗癌多肽药物的问题，本发明选择以P53突变蛋白作为抗癌药物的靶点蛋白，通过多肽芯片大规模筛选有潜力的多肽片段，再通过体内外筛选验证的手段，快速的筛选出了具有抗癌活性的多肽药物。本发明所述的iCXOncP11a、iCXOncP12a、iCXOncP13a、iCXOncP15a、iCXOncP17a、iCXOncP18a、iCXOncP19a、iCXOncP22a、iCXOncP31a、iCXOncP32a、iCXOncP33a、iCXOncP34a、iCXOncP35a、iCXOncP36a、iCXOncP16a、iCXOncP20a、iCXOncP21a、iCXOncP23a、iCXOncP24a、iCXOncP25a、iCXOncP27a和iCXOncP29a二十二种多肽(序列信息见表1)，展现出显著的抑制乳腺癌细胞生长效果，其中以iCXOncP12a、iCXOncP22a、iCXOncP16a、iCXOncP20a、iCXOncP21a、iCXOncP29a效果最为显著，可作为后续药物研发新的活性化合物发现提供思路。同时我们提供的全新的抗癌多肽序列，可以作为后续药物研发的先导化合物。
基于上述研究结果，申请人提出了本申请的一系列技术方案。在一种典型的实施例中，提供了一种靶向P53的多肽，该多肽特异性结合P53突变体，且多肽选自如下任意一条或多条：RRGPARVSQVPKHL(SEQ ID NO：1)、RRWLPPFGVFS(SEQ ID NO：2)、 RRNDYVLRLNKHS(SEQ ID NO：3)、RRYSRAPWSG(SEQ ID NO：4)、RRVEHENAFG(SEQ ID NO：5)、RRPNFPLAQSSD(SEQ ID NO：6)、RRFESKKRSG(SEQ ID NO：7)、RRYWYKNHLYHG(SEQ ID NO：8)、RRLLDAGPSEG(SEQ ID NO：9)、RREPAKSYWSQVE(SEQ ID NO：10)、RRPALGNRLWDAQVE(SEQ ID NO：11)、RRPNYLDQFADG(SEQ ID NO：12)、RRNHHQPADG(SEQ ID NO：13)、RRNAYGQVFD(SEQ ID NO：14)、RRFNFYRQVG(SEQ ID NO：15)、RRWRYYFWKED(SEQ ID NO：16)、RRGHEWPLDG(SEQ ID NO：17)、RNDEFAQKVS(SEQ ID NO：18)、RRFYQWKELED(SEQ ID NO：19)、RRHNVKWED(SEQ ID NO：20)、RRSRGGDG(SEQ ID NO：21)或RRSHPNAHED(SEQ ID NO：22)。
如上述，本发明以P53突变蛋白作为抗癌药物的靶点蛋白，通过多肽芯片大规模筛选有潜力的多肽片段，再通过体内外筛选验证的手段，针对现有治疗手段中存在的技术问题，寻找到了具有抗癌活性的全新多肽化合物；这些多肽作为药物具有靶标专一、低毒及低副作用的优势，因而具有开发为抗癌药物的潜在应用价值。上述多肽中，优选SEQ ID NO：2、SEQ ID NO：8、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：22所示的化合物，该化合物的抗癌活性最高，比如，对抗乳腺癌的活性较其他多肽活性高。
上述P53突变体为任何能够导致其抑癌功能丧失的突变体，在本申请中，包括但不限于如下至少一个位点突变的突变体：Y220C、R175H或R273H。
多肽具有一个显著的结构优势，既可以在不影响原有功能性多肽片段的基础上，通过固相合成或生物合成，在多肽的一端或两端引入新的功能性化学修饰，获得多功能性。这种多功能性化学修饰具有受体/配体结合能力的pH敏感脂质体进入细胞的机制，会诱导肿瘤组织细胞主动内吞脂质体继而释放药物。常见的多肽修饰物按照修饰位点，可以分为四大类：C末端修饰(酰胺化、硫酸酯化等)、N末端修饰(乙酰化、脂肪酸化等)、中间残基修饰(与Ser-、Tyr-、Asn-、Thr-结合的糖基化修饰；与Ser-、Tyr-、Thr-结合的磷酸化修饰等)以及环化修饰。
因此，根据药物开发所需，上述多肽在研制时还可以根据需要进行各种修饰。在一种优选的实施例中，该多肽为修饰后的肽段；优选地，修饰为化学基团修饰或氨基酸修饰。
比如，为进一步提高某些肽段的亲和性，在一种优选的实施例中，上述化学基团修饰为PEG修饰；优选地，PEG修饰为直链PEG修饰、具有单官能团的PEG修饰或者具有双官能团的PEG修饰；优选地，PEG修饰的位点选自多肽的N端、C端、Lys侧链和Cys侧链中的任意一个或多个；优选地，PEG修饰为分子量为500～40000的PEG修饰；优选地，氨基酸修饰为亲水性氨基酸修饰或半胱氨酸修饰；优选地，亲水性氨基酸修饰为在多肽的N端、C端或者NC两端添加1-4个亲水性氨基酸，更优选地，亲水性氨基酸为Glu、Lys、Ser或Gly；进一步优选地，1-4个亲水性氨基酸选自如下任意一种：Glu-Glu、Lys-Lys或Ser-Gly-Ser。
由于化学合成的多肽往往携带游离的氨基和游离的羧基，而多肽的序列往往代表的是其所在的母本蛋白的序列，因而，为了使合成的蛋白与母本蛋白在序列和活性方面更接近，通 常要对多肽的末端进行封闭，一般是对N端进行乙酰化和对C端进行酰胺化，这样修饰会减少多肽的总电荷，降低多肽的溶解度，进而也能够使多肽模拟其在母本蛋白中α氨基和羧基的原始状态。因此，在另一些实施例中，上述化学基团修饰为对多肽进行双端修饰，更优选地，对多肽的N端和C端分别进行乙酰化修饰及酰胺化修饰。
在某些实施例中，为了更好地实现对肽段的定向偶联，优选可以对多肽进行半胱氨酸修饰。具体地，包括但不限于在肽段的N端、C端或者NC两端添加，亦或者在多肽的肽链中间添加半胱氨酸。当在肽段的肽链中间添加半胱氨酸时，一个或多个半胱氨酸可以插入肽链中间(即插入两个氨基酸残基之间)，也可以将一个或多个半胱氨酸以支链形式连接于肽链的中间(即作为肽链中间的某个氨基酸的侧链)。
需要说明的是，上述多肽的修饰方法可以采用目前应用最为成熟且最为广泛的化学修饰方法，包括液相法和固相法。
在本申请第二种典型的实施例中，提供了一种多肽药物，多肽药物包括上述任一种多肽以及药学上可选的辅料。含有上述多肽分子的药物具有与P53突变体靶向结合能力高、抗癌活性高、低毒及低副作用的优势。
上述药学上可选的辅料，可以根据所欲制备的药物的剂型和/或给药方式、给药途径的不同，而有所不同，可以从现有药用辅料中合理选择。包括但不仅限于药学上可接受的载体、赋形剂或佐剂等辅料。而且，该多肽药物可以制成不同的剂型以适应多样化的给药途径，比特，给药途径包括口服给药、注射给药或经皮给药等。而药物的剂型包括胶囊、片剂、口服液、注射剂或透皮吸收剂等。
具体地，可以按照制药工业中已知的方法将本申请的药物制成片胶囊、片剂、口服液、注射剂或透皮吸收剂等。优选地，注射剂为静脉注射剂。在制备适于口服给药的胶囊剂、片剂、口服液时，可以使用蔗糖、乳糖、半乳糖、玉米淀粉、明胶、微晶纤维素、羧甲基纤维素等作为载体或赋形剂。另外，也可以使用制药工业中已知的方法和辅助成分将本申请的药物制成适于口服给药的溶液剂和悬浮剂。若制备适于胃肠道外途经给药的溶液剂和悬浮剂，可以使用蒸馏水、注射用水、等渗氯化钠或葡萄糖溶液，或者低浓度(例如1-100mm)磷酸盐缓冲液(PBS)作为载体或稀释剂。可以在这些胃肠道外给药的制剂中加入一种或多种其他辅助成分或添加剂，例如可使用抗坏血酸作为抗氧化剂，使用苯甲酸钠等作为防腐剂。在这些剂型的制剂中还可以含有其他的增溶剂、崩解剂、着色剂、分散剂或表面活性剂。
上述多肽药物由于具有抗癌活性，因而可以作为抗癌药物，优选为抗乳腺癌药物。上述多肽药物中，多肽的浓度根据剂型或给药方式的不同而有所不同。在一种优选的实施例中，多肽药物中多肽的浓度为0.1μM～100μM，在该浓度范围内均具有抗癌活性。更优选地，浓度为1μM～100μM，进一步优选为10μM～100μM。具体地，可以是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或100μM。
在本申请第三种典型的实施方式中，提供了上述任一种多肽在制备用于治疗癌症的药物中的应用。由于P53是肿瘤抑制蛋白，在抑制癌症发生机制上具有重要作用。因而，本申请筛选到的能够特异性与P53突变体结合的多肽在一定程度上都是具有潜在的治疗癌症的药用开发价值的。尤其是抗乳腺癌的药用价值。该抗乳腺癌的药应用于抑制人乳腺癌细胞增殖的功能，从而达到抗乳腺癌的目的。
上述癌症包括但不仅限于如下任意一种或多种：乳腺癌、肺癌、鼻咽癌、喉癌、胃癌、食道癌、肠癌、胰腺癌、胆囊癌、肾癌、膀胱癌、前列腺癌、白血病、淋巴癌、血管瘤、骨癌、宫颈癌、子官癌、卵巢癌、脂肪癌、脑瘤、鳞癌、皮肤癌、甲状腺癌、唇癌、黑色素癌、舌癌、胸腺癌以及中枢神经系统癌症；优选地，中枢神经系统癌症为脑癌。优选上述癌症为乳腺癌。
需要说明的是，上述治疗包括不同程度的抑制癌细胞增殖等效果，具体的抑制程度包括一种5％以上、10％以上、15％以上、20％以上、25％以上、30％以上、35％以上、40％以上、45％以上、50％以上、55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上、甚至100％的癌细胞的增殖。
在本申请第四种典型的实施方式中，提供了一种检测P53突变体的试剂盒，该试剂盒包括上述任一种多肽。由于上述多肽通过多肽芯片结合AI辅助筛选及实验验证了上述多肽具有特异性靶向结合P53突变体的能力，进而通过结合P53突变体而具有抗癌活性，尤其是SEQ ID NO：1-SEQ ID NO：22或其具有修饰的序列中任一条所示的多肽抗乳腺癌细胞的活性。因此，利用包含上述多肽的试剂盒能够特异地准确地检测P53突变体的存在与否以及表达量高低。
由于多肽分子相对较小，为提高检测能力，可以将上述多肽制备成多肽-载体蛋白偶联物的形式进行检测。根据多肽-载体蛋白偶联物制备的需求，可以选择具体合适的载体蛋白来形成多肽-载体蛋白偶联物。在一些优选的实施例中，多肽以多肽-蛋白偶联物的形式存在。本申请中的载体蛋白包括但不仅限于BSA(牛血清蛋白)、OVA(卵清白蛋白)、KLH(钥孔血蓝蛋白)或CS(酪蛋白)。根据不同多肽的氨基酸序列组成，为了便于与载体蛋白偶联，需要通过连接序列(又叫连接子或linker)与载体蛋白偶联。本申请中，连接序列优选CGSG。
需要说明的是，上述多肽-载体蛋白偶联物是重组合成表达的蛋白。上述重组表达的多肽-载体蛋白偶联物，除可以检测P53突变蛋白外，基于蛋白的同源性，在较大程度上也可以用于检测与P53蛋白同源异构蛋白或不同突变位点的蛋白。
根据多肽氨基酸的理化性质、所用的载体蛋白的不同及偶联方式的不同，每个载体蛋白所能偶联上的多肽的条数也有所不同。从偶联的效率及对抗体识别结合能力综合考虑，优选每个载体蛋白偶联2～50条多肽，更优选为3～45条、5～40条、5～35条、5～30条、8～30条、10～30条、12～30条、15～30条；或者，更优选为6～36条、8～32条、10～28条、10～26条、10～24条、10～22条、10～20条、10～18条、10～16条及10～15条中的任意一种情况。
上述试剂盒，根据具体需要可以制备成多种不同类型的检测试剂盒。但从方便检测，便于判断检测结果的角度考虑，试剂盒中的多肽可以设置为预包被于固相载体上的形式。具体的预包被的固相载体根据需要合理设计。更优选，固相载体包括酶标板(多为聚苯乙烯材料的)、膜载体或微球；进一步优选地，膜载体包括硝酸纤维素膜(使用最广泛)、玻璃纤维素膜或尼龙膜，更进一步优选地，膜载体上还包被有阳性对照物，多肽-载体蛋白偶联物和阳性对照物按检测顺序在硝酸纤维素膜上依次设置。
根据试剂盒的具体检测方法的不同，试剂盒中具体的配套试剂也相应有所不同，但均可以根据已知试剂盒的配制方式进行组合配套试剂。优选地，上述试剂盒中还包括以下至少之一：(1)酶标二抗，更优选酶标二抗为HRP标记的二抗(对应于ELISA检测试剂盒)；(2)胶体金结合垫，胶体金结合垫上包被有胶体金标记的多肽-载体蛋白偶联物和阳性对照物的特异性结合物(对应于免疫胶体金检测试剂盒)；(3)标记垫，标记垫上包被有荧光标记的微球，微球上负载有阳性对照物的特异性结合物(对应于免疫荧光检测试剂盒)。
上述免疫胶体金检测试剂盒和免疫荧光检测试剂盒检测相对更方便，只需要建立阳性对照的C线和检测样本的T线即可。阳性对照的C线处预包被的阳性对照物，只要是能够随着待测样本的血清层析过程携带过来的带有检测标记的特异性结合物结合即可，对具体的阳性对照物的具体抗原或抗体并无特殊限定。优选地，上述阳性对照物选自鼠免疫球蛋白、人免疫球蛋白、羊免疫球蛋白或兔免疫球蛋白，相应地，阳性对照物的特异性结合物选自抗鼠免疫球蛋白、抗人免疫球蛋白、抗羊免疫球蛋白或抗兔免疫球蛋白。
上述抗鼠免疫球蛋白根据免疫的对象的不同，可以是羊抗鼠的免疫球蛋白或兔抗鼠的免疫球蛋白，或者是其他可免疫的动物抗鼠的免疫球蛋白。同样地，抗人免疫球蛋白、抗羊免疫球蛋白或抗兔免疫球蛋白也可以根据免疫动物的不同，是不同物种来源的抗免疫球蛋白。上述免疫球蛋白可以是IgM、IgG、IgA、IgD或IgE中的任意一种。这些抗免疫球蛋白抗体可以是单克隆抗体或多克隆抗体。
上述试剂盒中，根据所需要检测的样本数量的多少，所使用的酶标板的规格也有所不同，可以在12～384孔酶标板中合理选择。预包被的酶标板中，根据不同多肽-载体蛋白偶联物中的检测对象的不同，每个孔中对多肽-载体蛋白偶联物的包被量也有所差异。类似地，多肽-载体蛋白偶联物在膜载体上(比如，硝酸纤维素膜)的包被量也不同。比如，可以为0.8～8μg/cm，更优选为0.8～7μg/cm，0.8～6μg/cm、0.8～5μg/cm、0.8～4μg/cm、0.8～3μg/cm、0.8～2μg/cm、0.8～1.8μg/cm、0.8～1.7g、0.8～1.6μg/cm、0.8～1.5μg/cm、0.8～1.4μg/cm或0.8～1.2μg/cm。
在一些优选的实施例中，该试剂盒包括芯片，芯片上预置有多肽，其中多肽由上述多肽组成多肽阵列。将上述多肽设置在多肽芯片上进行检测，不仅能利用该多肽对P53突变体的靶向结合能力提高检测的特异性，而且能利用阵列形式提高检测的通量，因而适用于癌症的大规模筛查。
下面将结合具体的实施例来进一步说明本申请的有益效果。需要说明的是，本申请的实施例主要包括以下几部分：
本发明通过对候选的抗癌多肽在不同浓度下(0.1μM，1μM，10μM，100μM)对MDA-MB-231细胞(人乳腺癌细胞)体外抑制活性IC 50值测定，确定了22种多肽的抗癌活性，即为所述抗乳腺癌iCXOncP11a、iCXOncP12a、iCXOncP13a、iCXOncP15a、iCXOncP17a、iCXOncP18a、iCXOncP19a、iCXOncP22a、iCXOncP31a、iCXOncP32a、iCXOncP33a、iCXOncP34a、iCXOncP35a、iCXOncP36a、iCXOncP16a、iCXOncP20a、iCXOncP21a、iCXOncP23a、iCXOncP24a、iCXOncP25a、iCXOncP27a和iCXOncP29a多肽。并且通过查证，根据目前公开的多肽库数据(NCBI多肽组)进行比对，我们发现本次申请的所有多肽为全新的序列。
具体实施方案：
(1)多肽芯片孵育筛选肽库。
(2)计算机docking。
(3)多肽候选序列的合成。
(4)各候选多肽生物学活性测定，基于对抗癌细胞的测定，检测多肽工作浓度，推测体外抑制活性IC 50值。
(5)使用293T细胞验证各候选多肽的细胞毒性。
实施例1
多肽芯片筛选肽库
1)结合肽集合筛选
A，选择候选Y220C、R175H、R273H的突变P53蛋白并对其进行荧光标记(试剂盒：Alexa
Figure PCTCN2022123235-appb-000001
555 Protein Labeling Kit，A20174)，其中，上述P53突变蛋白的来源如表1所示。
表1三种P53突变蛋白的来源：
蛋白 公司 货号 规格
P53 mutant Y220C Cusabio公司 CSB-BP024077HU(M1) 0.5mg
P53 mutant R175H Cusabio公司 CSB-BP024077HU(M2) 0.5mg
P53 mutant R273H Cusabio公司 CSB-BP024077HU(M3) 0.5mg
B，对荧光标记后的P53突变蛋白进行高通量短多肽阵列芯片检测。
a，将荧光标记好的样品按照1∶500、1∶1000、1∶5000、1∶10000、1∶50000、1∶500000及1∶5000000七个浓度梯度稀释，最终稀释浓度中位数在1：10000。
b，将芯片置于芯片水化用具中，加入超纯水没过芯片，在轨道摇床上55+5rpm/min，水化20min。然后用异丙醇喷洒芯片表面后将芯片放入离心机离心干燥。干燥好的芯片 按照实验设计的位置组装成assay cassette。
c，将稀释好的样本按照90μL/孔加入组装好的芯片上，置于恒温振荡仪上振荡孵育1小时。
d，将assay cassette置于洗板机进行清洗。
e，将assay cassette中的芯片进行拆卸、清洗、干燥后组装进imaging cassette，放入Molecular Device公司的ImageXpress micro 4成像仪进行扫描成像。最终每个检测样本得到一张TIFF图片文件，即为原始数据。
C，按照结合信号强度对多肽集合中的多肽序列进行排序；按照预定数量选取排名靠前的多肽序列作为靶点蛋白的候选多肽。
2)抗体孵育筛选有效肽
A，使用上述P53突变蛋白对IST芯片过饱和孵育，使突变蛋白与芯片中的肽段相互结合。
B，抗体PAB1620(Merck货号：MABE339)会结合正常结构的P53，检测PAB1620的结合情况可以说明P53蛋白结构恢复的情况，因此，使用PAB1620进行高通量短多肽阵列芯片检测，筛选出能够恢复P53结构活性的肽段。
C，通过分析免疫指征技术检测后的信号数据，找出特异性信号稳定的特异性结合肽段。
D，与初筛的肽段序列进行匹配，最终得出能够稳定结合突变P53并使其恢复结构活性的肽段。
实施例2
计算机docking(把实施例1的候选肽集进行模拟结合，进行进一步的筛选与验证)。
1)提取受体蛋白坐标：4MZR PDB文件中包含了蛋白、配体和水分子；首先提取出蛋白的坐标。
2)加氢：晶体结构中通常缺少氢原子的坐标(因为氢原子电子少，且质子核对电子吸引能力弱，因此很难定位)。但是在docking过程中，氢原子尤其是极性氢原子对计算静电作用是必须的。因此需要给蛋白加上氢原子。
3)定义配体结合的3D搜索空间：如果结合位点未知，理论上可以定义一个长方体盒子包含整个蛋白或者随便一个特定区域。
4)准备参考配体
从4MZR PDB结构中提取参考配体的原子位置。
与蛋白结构类似，配体的结构也缺少氢原子，因而需要添加氢原子并且定义哪些键是可以旋转的以用于柔性docking。
5)准备docking配置文件，Docking配置文件包含了输入的受体(蛋白)、配体(化合物)和默认搜索参数的信息。
6)Docking D及使用PyMol可视化Docking结果。
7)根据结合力的强弱，结合专业知识挑选潜在的功能肽段进行合成，进一步功能验证肽段的活性。相关结果见表2。
表2
序列号 结合力(kcal/mol)
SEQ ID NO：1 -164.6087
SEQ ID NO：2 -178.5362
SEQ ID NO：3 -170.6706
SEQ ID NO：4 -166.2695
SEQ ID NO：5 -143.2391
SEQ ID NO：6 -165.8533
SEQ ID NO：7 -145.4526
SEQ ID NO：8 -204.2022
SEQ ID NO：9 -120.7323
SEQ ID NO：10 -157.1535
SEQ ID NO：11 -172.8068
SEQ ID NO：12 -159.0396
SEQ ID NO：13 -155.3417
SEQ ID NO：14 -156.6235
SEQ ID NO：15 -186.8108
SEQ ID NO：16 -209.6496
SEQ ID NO：17 -158.2284
SEQ ID NO：18 -126.9338
SEQ ID NO：19 -171.5783
SEQ ID NO：20 -155.7704
SEQ ID NO：21 -109.194
SEQ ID NO：22 -146.3926
实施例3
多肽合成
委托吉尔生物合成候选肽段：末端进行myr修饰，详细信息见表3。
表3
序号 命名 多肽序列 相对分子质量
1 iCXOncP11a myr-RRGPARVSQVPKHL(myr修饰的SEQ ID NO：1) 1600.872
2 iCXOncP12a myr-RRWLPPFGVFS(myr修饰的SEQ ID NO：2) 1361.59
3 iCXOncP13a myr-RRNDYVLRLNKHS(myr修饰的SEQ ID NO：3) 1670.868
4 iCXOncP15a myr-RRYSRAPWSG(myr修饰的SEQ ID NO：4) 1235.346
5 iCXOncP17a myr-RRVEHENAFG(myr修饰的SEQ ID NO：5) 1214.296
6 iCXOncP18a myr-RRPNFPLAQSSD(myr修饰的SEQ ID NO：6) 1387.484
7 iCXOncP19a myr-RRFESKKRSG(myr修饰的SEQ ID NO：7) 1250.406
8 iCXOncP22a myr-RRYWYKNHLYHG(myr修饰的SEQ ID NO：8) 1692.894
9 iCXOncP31a myr-RRLLDAGPSEG(myr修饰的SEQ ID NO：9) 1170.26
10 iCXOncP32a myr-RREPAKSYWSQVE(myr修饰的SEQ ID NO：10) 1635.778
11 iCXOncP33a myr-RRPALGNRLWDAQVE(myr修饰的SEQ ID NO：11) 1780.976
12 iCXOncP34a myr-RRPNYLDQFADG(myr修饰的SEQ ID NO：12) 1451.534
13 iCXOncP35a myr-RRNHHQPADG(myr修饰的SEQ ID NO：13) 1187.236
14 iCXOncP36a myr-RRNAYGQVFD(myr修饰的SEQ ID NO：14) 1225.316
15 iCXOncP16a myr-RRFNFYRQVG(myr修饰的SEQ ID NO：15) 1342.516
16 iCXOncP20a myr-RRWRYYFWKED(myr修饰的SEQ ID NO：16) 1704.89
17 iCXOncP21a myr-RRGHEWPLDG(myr修饰的SEQ ID NO：17) 1222.316
18 iCXOncP23a myr-RRNDEFAQKVS(myr修饰的SEQ ID NO：18) 1349.45
19 iCXOncP24a myr-RRFYQWKELED(myr修饰的SEQ ID NO：19) 1569.72
20 iCXOncP25a myr-RRHNVKWED(myr修饰的SEQ ID NO：20) 1239.352
21 iCXOncP27a myr-RRSRGGDG(myr修饰的SEQ ID NO：21) 859.888
22 iCXOncP29a myr-RRSHPNAHED(myr修饰的SEQ ID NO：22) 1218.236
其中，SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：14、SEQ ID NO：16及SEQ ID NO：22所示的多肽的合成的HPLC图谱如图1a至图1g所示。
实施例4
肽段生物学功能验证试验
一、化学品和试剂：Dulbecco′s Modified Eagle Medium(DMEM)购自Invitrogen(CA，USA)。胎牛血清(FBS)来自HyClone Laboratories，Inc(USA)。二甲基亚砜(DMSO)购自Sigma，MTT试剂盒购于生工生物(E606334-0500)。
二、MTT实验步骤：
1.试剂盒中所有管子在打开之前需要先离心。
2.MTT溶液的配制：用MTT Solvent(组分B)溶解MTT Reagent(组分A)，配制成5mg/ml的MTT溶液。配制后即可使用，或直接-20℃避光保存，也可以根据需要适当分装后-20℃避光保存。
3.每孔加入10μLMTT溶液，使每个孔中的MTT得终浓度为0.5mg/ml。
4.轻轻混匀后，5％CO 2，37℃培养箱中孵育4小时。
5.小心吸取每个孔中的培养基已防止细胞单层破裂。
6.每孔加入100μL Formazan Solubilization Solution(组分C)。
7.将96孔板放在振荡器上轻轻震荡10分钟，直至在普通光学显微镜下观察发现甲臜全部溶解。
8.在酶标免疫检测仪570nm测定吸光度。
三、细胞培养：
1)将MDA-MB-231(来源：ATCC公司)人乳腺癌细胞细胞维持在含有10％胎牛血清(FBS)和1％青霉素和链霉素的Dulbecco′s改良伊格尔培养基(DMEM)中，并且在温度37℃以及二氧化碳(CO 2)含量5％的环境中培养。
2)体外检测多肽的生物活性。
采用MTT(USE)检测法检测细胞增殖情况，先将MDA-MB-231细胞种植于96孔板中，每孔中含5×10 3个细胞，每孔含有10％FBS的100μL DMEM，留空8个孔作为空白对照。孵育(37℃，5％CO 2)过夜，使细胞贴壁。加入不同浓度(0.1μM，1μM，10μM，100μM)精确溶于DMSO的设计多肽，每孔加入含2％FBS的DMEM。孵育(37℃，5％CO 2)48小时，使多肽生效。
每孔加入0.05％MTT溶液。孵育(37℃，5％CO 2)3小时，使MTT代谢。弃去培养基，在100μLDMSO中重悬甲臜(MTT代谢产物)，将甲臜混入溶剂中。读取570nm处的光密度，记录结果。以浓度为横坐标，细胞活力为纵坐标绘制生长曲线，结果如图2a、图2b和图2c所示。
由图2a结果显示七种多肽均在不同程度上显示出抗癌活性，其中尤其以iCXOncP12a的抗癌活性最为显著，在浓度为10～100μM可有效抑制癌细胞生长，由此计算iCXOncP11a的IC 50＝37.64μM，iCXOncP12a的IC 50＝12.98μM，iCXOncP13a的IC 50＝41.23μM，iCXOncP15a的IC 50＝40.95μM，iCXOncP17a的IC 50＝44.36μM，iCXOncP18a的IC 50＝48.52μM，iCXOncP19a的IC 50＝31.3μM，阳性对照多肽ReACp53的IC 50＝52.96μM(阳性对照参考文献：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.)。
由图2b结果显示另外七种多肽均在不同程度上显示出抗癌活性，其中尤其以iCXOncP22a的抗癌活性最为显著，在浓度为10～100μM可有效抑制癌细胞生长，由此计算iCXOncP22a的IC 50＝6.739μM，iCXOncP31a的IC 50＝20.82μM，iCXOncP32a的IC 50＝14.27μM，iCXOncP33a的IC 50＝47.64μM，iCXOncP34a的IC 50＝42.47μM，iCXOncP35a的IC 50＝72.62μM，iCXOncP36a的IC 50＝33.57μM，阳性对照多肽ReACp53的IC 50＝52.96μM。
由图2c结果显示八种多肽均在不同程度上显示出抗癌活性，其中尤其以iCXOncP20a的抗癌活性最为显著，在浓度为10～100μM可有效抑制癌细胞生长，由此计算iCXOncP16a的IC 50＝26.04μM，iCXOncP20a的IC 50＝8.652μM，iCXOncP21a的IC 50＝29.65μM，iCXOncP23a的IC 50＝32.92μM，iCXOncP24a的IC 50＝28.39μM，iCXOncP25a的IC 50＝30.45μM，iCXOncP27a的IC 50＝24.33μM，iCXOncP29a的IC 50＝29.34μM，阳性对照多肽ReACp53的IC 50＝52.96μM。
由此可以判断，本次申请保护的22种多肽均有一定的抗癌效果，且抗癌效果大多数都要好于阳性对照。后续可通过对上述多肽的研究，进一步确认上述多肽化合物的成药性。
上述IC 50值计算方法：基于不同浓度下，多肽对肿瘤细胞的抑制率，绘制横坐标为多肽浓度、纵坐标为肿瘤细胞的抑制率的回归曲线，得出回归方程式与R值，并以50％抑制率为y值，计算IC 50的值。
在另一实验中，通过采用不同浓度(1μM，10μM，100μM)的22种多肽处理乳腺癌细胞，结果如图3a、图3b和图3c所示，多肽处理后的癌细胞生长缓慢，且在100μM的处理下，癌细胞无存活。
实施例5
肽段细胞毒性检测
在不同浓度的多肽作用下，使用MTT检测肾上皮细胞293T(来源：ATCC公司)的细胞活力，并通过结果计算22种多肽对于293T细胞的LD 50浓度。结果如下表4。根据LD 50以及IC 50的结果，iCXOncP12a、iCXOncP13a、iCXOncP18a、iCXOncP22a、iCXOncP36a、iCXOncP20a、iCXOncP21a、iCXOncP23a、iCXOncP24a、iCXOncP25a、iCXOncP29a的IC 50均远小于LD 50，相较于其他的肽段具有较高的药用开发价值。
表4
多肽 IC 50(μM) LD 50(μM)
阳性对照(同实施例4) 52.96 69.47
iCXOncP11a 37.64 33.16
iCXOncP12a 12.98 32.71
iCXOncP13a 41.23 237.8
iCXOncP15a 40.95 19.94
iCXOncP17a 44.36 53.62
iCXOncP18a 48.52 419.4
iCXOncP19a 31.3 23.78
iCXOncP22a 6.739 24.56
iCXOncP31a 20.82 25.67
iCXOncP32a 14.27 20.18
iCXOncP33a 47.64 165.5
iCXOncP34a 42.47 192.2
iCXOncP35a 72.62 155.5
iCXOncP36a 33.57 193.1
iCXOncP16a 26.04 50.26
iCXOncP20a 8.652 57.83
iCXOncP21a 29.65 231.5
iCXOncP23a 32.92 426.7
iCXOncP24a 28.39 447.1
iCXOncP25a 30.45 125.7
iCXOncP27a 24.33 41.65
iCXOncP29a 29.34 793.7
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本发明选择以P53突变体作为抗癌药物的靶点蛋白，通过多肽芯片大规模筛选有潜力的多肽片段，再通过体内外筛选验证的手段，快速的筛选出了具有抗癌活性的多肽药物。本发明所述的iCXOncP11a、iCXOncP12a、iCXOncP13a、iCXOncP15a、iCXOncP17a、iCXOncP18a、iCXOncP19a、iCXOncP22a、iCXOncP31a、iCXOncP32a、iCXOncP33a、iCXOncP34a、iCXOncP35a、iCXOncP36a、iCXOncP16a、iCXOncP20a、iCXOncP21a、iCXOncP23a、iCXOncP24a、iCXOncP25a、iCXOncP27a和iCXOncP29a二十二种多肽(序列信息见表3)，展现出显著的抑制乳腺癌细胞生长效果，综合考虑各多肽的IC 50值的大小、IC 50与LD 50的大小比较，以及显微镜下拍摄的多肽处理后的乳腺癌细胞存活情况(如表5所列)，以iCXOncP12a、iCXOncP22a、iCXOncP16a、iCXOncP20a、iCXOncP21a、iCXOncP29a效果最为显著。
表5
序列号 多肽 IC 50(μM) LD 50(μM) 细胞图片
SEQ ID NO：2 iCXOncP12a 12.98 32.71 图3a
SEQ ID NO：8 iCXOncP22a 6.739 24.56 图3b
SEQ ID NO：15 iCXOncP16a 26.04 50.26 图3c
SEQ ID NO：16 iCXOncP20a 8.652 57.83 图3c
SEQ ID NO：17 iCXOncP21a 29.65 231.5 图3c
SEQ ID NO：22 iCXOncP29a 29.34 793.7 图3c
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种靶向P53突变体的多肽，其特征在于，所述多肽特异性结合P53突变体，且所述多肽选自如下任意一条或多条：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21及SEQ ID NO：22。
  2. 根据权利要求1所述的多肽，其特征在于，所述P53突变体为选自如下至少一个位点突变的突变体：Y220C、R175H或R273H；
    优选地，所述多肽为修饰后的肽段；
    优选地，所述修饰为化学基团修饰或氨基酸修饰；
    优选地，所述化学基团修饰为PEG修饰、乙酰化修饰或酰胺化修饰中的任意一种或多种；
    优选地，所述PEG修饰为直链PEG修饰、具有单官能团的PEG修饰或者具有双官能团的PEG修饰；
    优选地，所述PEG修饰的位点选自所述多肽的N端、C端、Lys侧链和Cys侧链中的任意一个或多个；
    优选地，所述PEG修饰为分子量为500～40000的PEG修饰；
    优选地，所述多肽的N端和C端分别具有所述乙酰化修饰和所述酰胺化修饰；
    优选地，所述多肽的N端具有脂肪酸修饰，更优选为Myr修饰；
    优选地，所述氨基酸修饰为亲水性氨基酸修饰或半胱氨酸修饰；
    优选地，所述亲水性氨基酸修饰为在所述多肽的N端、C端或者NC两端添加1-4个亲水性氨基酸，更优选地，所述亲水性氨基酸为Glu、Lys、Ser或Gly；进一步优选地，所述1-4个亲水性氨基酸选自如下任意一种：Glu-Glu、Lys-Lys或Ser-Gly-Ser；
    优选地，所述半胱氨酸修饰为在所述多肽的如下任一位置添加半胱氨酸：N端、C端、NC两端或肽链中间；
    更优选地，在所述肽链中间添加半胱氨酸包括在所述肽链中间插入一个或多个半胱氨酸，或者一个或多个半胱氨酸以支链形式连接于所述肽链的中间。
  3. 一种多肽药物，其特征在于，所述多肽药物包括权利要求1或2所的多肽以及药学上可选的辅料。
  4. 根据权利要求3所述的药物，其特征在于，所述多肽药物为抗癌药物，优选为抗乳腺癌药物；
    优选地，所述多肽药物中所述多肽的浓度为0.1μM～100μM。
  5. 权利要求1或2所的多肽在制备用于治疗个体肿瘤或癌症的药物中的应用，可选的，所述药物为多肽药物或联合药物。
  6. 根据权利要求5所述的应用，其特征在于，所述癌症包括如下任意一种或多种：乳腺癌、肺癌、鼻咽癌、喉癌、胃癌、肝癌、食道癌、肠癌、胰腺癌、胆囊癌、肾癌、膀胱癌、前列腺癌、白血病、淋巴癌、血管瘤、骨癌、宫颈癌、子官癌、卵巢癌、脂肪癌、脑瘤、鳞癌、皮肤癌、甲状腺癌、唇癌、黑色素癌、舌癌、胸腺癌以及中枢神经系统癌症；
    优选地，所述中枢神经系统癌症为脑癌；
    优选的，所述应用包括通过所述多肽抑制人乳腺癌细胞增殖的功能。
  7. 一种检测P53突变体的试剂盒，其特征在于，所述试剂盒包括利要求1或2所述的多肽。
  8. 根据权利要求7的试剂盒，其特征在于，所述多肽以多肽-蛋白偶联物的形式存在；
    优选地，所述多肽-蛋白偶联物中的蛋白选自如下任意一种：牛血清蛋白、卵清白蛋白、钥孔血蓝蛋白或酪蛋白；
    更优选，所述多肽通过连接序列与所述蛋白偶联形成所述多肽-蛋白偶联物，进一步优选地，所述连接序列为CGSG。
  9. 根据权利要求7或8的试剂盒，其特征在于，所述多肽被包被于固相载体上；
    优选地，所述固相载体包括酶标板、膜载体或微球；
    优选地，所述膜载体包括硝酸纤维素膜、玻璃纤维素膜或尼龙膜；
    优选地，所述膜载体上还包被有阳性对照物，所述多肽和所述阳性对照物按检测顺序在所述膜载体上依次设置；
    优选地，所述试剂盒还包括如下至少之一：
    (1)酶标二抗，更优选所述酶标二抗为HRP标记的二抗；
    (2)胶体金结合垫，所述胶体金结合垫上包被有胶体金标记的抗原和所述阳性对照物的特异性结合物；
    (3)标记垫，所述标记垫上包被有荧光标记的微球，所述微球上负载有所述阳性对照物的特异性结合物；
    优选地，所述阳性对照物选自鼠免疫球蛋白、人免疫球蛋白、羊免疫球蛋白或兔免疫球蛋白，相应地，所述阳性对照物的特异性结合物选自抗鼠免疫球蛋白、抗人免疫球蛋白、抗羊免疫球蛋白或抗兔免疫球蛋白。
  10. 根据权利要求7的试剂盒，其特征在于，所述试剂盒包括芯片，所述芯片上预置有所述多肽组成的多肽阵列。
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