CN118005730A - Antitumor polypeptide targeting P53 mutant protein, application thereof and antitumor pharmaceutical composition - Google Patents

Abstract

The invention provides an anti-tumor polypeptide, application thereof and an anti-cancer pharmaceutical composition, and relates to the technical field of polypeptide medicines. The antitumor polypeptide is a polypeptide with an amino acid sequence shown as seq_1, or a polypeptide with an amino acid sequence shown as seq_1 as an initial structure, and is obtained through further modification. The anti-tumor polypeptide takes p53 as an anti-cancer target point, has a brand new sequence, provides a new thought for developing new active compounds for subsequent medicaments, and can be used as a lead compound for developing the subsequent medicaments.

Description

Antitumor polypeptide targeting P53 mutant protein, application thereof and antitumor pharmaceutical composition

Technical Field

The invention relates to the technical field of polypeptide medicaments, in particular to an anti-tumor polypeptide, application thereof and an anti-cancer pharmaceutical composition.

Background

P53 is a transcription factor specific to DNA sequences and plays an important role in the regulation of a series of biological processes such as cell cycle arrest, apoptosis, DNA damage repair and cell senescence. The p53 protein can maintain the stability of the genome, avoid or reduce the occurrence of mutations, and is therefore also a powerful "genome daemon". The occurrence and development of cancer has a very close relationship with p 53. p53 can protect cells from various stress stimuli, such as oxidative stress, nutritional deprivation, hypoxia, DNA damage, telomere abrasion, oncogene expression and ribosomal dysfunction, through transcription dependent and independent mechanisms.

TP53 (gene encoding p 53) mutation is the most common gene mutation in cancer, and p53 is found to be mutated in most tumor cells by sequencing the genome of different human cancer tissues, and the total mutation rate reaches 50%. Loss of p53 function or mutation can lead to cancer cells escaping apoptosis to gain unlimited proliferation potential, converting from oncostatin to pro-oncoprotein. Such as breast cancer. Thus, drugs that activate wild-type p53 or restore mutant p53 are sought to exert anticancer effects.

Breast cancer is one of the most common malignant tumors of females, and according to the global latest cancer burden data in 2020 issued by the international cancer research Institute (IARC) of the world health organization, the global new breast cancer in 2020 reaches 226 ten thousand cases, and exceeds lung cancer (221 ten thousand cases) for the first time, and becomes the global first cancer. China is a large country of breast cancer, about 42 tens of thousands of new breast cancers occur in 2020, and almost 12 tens of thousands of deaths occur. Breast cancer usually occurs in mammary gland epithelial tissue, with women in large numbers, and men in 0.5% to 1% of all breast cancer patients. Methods of treatment for breast cancer include surgical excision, radiation therapy, chemotherapy, hormonal therapy, and the like. In recent years, molecular targeted therapy is taken as a new means for treating breast cancer, shows a certain curative effect in the treatment of breast cancer, and is increasingly receiving academic attention.

The molecular targeting treatment uses specific gene fragments in tumor cells as treatment sites, and the purpose of treating diseases is achieved by regulating or blocking the functions of the gene fragments. The breast cancer molecular targeting therapy refers to the treatment of signal pathways related to the occurrence and development of breast cancer and oncogene related expression products thereof. Molecular targeted drugs control changes in cellular gene expression by blocking signal transduction of tumor cells or related cells, thereby inhibiting or killing tumor cells. The targeting therapy has strong specificity and obvious effect, and does not damage normal tissues basically, so the tumor targeting therapy is the most promising scheme in tumor therapy.

Polypeptides are compounds of three or more amino acid molecules linked by peptide bonds. As one of important living things, peptide substances are widely present in living bodies to regulate the functional activities of various system organs and cells in the body. In recent years, polypeptide drugs have been paid attention to in the development of antitumor drugs by virtue of their high targeting property, low immunogenicity, high tissue permeability, safety and the like. In 2019, the worldwide market for anti-tumor polypeptide therapy has estimated at $86 billion and is expected to keep a steady trend in the next decade.

Therefore, how to develop a batch of polypeptide drug molecules with potential medicinal value and taking p53 as an anticancer target point also becomes a great hotspot problem for developing antitumor drugs.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an anti-tumor polypeptide which takes p53 as an anti-cancer target point, has a brand new sequence, provides a new thought for developing new active compounds for subsequent medicaments, and can be used as lead compounds for developing the subsequent medicaments.

It is another object of the present invention to provide the use of the above antitumor polypeptide and an antitumor pharmaceutical composition comprising the same.

In order to solve the technical problems, the invention adopts the following technical scheme:

According to one aspect of the present invention, there is provided an anti-tumour polypeptide, which is a polypeptide having an amino acid sequence as shown in seq_1, or a modified polypeptide having said amino acid sequence as shown in seq_1.

Preferably, the modification comprises one or more of amidation modification, sulfation modification, acetylation modification, fatty acidification modification, glycosylation modification, phosphorylation modification, PEG modification, amino acid modification, transmembrane peptide modification and cyclization modification;

Preferably, the modification site includes, but is not limited to, one or more of an N-terminal modification, a C-terminal modification, a side chain modification, a modification of an amino acid residue in the middle of the polypeptide, and a backbone modification;

Preferably, the anti-tumor polypeptide is a polypeptide with an amino acid sequence shown as seq_1, two arginines are added at the N end of the polypeptide, and the sequence shown as seq_2;

Preferably, the anti-tumor polypeptide is a polypeptide with an amino acid sequence shown as seq_1, wherein two arginines are added at the N end of the polypeptide, and the N end is modified by myristoylation, and the sequence is shown as seq_3.

According to another aspect of the invention, the invention also provides the use of the above anti-tumour polypeptide for targeting p53 mutants for non-diagnostic and therapeutic purposes, or for preparing products for targeting p53 mutants.

Preferably, the p53 mutant is a mutant of at least one site mutation selected from the group consisting of: S121F, V122G, C V, C141V, W146Y, C182S, V A, R209P, C229Y, H233Y, Y234F, N235K, Y F, T253V, N268D, E M or S315R.

According to another aspect of the invention, the invention also provides the use of the above anti-tumor polypeptide in the preparation of a medicament for preventing and/or treating cancer.

Preferably, the cancer comprises any one or more of the following: breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, stomach cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gall bladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, cancer of the uterine cervix, ovarian cancer, fat cancer, brain tumor, squamous carcinoma, skin cancer, thyroid cancer, lip cancer, melanin cancer, tongue cancer, thymus cancer, and central nervous system cancer;

Preferably, the central nervous system cancer is brain cancer;

Preferably, the use is in the manufacture of a medicament for the prevention and/or treatment of breast cancer;

Preferably, the anti-tumour polypeptide is selected from the group consisting of polypeptides having the amino acid sequence shown as seq_1, seq_2 or seq_3.

According to another aspect of the present invention, there is also provided an anticancer pharmaceutical composition comprising the above antitumor polypeptide, and pharmaceutically optional excipients.

Preferably, anticancer drugs and/or adjuvant therapeutic drugs are also included;

Preferably, the drug for treating cancer comprises one or more of methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecins, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxine, L-asparaginase, docetaxel and capecitabine.

Preferably, the anticancer pharmaceutical composition is for treating breast cancer, further comprising docetaxel;

compared with the prior art, the invention has the following beneficial effects:

The invention provides a novel polypeptide compound with anticancer activity, which is a polypeptide with an amino acid sequence shown as seq_1 (RPLLTRVTSRGP) or a polypeptide molecule with an amino acid sequence shown as seq_1 as an initial structure and obtained by further modification by taking p53 mutant protein as a target protein of an anticancer drug and combining AI aided design and screening means.

The polypeptide with the amino acid sequence shown as the seq_1 can be combined with the p53 mutant, so that the p53 mutant is prevented from being converted into a functional state from an aggregation state, and the cancer promotion function of the p53 mutant is restored to the cancer inhibition function of an unmutated wild type. The polypeptide molecules obtained by further modification can further improve the properties of the polypeptide compounds, such as stability, activity or film permeability, on the basis of not affecting the original functional polypeptide fragments.

The polypeptide which can be specifically combined with the p53 mutant and is screened by the invention has important value in the aspect of medicinal development for treating cancers. The antitumor polypeptide has the advantages of specific target, low toxicity and low side effect as a medicament, and overcomes the defects of the current treatment means to a certain extent. Meanwhile, the method has an important inspiring effect on the research of p53 mutant protein serving as an anticancer target drug, provides a new thought for the research of subsequent drugs to develop new active compounds, and can be used as a lead compound for the research of subsequent drugs.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is an HPLC chart of iCXOncP a;

FIG. 2 is a graph of MTT results of iCXOncP a on MDA-MB-231 cell proliferation, with the abscissa indicating polypeptide drug concentration, 0 indicating negative control without drug, and P <0.05, considered statistically different;

FIG. 3 is a graph showing the effect of iCXOncP a on MDA-MB-231 cell proliferation inhibition, wherein WT represents a wild-type, non-dosed negative control;

FIG. 4 is a graph showing the results of the weight change of mice in each experimental group in effect example 3;

FIG. 5 is a graph showing the results of tumor volume in mice of each experimental group in effect example 3 over time;

FIG. 6 is a graph showing the results of the change in tumor weight in mice of each experimental group in effect example 3 over 17 days.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references, as well as laboratory procedures and techniques thereof, are those well known and commonly used in the art.

It should be noted that:

In the present invention, all embodiments and preferred methods of implementation mentioned herein may be combined with each other to form new technical solutions, if not specifically stated; all technical features and preferred features mentioned herein may be combined with each other to form new solutions; the components involved or their preferred components can be combined with one another to form new technical solutions.

According to one aspect of the present invention, there is provided an antitumor polypeptide which is a polypeptide having an amino acid sequence shown as seq_1 or a polypeptide having a modified amino acid sequence shown as seq_1, i.e., a polypeptide molecule obtained by further modification with a polypeptide having an amino acid sequence shown as seq_1 as a starting structure.

The anti-tumor polypeptide is obtained based on a polypeptide chip technology, the polypeptide chip technology is a detection technology based on a polypeptide chip, various polypeptides on the polypeptide chip are used for being contacted with a sample, then each characteristic signal (particularly a fluorescent image carrying each characteristic signal) on the polypeptide chip is collected by using an image collection technology, and then the signal intensity of each characteristic in the chip, namely the detection result data of the polypeptide chip, is output. Based on the sample detection signal output by the detection result data of the polypeptide chip, the analysis of the analyte in the sample combined with the polypeptide on the polypeptide chip, the analysis of the sample and the like can be realized.

The invention uses p53 mutant as target protein of anticancer drug, combines AI aided design and screening means, and finds out polypeptide sequence with anticancer activity, namely sequence shown in seq_1. The polypeptide with the amino acid sequence shown as the seq_1 can be combined with the p53 mutant, so that the p53 mutant is prevented from being converted into a functional state from an aggregation state, and the cancer promotion function of the p53 mutant is restored to the cancer inhibition function of an unmutated wild type.

The p53 mutant is any mutant capable of causing the loss of cancer suppressing function of the p53 mutant protein, and the p53 mutant includes, but is not limited to, mutants with at least one of the following site mutations: S121F, V122G, C V, C141V, W146Y, C182S, V A, R209P, C229Y, H233Y, Y234F, N235K, Y F, T253V, N268D, E M or S315R.

The polypeptide with the sequence shown in the seq_1 has anticancer activity, is a brand new polypeptide compound, and has the advantages of target specificity, low toxicity and low side effect when being used as a medicament. The method overcomes the defects of the current therapeutic products to a certain extent, and has important inspiring effect on the research of p53 mutant protein serving as an anticancer target drug. The anti-tumor polypeptide taking the screened polypeptide sequence as a core shows remarkable effect of inhibiting the growth of breast cancer cells, and can provide a new thought for developing new active compounds for subsequent medicaments. Meanwhile, the brand new anticancer polypeptide sequence provided by the invention can be used as a lead compound for the research and development of subsequent medicaments.

The polypeptide has a remarkable structural advantage, and can be modified at one or two ends of the polypeptide on the basis of not affecting the original functional polypeptide fragment, so that the polypeptide has multiple functions, for example, some optional chemical modification mechanisms for leading pH sensitive liposome with receptor/ligand binding capacity to enter cells can induce tumor tissue cells to actively endocytose the liposome and then release medicines. Therefore, according to the requirement of drug development, the polypeptide can be modified according to the requirement, the invention is not limited to the above, and the modified polypeptide is based on the polypeptide with the amino acid sequence shown as seq_1, and the modified polypeptide also belongs to the protection scope of the invention.

Specific modifications are well known in the art and are intended to be included within the scope of the invention, as defined in the various general and more specific textbooks, references, handbooks, trade shows, standard documents, equipment specifications, and the like. Specific modifications include, but are not limited to: amidation modification, sulfation modification, acetylation modification, fatty acidification modification, glycosylation modification, phosphorylation modification, PEG modification, amino acid modification, membrane-penetrating peptide modification and cyclization modification. Modification introduction the present invention is not limited, and alternatively by solid phase synthesis or biosynthesis, the most well-established and widely used chemical modification methods currently available, including liquid phase methods and solid phase methods, may be employed.

Modification sites include, but are not limited to, one or more of N-terminal modification, C-terminal modification, side chain modification, modification of amino acid residues in the middle of the polypeptide, and backbone modification, specific examples include, but are not limited to: c-terminal modifications including, but not limited to, amidation and/or sulfation; n-terminal modifications including, but not limited to, acetylation and/or lipolysis; modifications of intermediate amino acid residues of polypeptides, including, but not limited to, glycosylation modifications that bind Ser-, tyr-, asn-, thr-; and one or more of phosphorylation modifications to Ser-, tyr-, thr-binding; cyclized modifications, including but not limited to side chain-to-side chain, terminal-to-side chain, or terminal-to-terminal or head-to-tail.

In some alternative embodiments, the PEG modification is a linear PEG modification, a PEG modification with a monofunctional group, or a PEG modification with a difunctional group; preferably, the site of PEG modification is selected from any one or more of the N-terminus, C-terminus, and side chain of the polypeptide; preferably, the PEG modification is one having a molecular weight of 500 to 40000.

Amino acid modification refers to the addition of an amino acid to a polypeptide having an amino acid sequence such as that shown in seq_1, where the addition site includes, but is not limited to, the N-terminus, the C-terminus, or both the N-and C-termini, or in the middle of the peptide chain of the polypeptide, where the addition in the middle of the peptide chain of the polypeptide includes insertion of the amino acid as a modification in the middle of the peptide chain or attachment in branched form in the middle of the peptide chain. The amino acid modification is preferably a hydrophilic amino acid modification or a cysteine modification.

In some alternative embodiments, the hydrophilic amino acid is modified to add 1-4 hydrophilic amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptide, more preferably, the hydrophilic amino acids are one or more of Glu (glutamic acid), lys (lysine), ser (serine), arg (arginine), and Gly (glycine); further preferably, 1 to 4 hydrophilic amino acids are selected from any one of the following: glu-Glu, lys-Lys, arg-Arg or Ser-Gly-Ser.

In some alternative embodiments, to facilitate the entry of the polypeptide into the cell across the cell membrane, arginine (Arg) may be added at the N-terminus on the basis of the polypeptide having an amino acid sequence as shown in seq_1, such that the polypeptide has a positive charge at the N-terminus. The sequence obtained by adding two arginines (Arg) to the N-terminal was RRRPLLTRVTSRGP (seq_2).

In some alternative embodiments, cysteine modifications may be made to the polypeptide in order to better achieve directional coupling of peptide fragments. Specifically, including but not limited to adding at the N-terminus, C-terminus, or both the N-and C-termini of a peptide fragment, or adding a cysteine in the middle of the peptide chain of a polypeptide. When cysteines are added in the middle of the peptide chain of a peptide fragment, one or more cysteines may be inserted in the middle of the peptide chain (i.e., between two amino acid residues), or one or more cysteines may be linked in branched form in the middle of the peptide chain (i.e., as a side chain of an amino acid in the middle of the peptide chain).

In some alternative embodiments, the fatty acid modifications include myristoylation modifications and palmitoylation modifications, and the fatty acid acylation N-terminal can allow the polypeptide or protein to bind to a cell membrane, thereby improving the membrane permeability of the polypeptide molecule.

In some alternative embodiments, the anti-tumor polypeptide is a polypeptide having an amino acid sequence as set forth in seq_1 that has been at least one of acetylated modified and amidated modified. Because chemically synthesized polypeptides often carry free amino groups and free carboxyl groups, and the sequence of the polypeptide often represents the sequence of the parent protein in which it is located, in order to make the synthesized protein more similar to the parent protein in terms of sequence and activity, the ends of the polypeptide are usually blocked, typically the N-terminus is acetylated and the C-terminus is amidated, so that the modification reduces the total charge of the polypeptide, reduces the solubility of the polypeptide, and thus also allows the polypeptide to mimic its original state of alpha amino groups and carboxyl groups in the parent protein. Thus, in other embodiments, the modifications are double-ended modifications to the polypeptide, more preferably, acetylation modifications and amidation modifications to the N-terminus and C-terminus of the polypeptide, respectively.

In some alternative embodiments, the anti-tumor polypeptide is in the form of a linear polypeptide with a transmembrane peptide or a cyclic polypeptide without a transmembrane peptide in order to more effectively enable the polypeptide having an amino acid sequence as shown in seq_1 to cross the cell membrane as a drug and enter the body to bind to the targeting of the p53 mutant. The transmembrane peptide is optionally disposed at the C-terminus of the polypeptide or at one end of a drug molecule, which can be brought into a particular type of cell where the molecule then undergoes a biochemical reaction. The transmembrane peptide may optionally be a peptide fragment rich in basic amino acids, including, but not limited to, arginine (Arg) or lysine (Lys).

In some alternative embodiments, the anti-tumor polypeptide is a polypeptide having an amino acid sequence as shown in seq_1 with two arginines added to the N-terminus of the peptide fragment, i.e., a sequence as shown in seq_2 (RRRPLLTRVTSRGP).

In some alternative embodiments, the anti-tumor polypeptide is a polypeptide with an amino acid sequence such as that shown in seq_1, two arginines added to the N-terminus of the peptide, and Myr (myristoylation) modified N-terminus, and a sequence such as that shown in seq_3 (Myr-RRRPLLTRVTSRGP).

Based on the fact that the polypeptide with the screened amino acid sequence shown as the seq_1 can be combined with the p53 mutant, the invention also provides application of the anti-tumor polypeptide in targeting the p53 mutant for non-diagnosis and treatment purposes or preparing a product for targeting the p53 mutant. Wherein the mutation site of the p53 mutant includes, but is not limited to, one or more of S121F, V122G, C135V, C V, W146Y, C182S, V203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268D, E294M and S315R. The non-diagnostic and therapeutic purpose of the p53 mutant, or products for targeting the p53 mutant, are optionally useful for studying p 53-associated signaling pathways or tumor-associated mechanisms.

According to another aspect of the invention, the invention also provides the use of the above anti-tumor polypeptide in the preparation of a medicament for preventing and/or treating cancer.

Since p53 is a tumor suppressor protein, it plays an important role in suppressing the mechanism of cancer occurrence. Thus, the polypeptides screened for the ability to specifically bind to p53 mutants of the invention are of great value in the development of pharmaceutical agents for the treatment of cancer. Cancers include, but are not limited to, breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gall bladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphatic cancer, hemangioma, bone cancer, cervical cancer, cancer of the uterine cervix, ovarian cancer, fat cancer, brain tumor, squamous carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymus cancer, and central nervous system cancer. Of these, the central nervous system cancer is preferably brain cancer. Preferably, the antitumor polypeptide is applied to the preparation of a medicament for preventing and/or treating breast cancer. It is further preferred that the antitumor polypeptide having an amino acid sequence as shown in seq_1, seq_2 or seq_3 is used for the preparation of a medicament for preventing and/or treating cancer.

Based on the above inventive concept of applying the anti-tumor polypeptide to the preparation of a medicament for preventing and/or treating cancer, the invention also provides an anti-cancer pharmaceutical composition, which comprises the anti-tumor polypeptide and pharmaceutically optional auxiliary materials. The medicine containing the antitumor polypeptide has the advantages of high targeting binding capacity with p53 mutant, high anticancer activity, low toxicity and low side effect.

The anticancer pharmaceutical composition can be prepared into different dosage forms to adapt to various administration routes, wherein the administration routes comprise oral administration, injection administration or transdermal administration. The dosage forms of the medicine include, but are not limited to, capsules, tablets, granules, oral solutions, oral suspensions, oral emulsions, injections, transdermal absorbents, suppositories, gels, paints, film coating agents or patches, and the like.

The pharmaceutically optional auxiliary materials can be selected reasonably from the existing medicinal auxiliary materials according to the dosage form and/or the administration mode and the administration route of the medicament to be prepared. Including but not limited to one or more of solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, bacteriostats, suspending agents, coating agents, film formers, fragrances, viscosity enhancers, anti-adhesion agents, antioxidants, anti-oxidant potentiators, chelating agents, pH adjusters, adsorbents, plasticizers, surfactants, thickeners, inclusion agents, protectants, humectants, softeners, absorbents, diluents, release modifiers, pressure sensitive adhesives, hardening agents, hollow capsules, matrices, and pharmaceutical carrier materials.

Specific examples the medicament of the present invention may be formulated into injections, capsules, tablets, oral solutions, oral suspensions or transdermal absorbents, etc. according to methods known in the pharmaceutical industry. Optionally, the injection is an intravenous injection; alternatively, sucrose, lactose, galactose, corn starch, gelatin, microcrystalline cellulose, carboxymethyl cellulose, etc. may be used as a carrier or excipient in the preparation of a capsule or tablet suitable for oral administration; alternatively, the anticancer pharmaceutical composition may be formulated into an oral solution or an oral suspension suitable for oral administration using methods and auxiliary ingredients known in the pharmaceutical industry; alternatively, if solutions and suspensions suitable for parenteral administration are prepared, distilled water, water for injection, isotonic sodium chloride or dextrose solution, or a low concentration (e.g., 1 to 100 mm) Phosphate Buffer (PBS) may be used as a carrier or diluent. One or more other auxiliary ingredients or additives may be added to these formulations for parenteral administration, for example, ascorbic acid may be used as an antioxidant, sodium benzoate and the like may be used as a preservative. Other solubilizing agents, disintegrants, colorants, dispersants or surfactants may also be included in the formulations of these dosage forms.

In some alternative embodiments, the anti-tumor polypeptide may also be used in combination with other drugs, including but not limited to anti-cancer drugs and/or adjunctive therapeutic drugs.

The anticancer drug may be selected according to the type of cancer diagnosed with the drug to be prepared, and the present invention is not limited thereto, including but not limited to one or more of methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecine, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxine, L-asparaginase, docetaxel and capecitabine.

The auxiliary therapeutic agents include, but are not limited to, agents that help promote absorption of the primary agent, prevent or reduce adverse reactions, or enhance body functions, and the auxiliary therapeutic agents may be selected from the following classes according to their function: enhancing one or more of tissue metabolism, blood circulation, neurotrophic, vitamins, electrolytes, free radical scavenger, immunomodulator and novel saccharide transfusion.

In some preferred embodiments, the anti-cancer pharmaceutical composition comprises an anti-tumor polypeptide having a sequence as shown in seq_1, seq_2 or seq_3, preferably an anti-tumor polypeptide having a sequence as shown in seq_3.

In some preferred embodiments, the anti-cancer pharmaceutical composition is for use in the treatment of breast cancer, comprising the anti-tumor polypeptide and docetaxel, preferably comprising an anti-tumor polypeptide and docetaxel having the sequences shown in seq_3. The mass ratio of docetaxel to the anti-tumor polypeptide sum is preferably 5: (20-80), for example, may be 5:20, 5:30, 5:40, 5:50, 5:60, 5:70 or 5:80. Experiments show that the anti-tumor polypeptide and docetaxel are combined for use, and the anti-cancer effect is better than that of docetaxel alone.

The advantageous effects of the present invention will be further described below in connection with specific examples. It should be noted that the embodiments of the present invention mainly include the following parts:

A. data analysis: screening binding peptide fragments with obvious difference between two groups of target protein and positive control polypeptide according to peptide library contained in the existing polypeptide chip;

Ai calculation: specific polypeptide characteristics are randomly sheared, binding force with p53 mutant proteins is calculated, and polypeptides with the binding force ranked at the front are screened and compared with the prior knowledge base;

C. and (3) experimental verification: the synthesis of potential polypeptides is carried out in cell experiments and animal experiments for polypeptide anticancer, and verification is carried out to screen active polypeptides.

Example 1

Polypeptide library screening and AI analysis

1. The binding peptide fragments with significant differences were initially screened as a first candidate polypeptide pool by using the crystal structure of P53 muteins in the polypeptide chip (see PDB document number and website below, specifically covering mutants involving any one or more of the following 17 mutation sites: S121F, V122G, C135V, C141V, W Y, C182 56203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268 294M or S315R, site alignment with the protein sequence numbered P04637D as wild-type P53 protein sequence), by comparing the binding energy with the binding energy of positive polypeptides in the peptide library. Aiming at the first candidate polypeptide set, the AutoDock CrankPep software is utilized to calculate the binding force between each candidate polypeptide and the p53 mutant protein based on the Docking strategy, and then a second candidate polypeptide set with higher binding force is obtained through screening.

1) Search of three-dimensional structure of p53 mutant protein: downloading the three-dimensional conformation file of the p53 mutant protein from a PDB database, wherein the PDB file number and the website are as follows:

[RCSB PDB-4MZR:Crystal structure of a polypeptide p53 mutant bound to DNA](https://www.rcsb.org/structure/4MZR).

2) Preparing a receptor protein: extracting receptor protein coordinates: the 4MZR PDB file contains proteins, ligands and water molecules; first, the coordinates of the protein are extracted.

Hydrogenation: the coordinates of the hydrogen atoms are usually lacking in the crystal structure (because the hydrogen atoms have few electrons and protons have weak electron-attracting ability and thus are difficult to locate). However, during the docking process, hydrogen atoms, especially polar hydrogen atoms, are necessary to account for the electrostatic effects, and therefore the addition of hydrogen atoms to proteins is required.

Defining a ligand-bound 3D search space: if the binding site is unknown, it is theoretically possible to define a cuboid box containing the entire protein or optionally a specific region.

3) Preparing a reference ligand: the atomic position of the reference ligand is extracted from the 4MZR PDB structure.

Similar to protein structures, the structure of the ligand also lacks hydrogen atoms, thus requiring the addition of hydrogen atoms and defining which bonds are rotatable for flexible docking.

4) Preparing a locking configuration file: docking profiles contain information of entered receptors (proteins), ligands (compounds) and default search parameters.

5) DockingD and visualising Docking results using PyMol.

2. According to the strength of binding force of candidate polypeptide, the length of polypeptide, the fluctuation degree of binding force and the average value of binding force are considered, and then the potential functional peptide fragments are selected to verify the anticancer activity by combining the related knowledge of known target proteins. And the polypeptide of the invention is found to be a brand new polypeptide sequence by comparison according to the presently disclosed polypeptide library data (NCBI polypeptide group).

It should be noted that, in order to facilitate the polypeptide to cross the cell membrane and enter the cell, an R amino acid may be added to the N-terminal based on the sequence of seq_1, so that the polypeptide has a positive charge at the N-terminal. Thus, in chemical synthesis, sequence RRRPLLTRVTSRGP (seq_2) with two additional R amino acids at the N-terminus was synthesized, and the binding energy of the polypeptide shown in seq_2 is: 187.71kcal/mol.

Example 2

Polypeptide synthesis

Entrusting Jil biosynthesis of candidate peptide and positive control polypeptide, the peptide synthesized in this example is modified by myr, the sequence is myr-RRRPLLTRVTSRGP (seq_3), the number is: iCXOncP7a, and performing HPLC analysis. The synthetic HPLC profile of the polypeptide shown in seq_3 is shown in FIG. 1. The myr modification is commonly known as myristoylation (N-Myristoylation) and is catalysed by N-myristoyltransferase (N-myristoyltransferase, NMT, EC 2.3.1.97). Most N-myristoylated proteins are membrane-bound and by such modification the ability of the polypeptide to penetrate the cell membrane is enhanced, thereby facilitating the binding of the polypeptide to the target protein.

Effect example 1

Peptide fragment biological function verification test

1. Chemicals and reagents, see table 1:

Table 1:

2. MTT assay procedure:

(1) All tubes need to be centrifuged before they are opened.

(2) Preparation of MTT solution: MTT REAGENT (component A) was dissolved in MTT Solvent (component B) to prepare a 5mg/ml MTT solution. Can be used after preparation, or stored at-20deg.C in dark place, or packaged appropriately according to requirement, and stored at-20deg.C in dark place.

(3) Mu.L of MTT solution was added to each well to give a final concentration of MTT in each well of 0.5mg/ml.

(4) After gentle mixing, 5% CO ₂ was incubated in an incubator at 37℃for 4 hours.

(5) The medium in each well was carefully aspirated to prevent rupture of the cell monolayer.

(6) 100 Μ L Formazan Solubilization Solution (component C) was added per well.

(7) The 96-well plate was gently shaken on a shaker for 10 minutes until complete dissolution of the formazan was observed under a common light microscope.

(8) The absorbance was measured at 570nm in an enzyme-labeled immunoassay instrument.

3. Inhibition of MDA-MB-231 (ATCC) cell Activity

(1) Cell culture: MDA-MB-231 (ATCC) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin, and placed in a humid, 5% CO ₂ environment at 37 ℃.

(2) In vitro detection of the biological Activity of the polypeptide: cell proliferation was examined by MTT (USE) assay, MDA-MB-231 (ATCC) cells were first seeded in 96-well plates containing 5X 10 ³ cells per well, and 1 00. Mu.L of DMEM containing 10% FBS was added per well, leaving 8 wells empty as a blank. Incubation (37 ℃,5% co ₂) was carried out overnight to allow cells to adhere to the wells. Different concentrations (0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M) of the designed polypeptide, precisely dissolved in DMSO, were added, as well as positive controls, DMEM with 2% FBS was added per well. Incubation (37 ℃,5% co ₂) was carried out for 48 hours to validate the polypeptide.

0.05% MTT solution was added per well. MTT was metabolized by incubation (37 ℃,5% CO ₂) for 3 hours. Media was discarded, formazan (MTT metabolite) was resuspended in 100 μl DMSO and mixed in solvent. The optical density at 570nm was read and the results recorded. The growth curve was plotted with the concentration on the abscissa and the cell survival on the ordinate, and the results are shown in fig. 2.

The results of fig. 2 show that: iCXOncP7a polypeptide can effectively inhibit the growth of cancer cells, and has obvious anticancer activity at the concentration of 10-100 mu M. IC ₅₀ =12.69 μm of iCXOncP a was calculated from the experimental data. From this, it was confirmed that the polypeptide having the sequence selected in example 1 had a certain anticancer effect, and the anticancer effect was superior to that of the positive control. The patentability of the polypeptide compound can be further confirmed by subsequent studies on the polypeptide.

IC ₅₀ =58.5 μm of positive control polypeptide ReACp (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.).

The IC ₅₀ value calculating method comprises the following steps: based on the inhibition rate of the polypeptide to the tumor cells at different concentrations, a regression curve of the abscissa-polypeptide concentration and the ordinate-inhibition rate of the tumor cells is drawn to obtain a regression equation and an R value, and the value of IC ₅₀ is calculated by taking the 50% inhibition rate as a y value.

Effect example 2

Microscopic observation of cell proliferation

By treating iCXOncP a with different concentrations (1. Mu.M, 10. Mu.M, 100. Mu.M) of the polypeptide, the results are shown in FIG. 3, in which cancer cells after polypeptide treatment grow slowly and are essentially non-viable at 10. Mu.M-100. Mu.M. This demonstrates that the above polypeptides have significant activity against breast cancer cells.

Effect example 3

Inhibition of tumors in mice

The quarantine-qualified mice are taken, the skin of the injection site of the mice is disinfected, and 0.1mL of cell suspension with the cell concentration of 5 multiplied by 10 ⁷/mL is extracted by a 1mL syringe and inoculated under the right armpit of the mice, namely, each mouse is inoculated with 5 multiplied by 10 ⁶ cells subcutaneously. When the average tumor volume grows to about 60mm ³, 64 random group administrations are carried out by selecting relatively uniform tumor volume.

① Model control group; ② Positive drug vehicle control group; ③ Docetaxel group; ④ Capecitabine group; ⑤ A low dose group of polypeptides; ⑥ Dose group in polypeptides; ⑦ A high dose group of polypeptides; ⑧ Docetaxel + polypeptide low dose group.

The model control group is subjected to polypeptide drug solvent (physiological saline) control treatment once a day according to the polypeptide administration treatment group; the positive medicine solvent control group is treated with the positive medicine solvent once a day, namely the solvent of docetaxel and capecitabine medicines, wherein the solvent of the specific capecitabine medicine is corn oil solution containing 2% of ethanol, the solvent of the docetaxel medicine is corn oil solution containing 2% of DMSO, and the solvents of the docetaxel medicine and the capecitabine medicine are used for mice simultaneously; docetaxel group was injected once daily with docetaxel (5 mg/kg); capecitabine groups were injected once daily with capecitabine (200 mg/kg); the polypeptide low dose group was injected once daily with low dose polypeptide (20 mg/kg); the polypeptide dose group was injected with medium dose of polypeptide (40 mg/kg) once daily; the polypeptide high dose group was injected once daily with high dose polypeptide (80 mg/kg); docetaxel + polypeptide low dose group docetaxel (5 mg/kg) +low dose polypeptide (20 mg/kg) was injected once daily. The dosing experimental groups and dose designs are shown in table 2. The tumor volume and body weight of the mice were measured once every two days, the mice were sacrificed after the end of treatment, the tumor weights were weighed, and the data were analyzed and plotted using GRAPHPAD PRISM software.

TABLE 2 administration protocol grouping and dose design table

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Remarks: [1] i.p. means intraperitoneal injection, i.g. means lavage, s.c. means subcutaneous injection;

[2] taking the first administration day as the first day, recording as D1, and the rest of the time and so on;

[3] Represents once daily administration for a total of 12 administrations;

[4] Once every 7 days, 2 times in total.

The body weight of the mice is plotted on the ordinate and the time is plotted on the abscissa. As shown in FIG. 4, iCXOncP a and the positive drug had no significant effect on the body weight of the mice compared to the model control group and the positive drug vehicle control group. Fig. 5 shows the change of tumor volume in mice of each experimental group over time, fig. 6 shows the change of tumor weight in mice of each experimental group over 17 days (each point in the figure represents the tumor mass of each mouse in the experimental group), and the result shows that iCXOncP a has a significant inhibition effect on tumor growth, has a significant difference in tumor volume and weight compared with a model control group, and is dose-dependent. Compared with the positive drug group (docetaxel group/capecitabine group), the inhibition effect of the polypeptide iCXOncP a on tumors is higher than that of the positive drug group (P < 0.05), and the inhibition effect of the docetaxel+polypeptide low-dose group on mice tumors is also obviously higher than that of the docetaxel group. Further demonstrating the safety and efficacy of iCXOncP a in treating breast cancer.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

1. An anti-tumor polypeptide, characterized in that the anti-tumor polypeptide is a polypeptide with an amino acid sequence shown as seq_1 or a modified polypeptide with an amino acid sequence shown as seq_1.

2. The anti-tumor polypeptide of claim 1, wherein the modification comprises one or more of amidation modification, sulfation modification, acetylation modification, fatty acidification modification, glycosylation modification, phosphorylation modification, PEG modification, amino acid modification, transmembrane peptide modification, and cyclization modification;

Preferably, the modification site includes, but is not limited to, one or more of an N-terminal modification, a C-terminal modification, a side chain modification, a modification of an amino acid residue in the middle of the polypeptide, and a backbone modification;

preferably, the PEG modification is a linear PEG modification, a PEG modification with a monofunctional group, or a PEG modification with a difunctional group;

Preferably, the site of PEG modification is selected from any one or more of the N-terminus, C-terminus and side chain of the polypeptide;

preferably, the PEG modification is PEG modification with molecular weight of 500-40000;

Preferably, the amino acid modification comprises a hydrophilic amino acid modification or a cysteine modification;

Preferably, the hydrophilic amino acid is modified to add 1-4 hydrophilic amino acids to the N-terminal and/or C-terminal of the polypeptide with the amino acid sequence shown as seq_1;

preferably, the hydrophilic amino acids include one or more of Glu, lys, ser, arg and Gly;

preferably, the 1 to 4 hydrophilic amino acids are selected from any one of the following: glu-Glu, lys-Lys, arg-Arg or Ser-Gly-Ser;

Preferably, the cysteine is modified to add a cysteine at a position of the polypeptide having the amino acid sequence shown as seq_1: at least one of the N-terminal, C-terminal and peptide chain intermediate;

Preferably, adding a cysteine in the middle of a polypeptide having an amino acid sequence as shown in seq_1 comprises inserting one or more cysteines in the middle of the polypeptide or one or more cysteines are linked in branched form in the middle of the polypeptide;

preferably, the fatty acid modification comprises a myristoylation modification or a palmitoylation modification.

3. The anti-tumor polypeptide according to claim 2, wherein the polypeptide having the amino acid sequence shown in seq_1 is modified by at least acetylation and/or amidation;

Preferably, the N end of the polypeptide with the amino acid sequence shown as the seq_1 is subjected to acetylation modification, and the C end is subjected to amidation modification;

preferably, the anti-tumor polypeptide is a linear polypeptide with a transmembrane peptide;

preferably, the anti-tumor polypeptide is a cyclic polypeptide without a transmembrane peptide;

Preferably, the anti-tumor polypeptide is a polypeptide with an amino acid sequence shown as seq_1, two arginines are added at the N end of the polypeptide, and the sequence shown as seq_2;

Preferably, the anti-tumor polypeptide is a polypeptide with an amino acid sequence shown as seq_1, wherein two arginines are added at the N end of the polypeptide, and the N end is modified by myristoylation, and the sequence is shown as seq_3.

4. An anti-tumour polypeptide according to any of claims 1 to3, wherein the anti-tumour polypeptide is selected from the group consisting of polypeptides having an amino acid sequence as set out in seq_1, seq_2 or seq_3.

5. Use of an anti-tumor polypeptide according to any one of claims 1-4 for targeting a p53 mutant for non-diagnostic and therapeutic purposes, or for the preparation of a product for targeting a p53 mutant;

Preferably, the p53 mutant is a mutant of at least one site mutation selected from the group consisting of: S121F, V122G, C V, C141V, W146Y, C182S, V A, R209P, C229Y, H233Y, Y234F, N235K, Y F, T253V, N268D, E M or S315R.

6. Use of an anti-tumor polypeptide according to any one of claims 1-4 in the manufacture of a medicament for the prevention and/or treatment of cancer.

7. The use of claim 6, wherein the cancer comprises any one or more of: breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, stomach cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gall bladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, cancer of the uterine cervix, ovarian cancer, fat cancer, brain tumor, squamous carcinoma, skin cancer, thyroid cancer, lip cancer, melanin cancer, tongue cancer, thymus cancer, and central nervous system cancer;

Preferably, the central nervous system cancer is brain cancer;

Preferably, the use is in the manufacture of a medicament for the prevention and/or treatment of breast cancer;

preferably, the anti-tumor polypeptide is an anti-tumor polypeptide according to claim 4.

8. An anti-cancer pharmaceutical composition comprising an anti-tumor polypeptide according to any one of claims 1-4, and pharmaceutically optional excipients.

9. The anticancer pharmaceutical composition of claim 8, further comprising an anticancer drug and/or an adjunctive therapeutic drug;

Preferably, the drug for treating cancer comprises one or more of methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecins, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxine, L-asparaginase, docetaxel and capecitabine.

10. The anticancer pharmaceutical composition of claim 9, wherein the anti-tumor polypeptide is the anti-tumor polypeptide of claim 4;

preferably, the anticancer pharmaceutical composition is for treating breast cancer, further comprising docetaxel;

preferably, the anticancer pharmaceutical composition comprises an antitumor polypeptide and docetaxel having sequences as shown in seq_3;

Preferably, the mass ratio of docetaxel to the antitumor polypeptide is 5 (20-80);

preferably, the mass ratio of docetaxel to the anti-tumor polypeptide is 5:20.