Polypeptides Targeting HDAC5 and Their Application in the Preparation of Drugs for Treating Cancer

Abstract

The present invention provides a polypeptide targeting HDAC5 and its application in the preparation of a drug for treating cancer. The polypeptide specifically binds to HDAC5, and the polypeptide is selected from any one or more of SEQ ID NOs: 1-5 and SEQ ID NOs: 11-15. By using HDAC5 as the target protein of the anti-cancer drug and combining AI-assisted design and screening methods, a number of novel polypeptide compounds with anti-cancer activity have been found. These polypeptides have the advantages of specific targeting, low toxicity and low side effects as drugs, and thus have potential application value for development as anti-cancer drugs. In particular, when the polypeptide shown in SEQ ID NO: 1 or 11 is used in cell experiments in the form of SEQ ID NO: 6 or 16 with a cell-penetrating peptide, it is found that its anti-hepatocellular carcinoma activity is the highest.

Description

Technical Field

[0001] The present invention relates to the field of anticancer drug development, and in particular to a polypeptide targeting HDAC5 and an application thereof in preparing a drug for treating cancer. Background Art

[0002] Histone deacetylases (HDACs) are a class of proteases that play a crucial role in chromatin structural modification and gene expression regulation. Normally, histone acetylation facilitates the dissociation of DNA from the histone octamer, loosening the nucleosome structure and enabling specific binding of various transcription factors and co-transcription factors to DNA binding sites, activating gene transcription. Histone deacetylation is a marker of epigenetic repression and plays a crucial role in transcriptional regulation, cell cycle progression, and developmental events. The HDAC5 protein is primarily responsible for deacetylation of N-terminal lysine residues on core histones (H2A, H2B, H3, and H4). Deacetylases function by forming large multiprotein complexes. In recent years, a growing number of studies have implicated HDAC5 in the development and progression of tumors.

[0003] HDAC5 is a novel anti-cancer target, particularly as a target molecule for liver cancer, which can effectively inhibit the development of cancer. Liver cancer, a malignant tumor that originates in the liver, has become one of the most common and devastating diseases worldwide. According to statistics, approximately 110,000 people die from liver cancer in China each year, accounting for 45% of liver cancer deaths worldwide. Currently, the most common clinical treatments for liver cancer include surgery (including liver transplantation), ablation, targeted therapy, radiotherapy, and chemotherapy. Surgical treatment is only effective when liver cancer is discovered early and the patient is healthy enough. However, many liver cancer patients are diagnosed in the middle or late stages of the disease, missing the best opportunity for a cure. Other treatments aim to kill tumor cells, but they also have significant side effects on normal cells. These side effects can increase the rate of cancer recurrence and affect healthy tissues or organs. Therefore, the development of safe, effective, and target-specific anti-liver cancer drugs is urgent.

[0004] A peptide is a compound consisting of three or more amino acid molecules linked by peptide bonds. As one of the most important biological substances, peptides are widely present in living organisms, regulating the functional activities of various organs and cells in the body. In recent years, peptide drugs have attracted considerable attention in the development of anti-tumor drugs due to their advantages such as high targeting, low immunogenicity, high tissue penetration, and safety. In 2019, the global market for anti-tumor peptide therapeutics was valued at US$8.6 billion, and is expected to maintain steady growth over the next decade.

[0005] Therefore, how to develop a group of peptide drug molecules with potential medicinal value using HDAC5 as an anti-cancer target has also become a hot issue in the development of anti-tumor drugs. Summary of the Invention

[0006] The main purpose of the present invention is to provide a polypeptide targeting HDAC5 and its use in the preparation of drugs for treating cancer, so as to provide more polypeptide drug molecules with potential medicinal value for the development of anti-cancer drugs.

[0007] To achieve the above objectives, according to one aspect of the present invention, a polypeptide targeting HDAC5 is provided, which specifically binds to HDAC5, 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: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0008] Furthermore, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification; preferably, the chemical group modification is any one or more of PEG modification, acetylation modification or amidation modification; preferably, the PEG modification is a linear PEG modification, a PEG modification with a single functional group or a PEG modification with a bifunctional group; preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain and Cys side chain of the polypeptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40,000; preferably, the N-terminus and C-terminus of the polypeptide have acetylation modification and amidation modification, respectively; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification; preferably, the hydrophilic amino acid modification is 1-4 hydrophilic amino acids are added to the N-terminus, C-terminus or NC-terminal ends of the peptide, more preferably, the hydrophilic amino acids are Glu, Lys, Ser or Gly; further preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys or Ser-Gly-Ser; preferably, the cysteine ​​modification is to add cysteine ​​at any of the following positions of the polypeptide: N-terminus, C-terminus, NC-terminal ends or in the middle of the peptide chain; more preferably, adding cysteine ​​in the middle of the peptide chain includes inserting one or more cysteines in the middle of the peptide chain, or one or more cysteines are connected to the middle of the peptide chain in the form of a branched chain; preferably, the polypeptide exists in the form of a linear polypeptide with a membrane-penetrating peptide or a cyclic polypeptide without a membrane-penetrating peptide; preferably, the amino acid sequence of the membrane-penetrating peptide is SEQ IDNO: 21: RKKRRQRRR; More preferably, the linear polypeptide with a membrane-penetrating peptide is selected from any one or more of the following: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1: 6, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

[0009] In order to achieve the above object, according to a second aspect of the present invention, a polypeptide drug is provided, which comprises the above polypeptide and pharmaceutically optional excipients.

[0010] Furthermore, the polypeptide drug is an anticancer drug, preferably an anti-liver cancer drug; preferably, the concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM.

[0011] According to a third aspect of the present invention, there is provided use of the above polypeptide in the preparation of a drug for treating cancer.

[0012] Furthermore, the cancer includes any one or more of the following: liver 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, breast 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.

[0013] To achieve the above object, according to one aspect of the present invention, a kit for detecting HDAC5 is provided, which comprises the above polypeptide.

[0014] Furthermore, the polypeptide exists in the form of a polypeptide-protein conjugate; preferably, the protein in the polypeptide-protein conjugate is selected from any one of the following: bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or casein; more preferably, the polypeptide is coupled to the protein via a linker sequence to form a polypeptide-protein conjugate, and further preferably, the linker sequence is CGSG.

[0015] Furthermore, the polypeptide is coated on a solid phase carrier; preferably, the solid phase carrier includes an enzyme labeling plate, a membrane carrier or a microsphere; preferably, the membrane carrier includes a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane; 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 in the detection order; preferably, the kit further includes at least one of the following: (1) an enzyme-labeled secondary antibody, more preferably the enzyme-labeled secondary antibody is an HRP-labeled secondary antibody; (2) a colloidal gold binding pad, the colloidal gold binding pad is coated with a colloidal gold-labeled antigen and a specific binding substance of the positive control substance; (3) a labeling pad, the labeling pad is coated with fluorescently labeled microspheres, and the microspheres are loaded with a specific binding substance of the positive control substance; preferably, the positive control substance is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin, and accordingly, the specific binding substance of the positive control substance is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

[0016] Furthermore, the kit includes a chip on which a polypeptide array composed of polypeptides is pre-installed.

[0017] By applying the technical solutions of the present invention, using HDAC5 as a target protein for anticancer drugs, combined with AI-assisted design and screening, several novel peptide compounds with anticancer activity have been identified. These peptides possess the advantages of target specificity, low toxicity, and minimal side effects, and therefore have the potential for development as anticancer drugs. In particular, the peptides represented by SEQ ID NOs: 1 and 11, when combined with the cell-penetrating peptides represented by SEQ ID NOs: 6 and 16, demonstrated the highest anticancer activity in cell-based experiments. For example, their activity against liver cancer was higher than that of other peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 A box plot showing the binding ability of the second candidate polypeptide screened in Example 1 of the present application to HDAC5 is shown;

[0020] Figure 2 Show peace Figure 3 The HPLC spectra of the synthesis of the polypeptides represented by SEQ ID NOs: 6 and 16 in Example 2 of the present application are shown respectively;

[0021] Figure 4 and Figure 5 The MTT results of the effects of the candidate anticancer polypeptides represented by SEQ ID NOs: 6-10 and 16-20 on the proliferation of HepG2 cells are shown respectively;

[0022] Figure 6 and Figure 7 The figures respectively show the inhibitory effects of the polypeptides represented by SEQ ID NOs: 6 and 16 in Example 4 of the present application on HepG2 cell proliferation. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0024] Explanation of terms:

[0025] Polypeptide: In this application, it refers to any peptide segment predicted or screened to be able to specifically bind to the target HDAC5.

[0026] Polypeptide-carrier protein conjugate: In this application, it refers to a conjugate formed by coupling a polypeptide to a carrier protein, wherein a carrier protein can be coupled to one or more polypeptides. When multiple polypeptides are coupled, the multiple polypeptides have the same amino acid sequence. Depending on the differences in the physicochemical properties of the specific coupled polypeptide sequence, the different types of specific carrier proteins, and the different coupling methods, the number of polypeptides coupled to each carrier protein varies. In this application, 2 to 50 polypeptides are preferred, and 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, and 15 to 30 polypeptides are more preferred; or, more preferably, any one of 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, and 10 to 15 polypeptides are more preferred.

[0027] Peptide chip technology is a detection technology based on peptide chips. It utilizes the contact between a wide variety of peptides on a peptide chip and a sample. Image acquisition technology is then used to capture the characteristic signals on the peptide chip (specifically, fluorescent images carrying each characteristic signal). The signal intensity of each characteristic on the chip is then output as the peptide chip detection result data. Based on the sample detection signal output from the peptide chip detection result data, analysis of analytes in the sample that have bound to the peptides on the peptide chip, as well as sample analysis, can be performed.

[0028] Motif: In biology, a motif is a mathematical statistical model based on data, typically a sequence, but can also be a structure, a sequence prediction for a specific group. For example, a DNA sequence can be defined as a transcription factor binding site, meaning that the sequence tends to be bound by transcription factors. For proteins, a sequence motif can be defined as a protein sequence belonging to a given protein family. A simple motif can be, for example, a pattern shared by all members of a group.

[0029] ROC curve: A curve that reflects the relationship between sensitivity and specificity. The X-axis represents 1 minus specificity, also known as the false positive rate. The closer the X-axis is to zero, the higher the accuracy. The Y-axis represents sensitivity, also known as the true positive rate. A larger Y-axis indicates better sensitivity. The graph is divided into two parts based on the position of the curve. The area below the curve is called the AUC (Area Under the Curve), which indicates predictive accuracy. A higher AUC value indicates higher predictive accuracy. The closer the curve is to the upper left corner (smaller X, larger Y), the higher the predictive accuracy.

[0030] As mentioned in the background, there is an urgent need to develop anticancer peptide drugs targeting different targets. This invention, using HDAC5 as a target protein for anticancer drugs and combining AI-assisted design and screening, has identified novel peptide compounds with anticancer activity. Experimental validation has also confirmed the anticancer activity of the selected peptide drugs. Leveraging the advantages of peptide drugs, such as specific target, low toxicity, and minimal side effects, the peptides screened in this invention address, to some extent, the shortcomings of current drug development and treatment methods.

[0031] Based on the above research results, the applicant has proposed a series of technical solutions of the present application. In a typical embodiment, a polypeptide targeting HDAC5 is provided, which specifically binds to HDAC5 and is selected from any one or more of the following: RPWLFPYHAFS (SEQ ID NO: 1), PYWRHRHPRYRVF (SEQ ID NO: 2), QFWRHPWLQD (SEQ ID NO: 3), WPHRHLFWKE (SEQ ID NO: 4), PLYHRHPWFG (SEQ ID NO: 5), YRFFFPYHLD (SEQ ID NO: 11), PQRFPYHYDNRLD (SEQ ID NO: 12), FWNHRHPHSD (SEQ ID NO: 13), WLHRHRFSG (SEQ ID NO: 14), and QFPFPYQYRG (SEQ ID NO: 15).

[0032] As described above, this application uses HDAC5 as a target protein for anticancer drugs, combined with AI-assisted design and screening methods, to find the above-mentioned new polypeptide compounds with anticancer activity. These polypeptides have the advantages of target specificity, low toxicity and low side effects as drugs, and therefore have potential application value for development as anticancer drugs. Among the above-mentioned polypeptides, the polypeptide represented by SEQ ID NO: 1 or 11 is preferred. When it is used in the form of SEQ ID NO: 6 or 16 with a cell-penetrating peptide in cell experiments, it is found that the polypeptide has the highest anticancer activity, for example, the activity against liver cancer is higher than that of other polypeptides.

[0033] The polypeptides can be modified as needed during drug development. In a preferred embodiment, the polypeptides are modified peptides; preferably, the modifications are chemical group modifications or amino acid modifications.

[0034] For example, in order to further improve the affinity of certain peptide segments, in a preferred embodiment, the above-mentioned chemical group modification is PEG modification; preferably, the PEG modification is a straight-chain PEG modification, a PEG modification with a single functional group, or a PEG modification with a bifunctional group; preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain, and Cys side chain of the polypeptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40,000; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification; preferably, the hydrophilic amino acid modification is the addition of 1-4 hydrophilic amino acids to the N-terminus, C-terminus, or both ends of the NC of the polypeptide, more preferably, the hydrophilic amino acid is Glu, Lys, Ser, or Gly; further preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys, or Ser-Gly-Ser.

[0035] Since chemically synthesized polypeptides often carry free amino and carboxyl groups, and the sequence of a polypeptide often represents the sequence of the parent protein in which it is derived, 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, generally by acetylation of the N-terminus and amidation of the C-terminus. Such modifications reduce the overall charge of the polypeptide, lowering its solubility, and further allowing the polypeptide to mimic the original state of the α-amino and carboxyl groups in the parent protein. Therefore, in other embodiments, the above-mentioned chemical group modification is a double-terminal modification of the polypeptide, more preferably, the N-terminus and C-terminus of the polypeptide are acetylated and amidated, respectively.

[0036] In certain embodiments, in order to better achieve directional coupling of peptide segments, it is preferred to modify the polypeptide with cysteine. Specifically, this includes but is not limited to adding cysteine ​​at the N-terminus, C-terminus, or both ends of the NC of the peptide segment, or adding cysteine ​​in the middle of the peptide chain of the polypeptide. When adding cysteine ​​in the middle of the peptide chain of the peptide segment, one or more cysteines can be inserted in the middle of the peptide chain (i.e., inserted between two amino acid residues), or one or more cysteines can be connected to the middle of the peptide chain in the form of a branched chain (i.e., as a side chain of an amino acid in the middle of the peptide chain).

[0037] In certain embodiments, in order to more effectively enable the above-mentioned polypeptide molecules to pass through the cell membrane as drugs, enter the body and bind to the HDAC5 target, preferably, the polypeptide is present in the form of a linear polypeptide with a penetrating peptide or a cyclic polypeptide without a penetrating peptide. The penetrating peptide is usually set at the C-terminus of the polypeptide or one end of the drug molecule, and can be used to bring the polypeptide molecule or drug molecule into a specific type of cell, and then the molecule undergoes a biochemical reaction in the cell. The penetrating peptide is usually rich in basic amino acids such as arginine (Arg) or lysine (Lys). Examples of more commonly used penetrating peptides are as follows: (1) H7R8: HHHHHHHRRRRRRRR (SEQ ID NO: 23); (2) YGRKKRRQRRR (SEQ ID NO: 24); or (3) GRRRRRRRRRPPQ (SEQ ID NO: 25).

[0038] In a preferred embodiment, the amino acid sequence of the cell-penetrating peptide is RKKRRQRRR (SEQ ID NO: 21). Specifically, the amino acid sequence of the linear polypeptide with a membrane-penetrating peptide is RKKRRQRRR-RPWLFPYHAFS (SEQ ID NO: 6), RKKRRQRRR-PYWRHRHPRYRVF (SEQ ID NO: 7), RKKRRQRRRQFWRHPWLQD (SEQ ID NO: 8), RKKRRQRRR-WPHRHLFWKE (SEQ ID NO: 9), RKKRRQRRR-PLYHRHPWFG (SEQ ID NO: 10), RKKRRQRRRYRFFFPYHLD (SEQ ID NO: 16), RKKRRQRRRPQRFPYHYDNRLD (SEQ ID NO: 17), RKKRRQRRRFWNHRHPHSD (SEQ ID NO: 18), RKKRRQRRRWLHRHRFSG (SEQ ID NO: 19) or RKKRRQRRRQFPFPYQYRG (SEQ ID NO: 20).

[0039] The cell-penetrating peptide shown in SEQ ID NO: 21 is TAT-modified, which is a well-known and mature method for modifying cell-penetrating peptides. This modification of the cell-penetrating peptide can enhance the membrane-penetrating performance of the polypeptide.

[0040] In a second exemplary embodiment of the present application, a polypeptide drug is provided, comprising any of the above polypeptides and pharmaceutically optional excipients. The drug containing the above polypeptide molecule has the advantages of high targeting ability to bind to HDAC5, high anticancer activity, low toxicity, and few side effects.

[0041] The aforementioned pharmaceutically acceptable excipients may vary depending on the desired dosage form and / or mode and route of administration of the drug, and may be appropriately selected from existing pharmaceutical excipients. These include, but are not limited to, pharmaceutically acceptable carriers, excipients, adjuvants, and other excipients. Furthermore, the polypeptide drug may be formulated into various dosage forms to accommodate diverse routes of administration, including oral, parenteral, and transdermal administration. Drug dosage forms include capsules, tablets, oral solutions, injections, and transdermal formulations.

[0042] Specifically, the medicine of the present application can be made into capsules, tablets, oral liquids, injections or transdermal absorbents according to methods known in the pharmaceutical industry. Preferably, the injection is an intravenous injection. When preparing capsules, tablets, and oral liquids suitable for oral administration, sucrose, lactose, galactose, corn starch, gelatin, microcrystalline cellulose, carboxymethyl cellulose, etc. can be used as carriers or excipients. In addition, the medicine of the present application can also be made into solutions and suspensions suitable for oral administration using methods and auxiliary ingredients known in the pharmaceutical industry. If solutions and suspensions suitable for parenteral administration are prepared, distilled water, water for injection, isotonic sodium chloride or glucose solution, or low concentration (e.g., 1-100 mm) phosphate buffered saline (PBS) can be used as carriers or diluents. One or more other auxiliary ingredients or additives can be added to these parenteral preparations, for example, ascorbic acid can be used as an antioxidant, and sodium benzoate, etc. can be used as a preservative. Other solubilizers, disintegrants, colorants, dispersants, or surfactants can also be contained in the preparations of these dosage forms.

[0043] The above-mentioned polypeptide drug can be used as an anticancer drug, preferably an anti-liver cancer drug, because of its anti-cancer activity. In certain embodiments, the polypeptide exists in the form of a linear polypeptide with a membrane-penetrating peptide to further improve the membrane-penetrating performance of the polypeptide drug. In other embodiments, the polypeptide exists in the form of a cyclic or linear polypeptide without a membrane-penetrating peptide, which has certain anti-cancer activity, especially anti-liver cancer activity. Further preferably, the membrane-penetrating peptide is selected from the amino acid sequence shown in SEQ ID NO: 21 above. The polypeptide with a membrane-penetrating peptide is shown in SEQ ID NOs: 6-10 or SEQ ID NOs: 16-20 above, more preferably the sequence shown in SEQ ID NO: 6 or 16.

[0044] In the above-mentioned polypeptide drugs, the concentration of the polypeptide varies depending on the dosage form or administration method. In a preferred embodiment, the concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM, and the anticancer activity is maintained within this concentration range. More preferably, the concentration is 1 μM to 100 μM, and even more preferably 10 μM to 100 μM. Specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 μM.

[0045] In a third exemplary embodiment of the present application, the use of any of the above-mentioned polypeptides in the preparation of a drug for treating cancer is provided. Since HDAC5 deacetylates the N-terminal lysine residues of core histones (H2A, H2B, H3, and H4) in nucleosomes, it is closely related to the occurrence and development of various tumors. Therefore, the polypeptides that can specifically bind to HDAC5 identified in this application all have potential pharmaceutical development value for treating cancer, particularly for liver cancer.

[0046] The aforementioned cancers include, but are not limited to, any one or more of the following: liver cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, stomach 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, fatty liver cancer, breast 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. Preferably, the aforementioned cancer is liver cancer.

[0047] It should be noted that the above-mentioned treatment includes different degrees of inhibition of cancer cell proliferation and other effects. The specific inhibition degrees include more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, 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.

[0048] In a fourth exemplary embodiment of the present application, a kit for detecting HDAC5 is provided, comprising any of the above-described polypeptides. Because the above-described polypeptides have been verified through peptide microarrays combined with AI-assisted screening and experimental validation to have the ability to specifically target and bind to HDAC5, and thus to exhibit anti-cancer activity by binding to HDAC5, particularly the anti-liver cancer cell activity of the polypeptides represented by SEQ ID NOs: 1, 11, or SEQ ID NOs: 6 or 16, using a kit comprising the above-described polypeptides, the presence and expression level of HDAC5 can be specifically and accurately detected.

[0049] Since the polypeptide molecules are relatively small, in order to improve the detection capability, the above-mentioned polypeptides can be prepared into the form of polypeptide-carrier protein conjugates for detection. According to the requirements of the preparation of the polypeptide-carrier protein conjugate, a specific suitable carrier protein can be selected to form the polypeptide-carrier protein conjugate. In some preferred embodiments, the polypeptide exists in the form of a polypeptide-protein conjugate. The carrier proteins in this application include but are not limited to BSA (bovine serum albumin), OVA (ovalbumin), KLH (keyhole limpet hemocyanin) or CS (casein). According to the amino acid sequence composition of different polypeptides, in order to facilitate coupling with the carrier protein, it is necessary to couple with the carrier protein through a connecting sequence (also called a linker or linker). In this application, the connecting sequence is preferably CGSG.

[0050] It should be noted that the aforementioned polypeptide-carrier protein conjugates are recombinantly synthesized and expressed proteins. In addition to detecting HDAC5, based on protein homology, these recombinantly expressed polypeptide-carrier protein conjugates can also be used to detect other proteins homologous to HDAC5, or other recombinant proteins in the HDAC protein family, to a large extent.

[0051] The number of polypeptides that can be coupled to each carrier protein varies depending on the physicochemical properties of the polypeptide amino acids, the different carrier proteins used, and the different coupling methods. Taking into account the coupling efficiency and the antibody recognition and binding ability, it is preferred that each carrier protein be coupled to 2 to 50 polypeptides, more preferably 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, or 15 to 30; or more preferably any one of 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, and 10 to 15.

[0052] The above-mentioned kit can be prepared into a variety of different types of detection kits according to specific needs. However, from the perspective of convenient detection and easy judgment of the test results, the polypeptide in the kit can be set to be pre-coated on a solid phase carrier. The specific pre-coated solid phase carrier is reasonably designed according to needs. More preferably, the solid phase carrier includes an ELISA plate (mostly made of polystyrene materials), a membrane carrier or a microsphere; further preferably, the membrane carrier includes a nitrocellulose membrane (the most widely used), a glass cellulose membrane or a nylon membrane, and further preferably, 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 in the order of detection.

[0053] Depending on the specific detection method of the kit, the specific supporting reagents in the kit will also vary accordingly, but all supporting reagents can be combined according to the preparation method of known kits. Preferably, the above kit also includes at least one of the following: (1) an enzyme-labeled secondary antibody, more preferably an HRP-labeled secondary antibody (corresponding to an ELISA detection kit); (2) a colloidal gold conjugate pad coated with a colloidal gold-labeled polypeptide-carrier protein conjugate and a specific binding substance of the positive control substance (corresponding to an immunocolloidal gold detection kit); (3) a labeling pad coated with fluorescently labeled microspheres, and the microspheres are loaded with a specific binding substance of the positive control substance (corresponding to an immunofluorescence detection kit).

[0054] The above-mentioned immunocolloidal gold detection kit and immunofluorescence detection kit are relatively more convenient for detection, and only the C line of the positive control and the T line of the test sample need to be established. The positive control substance pre-coated at the C line of the positive control can be a specific binding substance with a detection label that can be carried over during the serum chromatography process of the test sample, and there is no special limitation on the specific antigen or antibody of the specific positive control substance. Preferably, the above-mentioned positive control substance is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin, and accordingly, the specific binding substance of the positive control substance is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

[0055] The anti-mouse immunoglobulin can be a goat anti-mouse immunoglobulin or a rabbit anti-mouse immunoglobulin, or an anti-mouse immunoglobulin from another immunizable animal, depending on the target of the immunization. Similarly, the anti-human immunoglobulin, anti-goat immunoglobulin, or anti-rabbit immunoglobulin can also be immunoglobulins from different species, depending on the animal being immunized. The immunoglobulin can be any one of IgM, IgG, IgA, IgD, or IgE. These anti-immunoglobulin antibodies can be monoclonal antibodies or polyclonal antibodies.

[0056] In the above-mentioned kits, the specifications of the ELISA plates used vary depending on the number of samples to be tested, and can be appropriately selected from 12- to 384-well ELISA plates. In pre-coated ELISA plates, the amount of peptide-carrier protein conjugate coated per well also varies depending on the target of detection in the peptide-carrier protein conjugate. Similarly, the amount of peptide-carrier protein conjugate coated on a membrane support (e.g., nitrocellulose membrane) also varies. For example, it can be 0.8-8 μg / cm, more preferably 0.8-7 μg / cm, 0.8-6 μg / cm, 0.8-5 μg / cm, 0.8-4 μg / cm, 0.8-3 μg / cm, 0.8-2 μg / cm, 0.8-1.8 μg / cm, 0.8-1.7 μg / cm, 0.8-1.6 μg / cm, 0.8-1.5 μg / cm, 0.8-1.4 μg / cm or 0.8-1.2 μg / cm.

[0057] In some preferred embodiments, the kit includes a chip pre-loaded with polypeptides, wherein the polypeptides comprise a polypeptide array comprising the aforementioned polypeptides. Detection of the aforementioned polypeptides on the polypeptide chip not only enhances detection specificity by leveraging the polypeptides' targeted binding ability to HDAC5, but also increases detection throughput using the array format, making it suitable for large-scale cancer screening.

[0058] The beneficial effects of the present application will be further illustrated below with reference to specific embodiments. It should be noted that the embodiments of the present application mainly include the following parts:

[0059] A. Data Analysis: Based on the peptide library contained in the existing peptide array, screen for binding peptides that show significant differences between the target protein and negative serum samples;

[0060] B. AI calculation: Randomly cut specific peptide features, calculate the binding affinity with HDAC5 protein, select the peptides with the highest binding affinity, and compare them with the existing knowledge base;

[0061] C. Experimental verification: Synthesize potential peptides for cell experiments on peptide anti-cancer effects to screen for active peptides.

[0062] Example 1: Peptide library screening and AI analysis

[0063] 1. Using a peptide array, we used a commercially available recombinant purified HDAC5 protein (manufacturer: BPSBioscience; catalog number: Q9UQL6) and compared it to negative serum to initially identify peptides with significant differences between the two groups. These peptides served as the first candidate peptide set. For this first candidate peptide set, we used AutoDock CrankPep software, based on a docking strategy, to calculate the binding affinity between each candidate peptide and HDAC5 protein, and then screened the second candidate peptide set.

[0064] 1) Search for the three-dimensional structure of HDAC5 protein

[0065] Download the three-dimensional conformation file of HDAC5 protein from the PDB database [RCSB PDB-5UWI: Crystal Structure of HDAC5 NES Peptide in complex with CRM1-Ran-RanBP1] (specific website: https: / / www.rcsb.org / structure / 5UWI).

[0066] 2) Prepare receptor protein

[0067] Extracting receptor protein coordinates: The 5UWI PDB file contains proteins, ligands, and water molecules; first extract the protein coordinates.

[0068] Hydrogenation: Crystal structures often lack coordinates for hydrogen atoms (because hydrogen atoms have few electrons and their protons have a weak attraction to electrons, making them difficult to locate). However, during docking, hydrogen atoms, especially polar hydrogen atoms, are essential for calculating electrostatic interactions. Therefore, it is necessary to add hydrogen atoms to the protein.

[0069] Define the 3D search space for ligand binding: If the binding site is unknown, theoretically you can define a rectangular box that contains the entire protein or any specific region.

[0070] 3) Prepare reference ligand

[0071] The atomic positions of the reference ligands were extracted from the 5UWI PDB structure.

[0072] Similar to protein structures, ligand structures also lack hydrogen atoms, so it is necessary to add hydrogen atoms and define which bonds are rotatable for flexible docking.

[0073] 4) Prepare the docking configuration file

[0074] The Docking configuration file contains the input information of receptors (proteins), ligands (compounds) and default search parameters.

[0075] DockingD and using PyMol to visualize Docking results.

[0076] See the results Figure 1 . Figure 1 Shown is a box plot of the binding ability between the second candidate polypeptide obtained by screening and the target protein HDAC5 (the horizontal axis represents the binding energy, and the larger the absolute value, the stronger the binding ability).

[0077] 2. According to Figure 1 The strength of the binding affinity was determined by combining known knowledge of the target protein HDAC5 with the selection of potential functional peptides for synthesis. This application selected 10 peptides (see Table 1) to further validate the peptide activity. Furthermore, based on comparisons with currently available peptide database data (NCBI Peptide Group), all peptides in this application are novel sequences.

[0078] Table 1:

[0079] Peptide sequence Binding energy kcal / mol RPWLFPYHAFS (SEQ ID NO: 1) -115.7246 PYWRHRHPRYRVF (SEQ ID NO: 2) -119.1618 QFWRHPWLQD (SEQ ID NO: 3) -116.8866 WPHRHLFWKE (SEQ ID NO: 4) -113.7353 PLYHRHPWFG (SEQ ID NO: 5) -113.0858 YRFFFPYHLD (SEQ ID NO: 11) -113.0858 PQRFPYHYDNRLD (SEQ ID NO: 12) -111.5115 FWNHRHPHSD (SEQ ID NO: 13) -110.8541 WLHRHRFSG (SEQ ID NO: 14) -110.2346 QFPFPYQYRG (SEQ ID NO: 15) -110.0055

[0080] Note: The larger the absolute value of binding energy in the table, the higher the peptide binding force may be.

[0081] Example 2: Peptide Synthesis

[0082] 1) Entrust Jier Bio to synthesize the following candidate peptides and positive control peptides:

[0083] Table 2:

[0084]

[0085] The HPLC spectra of the polypeptides shown in SEQ ID NO: 6 and 16 are shown in FIG. Figure 2 and Figure 3 shown.

[0086] Example 3: Peptide biological function verification test

[0087] 1. Chemicals and reagents:

[0088] Table 3:

[0089] Reagents source Dulbecco's Modified Eagle Medium(DMEM) Invitrogen (CA, USA) Fetal bovine serum (FBS) HyClone Laboratories, Inc (USA) Dimethyl sulfoxide (DMSO) Sigma MTT kit Bioengineering Biotechnology (E606334-0500)

[0090] 2. MTT assay steps:

[0091] 1. All tubes need to be centrifuged before opening.

[0092] 2. Preparation of MTT Solution: Dissolve MTT Reagent (Component A) in MTT Solvent (Component B) to prepare a 5 mg / ml MTT solution. Use immediately or store at -20°C in the dark. Alternatively, aliquot as needed and store at -20°C in the dark.

[0093] 3. Add 10 μL of MTT solution to each well to make the final concentration of MTT in each well be 0.5 mg / ml.

[0094] 4. After gently mixing, place in a 5% CO2, 37℃ incubator and incubate for 4 hours.

[0095] 5. Carefully aspirate the medium from each well to prevent the cell monolayer from breaking.

[0096] 6. Add 100 μL Formazan Solubilization Solution (Component C) to each well

[0097] 7. Place the 96-well plate on a shaker and gently shake for 10 minutes until the formazan is completely dissolved as observed under a normal optical microscope.

[0098] 8. Measure the absorbance at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0099] 3. Inhibition of HepG2 cell activity

[0100] (1) Cell culture: HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a humidified atmosphere at 37°C containing 5% carbon dioxide.

[0101] (2) In vitro detection of the biological activity of the peptide: The MTT (USE) assay was used to detect cell proliferation. HepG2 (ATCC) cells were first seeded in a 96-well plate, with 5×10 3 Cells were plated in 100 μL of DMEM containing 10% FBS per well, with 8 wells left empty as blank controls. The cells were incubated overnight (37°C, 5% CO2) to allow the cells to attach to the wells. Different concentrations (0.1 μM, 1 μM, 10 μM, 100 μM) of the designed peptides (iCXOncH1a, iCXOncH2a, iCXOncH3a, iCXOncH4a, iCXOncH5a, iCXOncH6a, iCXOncH7a, iCXOncH8a, iCXOncH9a, iCXOncH10a) dissolved in DMSO were added, along with a positive control. Each well was incubated in DMEM containing 2% FBS for 48 hours (37°C, 5% CO2) to allow the peptides to take effect.

[0102] Add 0.05% MTT solution to each well. Incubate (37°C, 5% CO2) for 3 hours to allow MTT to metabolize. Discard the culture medium and resuspend formazan (MTT metabolite) in 100 μL DMSO. Mix the formazan into the solvent. Read the optical density at 570 nm and record the result. Draw a growth curve with concentration as the horizontal axis and cell survival rate as the vertical axis. The results are shown in Figure 2. Figure 4 and Figure 5 shown.

[0103] Figure 4 The results showed that the peptide iCXOncH3a has significant anticancer activity and can effectively inhibit the growth of cancer cells at a concentration of 10μM to 100μM. At 100μM, the inhibitory activity is even higher, significantly better than the positive control peptide at the same concentration. The IC value of iCXOncH3a was calculated based on the experimental data. 50 =121.3 μM, IC of positive control 50 = 218.1 μM. This shows that the polypeptide of the present application is more active than the positive control peptide and can be used as an active compound for subsequent development.

[0104] Figure 5 The results showed that the peptide iCXOncH6a has significant anticancer activity and can effectively inhibit the growth of cancer cells at a concentration of 10μM to 100μM. At 100μM, the inhibitory activity is even higher, and the inhibition rate is significantly better than that of the positive control peptide at the same concentration. The IC value of iCXOncH6a was calculated based on the experimental data. 50 =39.5 μM, IC of positive control 50 =218.1μM.

[0105] IC 50 IC value calculation method: According to the inhibition rate of peptide on tumor cells at different concentrations, draw a regression curve with peptide concentration as the horizontal axis and the inhibition rate of tumor cells as the vertical axis, obtain the regression equation and R value, and calculate the peptide concentration when the inhibition rate is 50%, which is the IC value. 50 value.

[0106] Example 4: Microscopic observation of cell proliferation

[0107] The cells were treated with peptides of iCXOncH3a and iCXOncH6a at different concentrations (1 μM, 10 μM, 100 μM). Figure 6 and Figure 7 As shown, cancer cells grew slowly after polypeptide treatment, and no cancer cells survived under treatment with 100 μM.

[0108] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: through peptide chip combined with AI-assisted screening and experimental verification, the above polypeptides have the ability to specifically target and bind to HDAC5, and thus have anti-cancer activity by binding to HDAC5. In particular, the polypeptides represented by SEQ ID NO: 1 or 11 and the polypeptides with cell-penetrating peptides represented by SEQ ID NO: 6 or 16 have significant anti-liver cancer cell activity.

[0109] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention. Sequence Listing <110> Zhuhai Carbon Cloud Intelligent Technology Co., Ltd. <120> Polypeptides targeting HDAC5 and their use in preparing drugs for treating cancer <130> PN161915SZTY <160> 25 <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> HDAC5 binding peptides <400> 1 Arg Pro Trp Leu Phe Pro Tyr His Ala Phe Ser 1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> HDAC5 binding peptides <400> 2 Pro Tyr Trp Arg His Arg His Pro Arg Tyr Arg Val Phe 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> HDAC5 binding peptides <400> 3 Gln Phe Trp Arg His Pro Trp Leu Gln Asp 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> HDAC5 binding peptides <400> 4 Trp Pro His Arg His Leu Phe Trp Lys Glu 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> HDAC5 binding peptides <400> 5 Pro Leu Tyr His Arg His Pro Trp Phe Gly 1 5 10 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(20) <223> HDAC5 binding peptide iCXOncH3a with cell-penetrating peptide <400> 6 Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Pro Trp Leu Phe Pro Tyr 1 5 10 15 His Ala Phe Ser 20 <210> 7 <211> twenty two <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(22) <223> iCXOncH1a <400> 7 Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Tyr Trp Arg His Arg His 1 5 10 15 Pro Arg Tyr Arg Val Phe 20 <210> 8 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(19) <223> iCXOncH2a <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Phe Trp Arg His Pro Trp 1 5 10 15 Leu Gln Asp <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(19) <223> iCXOncH4a <400> 9 Arg Lys Lys Arg Arg Gln Arg Arg Arg Trp Pro His Arg His Leu Phe 1 5 10 15 Trp Lys Glu <210> 10 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(19) <223> iCXOncH5a <400> 10 Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Leu Tyr His Arg His Pro 1 5 10 15 Trp Phe Gly <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> HDAC5 binding peptides <400> 11 Tyr Arg Phe Phe Phe Pro Tyr His Leu Asp 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> HDAC5 binding peptides <400> 12 Pro Gln Arg Phe Pro Tyr His Tyr Asp Asn Arg Leu Asp 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> HDAC5 binding peptides <400> 13 Phe Trp Asn His Arg His Pro His Ser Asp 1 5 10 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> HDAC5 binding peptides <400> 14 Trp Leu His Arg His Arg Phe Ser Gly 1 5 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> HDAC5 binding peptides <400> 15 Gln Phe Pro Phe Pro Tyr Gln Tyr Arg Gly 1 5 10 <210> 16 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(19) <223> iCXOncH6a <400> 16 Arg Lys Lys Arg Arg Gln Arg Arg Arg Tyr Arg Phe Phe Phe Pro Tyr 1 5 10 15 His Leu Asp <210> 17 <211> twenty two <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(22) <223> iCXOncH7a <400> 17 Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln Arg Phe Pro Tyr His 1 5 10 15 Tyr Asp Asn Arg Leu Asp 20 <210> 18 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(19) <223> iCXOncH8a <400> 18 Arg Lys Lys Arg Arg Gln Arg Arg Arg Phe Trp Asn His Arg His Pro 1 5 10 15 His Ser Asp <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(18) <223> iCXOncH9a <400> 19 Arg Lys Lys Arg Arg Gln Arg Arg Arg Trp Leu His Arg His Arg Phe 1 5 10 15 Ser Gly <210> 20 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(19) <223> iCXOncH10a <400> 20 Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Phe Pro Phe Pro Tyr Gln 1 5 10 15 Tyr Arg Gly <210> twenty one <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Cell-penetrating peptides <400> twenty one Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 <210> twenty two <211> 5 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(5) <223> Head-to-tail cyclic pentapeptide <400> twenty two Arg Gly Asp Phe Lys 1 5 <210> twenty three <211> 15 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(15) <223> Cell-penetrating peptide H7R8 <400> twenty three His His His His His His Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 <210> twenty four <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Cell-penetrating peptides <400> twenty four Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 25 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> Cell-penetrating peptides <400> 25 Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Gln 1 5 10

Claims

1. A polypeptide targeting HDAC5, characterized in that, The polypeptide specifically binds to HDAC5, and the polypeptide is SEQ ID NO: 11: YRFFFPYHLD.

2. A linear polypeptide with a transmembrane peptide, characterized in that, The linear polypeptide is a peptide with a transmembrane peptide based on the peptide with the amino acid sequence of SEQ ID NO:

11.

3. The linear polypeptide according to claim 2, wherein The amino acid sequence of the transmembrane peptide is SEQ ID NO: 21: RKKRRQRRR.

4. The linear polypeptide according to claim 3, wherein The linear polypeptide with a transmembrane peptide is: SEQ ID NO: 16: RKKRRQRRRYRFFFPYHLD.

5. A polypeptide drug, characterized in that, The polypeptide drug comprises the polypeptide targeting HDAC5 as claimed in claim 1 or the linear polypeptide with a transmembrane peptide as claimed in any one of claims 2 - 4, and a pharmaceutically optional excipient.

6. The polypeptide drug according to claim 5, wherein, The concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM.

7. Use of the polypeptide targeting HDAC5 as claimed in claim 1 or the linear polypeptide with a transmembrane peptide as claimed in any one of claims 2 - 4 in the preparation of a drug for treating liver cancer.

8. A kit for detecting HDAC5, characterized in that, The kit comprises the polypeptide targeting HDAC5 as claimed in claim 1 or the linear polypeptide with a transmembrane peptide as claimed in any one of claims 2 - 4.

9. The kit according to claim 8, wherein The polypeptide is coated on a solid-phase carrier.

10. The kit according to claim 9, characterized in that, The solid-phase carrier includes an enzyme-linked immunosorbent assay (ELISA) plate, a membrane carrier or a microsphere.

11. The kit according to claim 10, wherein The membrane carrier includes a nitrocellulose membrane, a glass fiber membrane or a nylon membrane.

12. The kit according to claim 11, characterized in that, A positive control is further coated on the membrane carrier, and the polypeptide and the positive control are sequentially arranged on the membrane carrier in the detection order.

13. The kit according to claim 12, wherein The kit further comprises at least one of the following: (1) An enzyme-labeled secondary antibody; (2) A colloidal gold conjugate pad, on which a colloidal gold-labeled antigen and a specific binding substance of the positive control are coated; (3) A labeled pad, on which fluorescently labeled microspheres are coated, and the microspheres are loaded with a specific binding substance of the positive control.

14. The kit according to claim 13, wherein, The enzyme-labeled secondary antibody is an HRP-labeled secondary antibody.

15. The kit according to claim 14, wherein, The positive control is selected from murine immunoglobulin, human immunoglobulin, ovine immunoglobulin or rabbit immunoglobulin, and the specific binding substance of the positive control is selected from anti-murine immunoglobulin, anti-human immunoglobulin, anti-ovine immunoglobulin or anti-rabbit immunoglobulin.

16. The kit according to claim 8, wherein The kit comprises a chip, on which a polypeptide array composed of the polypeptide is pre-set.

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