Polypeptide for selectively inhibiting interaction of ZDHHC1-p53C135 and application thereof

Abstract

The invention provides a polypeptide capable of selectively inhibiting interaction of ZDHHC1-p53C135, and the polypeptide comprises the following core sequence: SEQ ID No.1: NKMFCQLAK. The polypeptide can selectively inhibit interaction of ZDHHC1-p53C135, promote degradation of mutant p53 and inhibit growth of various tumor cells, has the characteristics of high specificity, low toxic and side effects, broad-spectrum anti-tumor activity and the like, and can be used for preparing an anti-tumor drug. A new drug candidate is provided for clinical treatment of the p53 mutation tumor, and an important reference is provided for clinical treatment of the p53 mutation tumor.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polypeptide that selectively inhibits the interaction between ZDHHC1-p53C135 and its applications. Background Technology

[0002] TP53, an important tumor suppressor gene, encodes the p53 protein, a DNA-binding transcription factor. When cells face stress, it plays a crucial role by precisely regulating cell growth arrest, inducing senescence, and initiating apoptotic cell death (Liu Y, et al. Cancer Cell. 2024;42(6):946-967.). This function mainly relies on transcriptional regulation of multiple target genes, including p21, which arrests cell cycle progression, and Bax and Puma genes, which promote apoptosis. In the early stages of tumor development, stress responses triggered by oncogene activation or cell cycle dysregulation are a significant characteristic of tumor evolution. At this point, DNA replication errors caused by carcinogenic factors trigger a series of cascade reactions involving the activation of ATM, Chk1, Chk2 kinases and p53, which in turn induce senescence or apoptosis (Dsouza R, et al. Mol Biol Rep. 2025;52 (1):333; Li Q, et al. Signal Transduct Target Ther.2023;8 (1):338.). This p53-dependent response mechanism is the core defense line for the body to clear newly developed tumor cells or damaged cells and maintain genomic stability.

[0003] Research data shows that nearly 50% of human cancer cases involve TP53 gene mutations (Khan AA, et al. Cancer Metastasis Rev. 2021;40(1):245-272.), of which about 90% are missense mutations, resulting in abnormal p53 proteins carrying single-point mutations. From a protein structure perspective, about 28% of high-frequency mutation sites (such as R175, G245, R248, R249, R273, R282) are concentrated in the DNA-binding domain (DBD) of p53, and these mutations lead to adaptive changes in protein structure (Voskarides K, et al. Cells. 2023;12(3):512.). In terms of function, mutant p53 can exhibit loss of transcriptional activity and tumor suppressor function (LOF) of wild-type p53, and can also interfere with and inhibit the normal function of wild-type p53 through a "dominant negative effect". More complexly, some mutant p53 even acquires entirely new pro-tumor biological functions, known as "gain-of-functions (GOF)" (Peuget S, et al. Nat Rev Cancer. 2024;24(3):192-215.). Through the GOF effect, mutant p53 can regulate the occurrence and development of tumors in multiple dimensions, promote immune escape, and enhance cancer cell stemness, etc. (Song H, et al. Cancer Discov. 2024;14(11):2055-2060.). Experimental animal models have further confirmed that, compared with p53-deficient or wild-type mice, mice carrying mutant p53 exhibit a more aggressive tumor phenotype and a higher tendency to metastasize (Wang Z, et al. Cancer Discov. 2024;14(2):362-379; Lane D, et al. Cancer Discov. 2024;14(2):211-213). Therefore, p53 is considered a highly promising target in the field of cancer treatment.

[0004] To address the issue of p53 wild-type functional loss, several strategies exist. First, the complete p53 gene can be introduced to replace the mutated gene, restoring its transtranscriptional activation function. For example, the recombinant adenovirus vector-mediated wild-type p53 expression system (rAd5-p53), marketed as Gendicine™, was approved by the China Food and Drug Administration (CFDA) in 2003 for the treatment of head and neck malignancies. Another similar adenovirus vector, Ad-p53, is currently undergoing clinical trials (NCT03544723) for recurrent or metastatic head and neck cancer. However, because viral vectors struggle to effectively transduce all tumor cells, tumor recurrence after treatment is common. Second, targeting and interfering with the p53-MDM2 / MDM4 complex, using small molecules to alter the conformation of mutated p53. Therefore, inhibiting MDM and MDMX is also considered a key target for cancer treatment. The first MDM2 inhibitor, Nutlin 3a, was developed by Roche. It binds to MDM2, blocking the interaction between p53 and MDM2, promoting p53 accumulation, and enhancing its transcriptional activity. Currently, Nutlin 3a has been shown to induce cell cycle arrest and apoptosis in cancer cells in vitro and in vivo. Based on this, Roche further developed RG7112 (RO5045337) and RG7388 (RO5503781). In addition, other companies and institutions have also developed MDM2 inhibitors, such as Kartos' AMG232, Sanofi's SAR405838 (MI-77301), and Merck's MK-8242 (SCH-900242). However, significant blood toxicity has been observed in clinical trials.

[0005] To address the issue of mutant p53, several strategies have been employed: One is to achieve anti-tumor effects by restoring wild-type p53 activity. In 1999, Pfizer screened out the first compound, CP31398, which stabilized the wild-type conformation of p53. However, CP31398 intercalated into DNA, causing non-specific toxicity and failing to enter clinical trials. Nevertheless, this drug marked the beginning of the small-molecule era targeting mutant p53. Following CP-31398, compounds inducing apoptosis in mutant p53 cells, such as MIRA-1 and STIMA-1, were developed, but due to solubility issues and toxicity to normal cells, they did not enter clinical trials. Additionally, through rational design methods based on the p53 structure, small-molecule compounds PK083 and PK7080 were found to bind to the Y220C mutant, restoring the wild-type conformation and inducing Y220C-dependent cell cycle arrest and apoptosis. However, their specific mechanisms of action remain unclear, and they only target the Y220C mutation site. Some inhibitors, such as Geldanamycin, have the potential to disrupt the stability of mutant p53 by reducing its instability. Furthermore, focusing on synthetic lethality strategies, researchers have conducted systematic screening for synthetic lethality in cancer cells with TP53 mutations or deletions, identifying a series of candidate targets for p53-deficient synthetic lethality, such as PLK1, PLK4, CDK1, CDK16, mTOR, and AURKA. Algorithm-based analysis has also recommended kinases such as WEE1, AURKA, and FYN as potentially having a synthetic lethal relationship with p53. However, given the high heterogeneity of tumor cells at the genetic and epigenetic levels, the actual efficacy of synthetic lethality strategies cannot yet be confirmed. To date, no inhibitor targeting mutant p53 has been approved by the U.S. Food and Drug Administration (FDA).

[0006] In summary, p53 plays a central role in cellular responses to oncogenic stress and is the most frequently mutated gene in cancer. Missense mutant p53 is highly expressed in many tumors and exhibits gain-of-function activity that promotes tumorigenesis, making p53 a key target for cancer treatment. The inventors' previous research revealed a key post-translational regulatory mechanism of p53: palmitoylation of the cysteine ​​C135 site (p53C135). This palmitoylation directly affects the nuclear localization ability of p53, and the realization of this modification depends on the specific interaction between the palmitotransferase ZDHHC1 and p53C135. Mutant p53 achieves stable nuclear localization through palmitate modification of ZDHHC1, ultimately accumulating and exerting the GOF effect (Tang J, et al. Oncogene. 2021;40(35):5416-5426.). Based on this, the inventors specifically developed a polypeptide sequence that targets the interaction between ZDHHC1-p53C135 and demonstrated that it can promote the degradation of mutant p53 and inhibit the growth of various tumor cells. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a polypeptide that can selectively inhibit the interaction between ZDHHC1 and p53C135 and its application. The polypeptide can selectively inhibit the interaction between ZDHHC1 and p53C135, promote the degradation of mutant p53, and inhibit the growth of various tumor cells. It has the characteristics of high specificity, low toxicity and side effects, and broad-spectrum anti-tumor activity, providing an important reference for the clinical treatment of p53 mutant tumors.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a polypeptide that selectively inhibits the interaction between ZDHHC1 and p53C135, characterized in that the polypeptide contains the following core sequence: SEQ ID No.1: NKMFCQLAK.

[0009] Preferably, the amino acid sequence of the polypeptide is one of the following: (1) SEQ ID No.2: RRRRRRRR-NKMFCQLAK-NH2; (2) SEQ ID No.3: Ac-NKMFCQLAK-Ahx-RRRRRRRR-NH2, where Ac is an N-terminal acetyl group, Ahx is a 6-aminohexanoic acid linker, and -NH2 is a C-terminal amino group.

[0010] The present invention also provides the use of the polypeptide described in any of the above claims in the preparation of a tumor therapeutic drug, wherein the tumor is a p53 mutation-related tumor.

[0011] Preferably, the p53 mutation-related tumors include, but are not limited to, human metastatic pancreatic adenocarcinoma, human colorectal adenocarcinoma, human esophageal squamous cell carcinoma, human breast adenocarcinoma, human pancreatic ductal carcinoma, human renal clear cell adenocarcinoma, human non-small cell lung cancer, and human hepatocellular carcinoma.

[0012] Furthermore, the drug promotes the degradation of mutant p53 by inhibiting the interaction between ZDHHC1 and p53C135, thereby inhibiting tumor cell proliferation, inducing tumor cell apoptosis, arresting the tumor cell cycle, and reducing the migration and invasion capabilities of tumor cells.

[0013] The present invention also provides a pharmaceutical composition comprising a polypeptide comprising a therapeutically effective dose of any of the above-described polypeptides.

[0014] Furthermore, the dosage form of the pharmaceutical composition includes, but is not limited to, injections, oral preparations, or topical preparations. The injections include, but are not limited to, intravenous injections and subcutaneous injections. The oral preparations include, but are not limited to, tablets, capsules, and oral liquids. The topical preparations include, but are not limited to, ointments and gels.

[0015] The beneficial effects of the present invention are as follows: The present invention provides a polypeptide that can selectively inhibit the interaction between ZDHHC1 and p53C135, the polypeptide containing the following core sequence: SEQ ID No.1: NKMFCQLAK, the polypeptide can selectively inhibit the interaction between ZDHHC1 and p53C135, promote the degradation of mutant p53, inhibit the growth of various tumor cells, and has the characteristics of high specificity, low toxicity and side effects, and broad-spectrum anti-tumor activity. This peptide exhibits significant killing effects at the cellular level against various types of p53-mutant tumor cells, including human metastatic pancreatic adenocarcinoma AsPC-1 cells, human colorectal adenocarcinoma HCT-15 and HT-29 cells, human esophageal squamous cell carcinoma KYSE150 / KYSE410 / KYSE510 cells, human breast adenocarcinoma SK-BR-3 cells, human pancreatic ductal carcinoma PANC-1 cells, human renal clear cell adenocarcinoma 786-O cells, human non-small cell lung cancer H1975 and CAL-12T cells, and human hepatocellular carcinoma PLC / PRF / 5 cells. Furthermore, at the same concentration, this peptide selectively targets tumor cells, showing no significant toxic side effects on normal cells (293T cells and the bronchial epithelial cell line BEAS-2B). The peptide described in this invention provides a novel drug candidate for the clinical treatment of p53-mutant tumors and offers important reference for achieving clinical treatment of p53-mutant tumors. Attached Figure Description

[0016] Figure 1The figures are the HPLC results for XY-0913 and XY-0913-2, where A is the HPLC result for XY-0913 and B is the HPLC result for XY-0913-2. Figure 2 The MS results are for XY-0913 and XY-0913-2, where A is the MS result for XY-0913 and B is the MS result for XY-0913-2. Figure 3 CCK8 analysis results for the proliferation of XY-0913 mutant p53 cells (AsPC-1, HCT15, KYSE150, SK-BR-3, PANC-1 and 786-O) and normal cells 293T and BEAS-2B; Figure 4 The effect of XY-0913 peptide on colony formation in mutant p53 cells (AsPC-1, HCT15, KYSE150, SK-BR-3, PANC-1, 786-O, KYSE410, and H1975) is shown in Figure A, which is a visual morphological diagram of colony formation; and Figure B is a bar chart of quantitative statistics on colony formation rate. Figure 5 The effects of XY-0913 peptide on apoptosis in mutant p53 cells (AsPC-1, HCT15, KYSE150, SK-BR-3, KYSE510, H1975, and CAL-12T) and normal 293T cells were analyzed. In this study, A is a visual morphological diagram of apoptosis; B is a statistical column for the quantitative apoptosis rate. Figure 6 The study analyzed the effects of XY-0913 peptide on the cell cycle of mutant p53 cells (AsPC-1, HCT15, KYSE150, and SK-BR-3) and normal 293T cells. In the figure, A is the original cell cycle map obtained by flow cytometry; B is the quantitative bar chart of tumor cell cycle distribution; and C is the comparison and verification chart of normal 293T cell cycle. Figure 7 This study analyzed the wound healing ability of XY-0913 peptide on mutant p53 cells (AsPC-1, HCT15, KYSE150, SK-BR-3, CAL-12T, PLC / PRF / 5, KYSE410) and normal 293T cells. A is a bar chart quantifying cell scratch healing; B is a visual representation of the cell scratch healing process. Figure 8 The cell invasion ability of XY-0913 against mutant p53 cells (AsPC-1, KYSE510, KYSE410, SK-BR-3, H1975, HT-29) was analyzed. In the figure, A is a visual situation diagram; B is a quantitative statistical bar chart. Figure 9This study analyzed the differences in proliferation, colony formation, and invasion capabilities of XY-0913 and XY-0913-02 mutant p53 cells (AsPC-1 and PANC-1). A represents the results of the difference analysis in proliferation capabilities between AsPC-1 and PANC-1 cells; B is a visual representation of colony formation; C is a quantitative statistical bar chart of colony formation rate; D is a visual representation of tumor cell invasion capability; and E is a quantitative statistical bar chart of tumor cell invasion capability. Figure 10 The effects of XY-0913 and XY-0913-02 on the proliferative capacity of human organs from lung cancer patients are shown in Figure A. A represents the quantitative results of proliferative activity; B represents the histological staining results; and C represents a visual representation of the proliferative activity. Figure 11 The image shows the inhibitory effect of cells carrying polypeptide sequences on solid tumors in lung cancer A549-bearing mice; where A is a comparison of solid tumors from tumor-bearing mice; B is a bar chart of tumor weight quantification; and C is a tumor growth curve. Figure 12 The results show the effects of XY-0913 on inhibiting the interaction between ZDHHC1 and p53C135 and suppressing mutant p53 protein. Among them, A is a bar chart of WB quantification of mutant p53 protein level; B is a Co-IP electrophoresis diagram; C is a visual diagram of laser focusing imaging; and D is a graph of FRET efficiency quantification results. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. It should be noted that the experimental materials whose source is not specified in the embodiments of the present invention are all commercially available, and the experimental methods whose specific conditions are not specified in the embodiments of the present invention are generally performed according to conventional experimental methods or according to the methods recommended by the experimental material manufacturers.

[0018] Example 1 Preparation of polypeptides 1. Preparation of XY0913 (SEQ ID No.2: RRRRRRRR-NKMFCQLAK-NH2) The peptide RRRRRRR-NKMFCQLAK was synthesized according to the conventional methods and procedures for peptide synthesis. After the synthesis of peptide RRRRRRR-NKMFCQLAK, C-terminal amidation modification was performed using Rink Amide Resin. The C-terminus amidation modification was completed when the crude peptide was cleaved from the resin. The HPLC purity was 95.43%. Figure 1 A); [M+3H) 3+:m / z=778.05;[M+4H] 4+ :m / z=583.70;[M+5H] 5+ :m / z=467.17. Figure 2 A).

[0019] 2. Preparation of XY0913-02 (SEQ ID No.3: Ac-NKMFCQLAK-Ahx-RRRRRRRR-NH2) The peptide NKMFCQLAK-Ahx-RRRRRRRR was synthesized according to standard methods and procedures. The final step in the synthesis of NKMFCQLAK-Ahx-RRRRRRRR involved acetylation of the N-terminal amino group using acetic anhydride and pyridine (or DIEA). C-terminal amidation was performed using Rink Amide Resin; the amidation was completed when the crude peptide was cleaved from the resin. The HPLC purity was 96%. Figure 1 B); [M+3H) 3+ :m / z=829.78;[M+4H] 4+ :m / z=622.61. ( Figure 2 B).

[0020] 2 XY-0913 was used to analyze the proliferative toxicity of various tumor cells. The inhibitory effect of XY-0913 on the proliferation of six different tumor cell types (AsPC-1, HCT15, KYSE150, SK-BR-3, PANC-1, and 786-O) and two normal cell types (293T and BEAS-2B) was evaluated at the cellular level. Specifically, cells in the logarithmic growth phase were selected, the original culture medium was removed, and the cells were washed twice with PBS buffer. After trypsin digestion, the cells were centrifuged to obtain cell pellets, which were then counted and seeded into 96-well plates. After cell attachment, XY-0913 at concentrations of 0, 2.5, 5, 10, and 20 μM were added to each well, and treatment was performed at different time points (0, 24, 48, and 72 h). After treatment, CCK8 assay reagent was added, and the absorbance at OD450 nm was measured for each well. The results showed that XY-0913 significantly inhibited the growth of tumor cells, but at the same concentration, it did not significantly affect the proliferation of normal cells. (See attached table for details.) Figure 3 .

[0021] Example 3: Effects of XY-0913 on the clonogenic ability of various tumor cells To evaluate the effect of XY-0913 on the colony-forming ability of different cell types at the cellular level, eight tumor cell lines (AsPC-1, HCT15, KYSE150, SK-BR-3, PANC-1, 786-O, KYSE410, and H1975) were included in the experiment. Cells in the logarithmic growth phase were harvested, the culture medium was discarded, and the cells were washed twice with PBS buffer. After trypsin digestion and centrifugation, the cell pellet was collected, counted, and seeded into 6-well plates. After cell adhesion, XY-0913 at concentrations of 0 and 20 μM was added to the cells, respectively. When the cells grew to the point where there were obvious colonies and the cell number was greater than 50, the culture was terminated, and the cells were fixed with 4% paraformaldehyde for 30 min. After discarding the fixative, the cells were washed once with PBS, stained with crystal violet solution for 30 min, rinsed with tap water, air-dried, and photographed. The results showed that, compared with the control group, XY-0913 significantly inhibited tumor cell colony formation. (See attached figures). Figure 4 .

[0022] Effects of 4XY-0913 on apoptosis in various tumor cells The effects of XY-0913 on apoptosis levels in various cell types were examined at the cellular level, including seven tumor cell types: AsPC-1, HCT15, KYSE150, SK-BR-3, KYSE510, CAL-12T, and H1975, and normal cell type 293T. Cells in logarithmic growth phase were harvested, discarded from the culture medium, washed twice with PBS, digested with trypsin, centrifuged, and the resulting cell pellet was counted and seeded into 6-well plates. The following day, 0 and 10 μM of XY-0913 were added to the wells, and the cells were cultured for 48 h. After trypsin digestion, the cells were washed twice with PBS, stained with Annexin V and PI for 15 min, and then apoptosis levels were detected by flow cytometry. The results showed that, compared with the control group, XY-0913 significantly promoted tumor cell apoptosis but did not affect the apoptosis levels of normal cells. (See attached figures). Figure 5 .

[0023] Effects of 5XY-0913 on the cell cycle of various tumor cells The effects of XY-0913 on various cell cycles were investigated at the cellular level, including four tumor cell types: AsPC-1, HCT15, KYSE150, and SK-BR-3, and normal cells 293T. Cells in the logarithmic growth phase were harvested, discarded from the culture medium, washed twice with PBS, digested with trypsin, centrifuged, and the resulting cell pellet was counted and seeded into 6 cm dishes. The following day, 0 and 10 μM XY-0913 were added to the wells, and the cells were cultured for 48 h. After trypsin digestion, the cells were washed twice with PBS, fixed with 70% pre-chilled ethanol at 4°C for at least 4 h, stained with propidium iodide for 30 min, and then analyzed by flow cytometry to determine the cell cycle. The results showed that, compared with the control group, XY-0913 significantly inhibited the tumor cell cycle but did not affect the normal cell cycle. (See attached table for details.) Figure 6 .

[0024] Example 6: Effects of XY-0913 on the wound healing ability of various tumor cells The effect of XY-0913 on the wound healing ability of various cells was examined at the cellular level, including seven tumor cell types: AsPC-1, HCT15, KYSE150, SK-BR-3, CAL-12T, PLC / PRF / 5, and KYSE410, as well as normal cells 293T. Cells in the logarithmic growth phase were harvested, discarded from the culture medium, washed twice with PBS, digested with trypsin, centrifuged, and the resulting cell pellet was counted and seeded into 12-well plates. The next day, 0 and 10 μM XY-0913 were added, and the cells were cultured for 48 h. After trypsin digestion, 5 × 10⁶ cells / well were added to the 12-well plates. 5 Cells were seeded according to the principle that the confluence rate reached approximately 100% overnight. Before plating, three parallel horizontal lines were evenly drawn on the back of a 12-well plate using a marker pen, passing through the wells. The cells were then marked with a pipette tip perpendicular to the cell plane and the marking lines on the back of the plate. After marking, the cells were washed three times with sterile PBS to remove non-adherent cells, and then replaced with fresh serum-free culture medium. The cells were incubated at 37°C with 5% CO2 for 24 hours, and then observed and photographed. The experimental results showed that, compared with the control group, XY-0913 significantly inhibited tumor cell wound healing. (See attached results). Figure 7 .

[0025] Example 7: Effects of XY-0913 on the invasive ability of various tumor cells The effects of XY-0913 on the invasive abilities of various cell types were evaluated at the cellular level, including six tumor cell lines: AsPC-1, KYSE510, KYSE410, SK-BR-3, H1975, and HT-29. Cells in the logarithmic growth phase were harvested, discarded from the culture medium, washed twice with PBS, digested with trypsin, centrifuged, and the resulting cell pellet was seeded into 12-well plates. The next day, 0 and 10 μM XY-0913 were added, and the cells were cultured for 48 h. After trypsin digestion, 5 × 10⁶ cells were harvested.4 Cell culture medium suspension was added to 8 mm chambers for culture. After cells emerged from the chambers, they were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet for 30 minutes, washed with tap water, air-dried, photographed, and the number of cells that had emerged from the chambers was counted. The experimental results showed that, compared with the control group, XY-0913 significantly inhibited the invasive ability of tumor cells. See the results below. Figure 8 .

[0026] Example 8: Differences in tumor cell proliferation between XY-0913 and XY-0913-02 At the cellular level, the effects of XY-0913 and XY-0913-02 on the proliferation, colony formation, and invasion of two types of tumor cells: AsPC-1 and PANC-1 were compared and analyzed. Cells in the logarithmic growth phase were harvested, the culture medium was discarded, and the cells were washed twice with PBS. After trypsin digestion, the cells were centrifuged to obtain cell pellets, which were then counted and seeded into 96-well plates. After cell attachment, the two peptides at concentrations of 0, 10, and 20 μM were added to the cells, and the treatment was carried out for 0, 24, 48, and 72 h, respectively. The absorbance at OD450 nm was then measured using CCK8 assay. The results showed that XY-0913-02 significantly inhibited the proliferation of both types of tumor cells better than XY-0913. Simultaneously, the effects of the two peptides on cell colony formation and invasion were detected using the methods described in Examples 3 and 4. The results showed that XY-0913-02 significantly inhibited the colony formation and invasion of both types of tumor cells better than XY-0913. (See attached figures). Figure 9 .

[0027] Example 9: Effect of XY-0913 on organ proliferation in lung cancer patients "Miniature organoids" formed by the 3D self-assembly of stem cells precisely replicate the tissue microenvironment and cell-cell interaction mechanisms, overcoming the limitations of traditional 2D culture. They can carry patient-specific gene mutation characteristics, enabling personalized drug efficacy prediction and customized medical plan development, with a correlation of up to 89% with actual patient efficacy, significantly superior to conventional cell line models. Therefore, this study investigated the effects of two peptides, XY-0913 and XY-0913-02, on the proliferative capacity of human lung cancer organs at the organoid level. Collect organoids in good growth condition, discard the original culture medium, add 1 ml of trypsin to each well, pipette and transfer to 15 ml centrifuge tubes (pipette 20 times again), place in a water bath for 20 min to dissociate; after centrifugation, discard the supernatant, add 10 ml of ad DMEM / F12+++ to the pellet, pipette 20 times to remove matrix gel, centrifuge at 4°C and discard the supernatant; pipette 10 ml of ad DMEM / F12+++, filter through a 70 μm cell filter, centrifuge at 4°C and discard the supernatant; add 3 ml of ad DMEM / F12+++ to resuspend, count, take an appropriate amount of cells and resuspend in GCM containing 5% Matrigel, plate to 384 wells, and incubate at 37°C in a 5% CO2 incubator. Add peptide drugs to the 384-well plates according to a concentration gradient, the final volume of the 384-well plate is 40 μL, and the final concentrations of peptide drugs are 0, 2.5, 5, 10, and 20 μM. The proliferative activity of organoids after treatment with peptide drugs for 0, 24, 48, and 72 hours was detected using CellTiter-Glo® 3D Reagent reagent. The results showed that, with increasing peptide concentration and incubation time, peptides XY-0913 and XY-0913-02 significantly inhibited the proliferation of tumor-bearing human organoids compared to the control group, and XY-0913-02 showed significantly better inhibition of tumor organoid proliferation than XY-0913. (See attached results). Figure 10 .

[0028] Example 10: Antitumor effect of XY-0913 on tumor-bearing mice with lung cancer A549 cells A mouse model of lung cancer was established: Large numbers of A549 cells stably transfected with plasmids carrying Vector and p53 polypeptide sequences (XY-0913) were cultured. Logarithmic growth phase cells were collected, the culture medium was removed, and the cells were washed twice with PBS. The cell pellet was collected and counted. Approximately 0.1 mL of 5 × 10⁵ cells was subcutaneously injected into nude mice. 6 Cells were used to regularly observe subcutaneous tumor formation and measure tumor size. Comparison of tumor size between the peptide plasmid group and the control group revealed that tumor growth in mice transfected with the XY-0913 peptide sequence was significantly smaller than that in the control group. Results are shown below. Figure 11 .

[0029] Example 11: Effect of XY-0913 on mutant p53 At the cellular level, the effects of XY-0913 on p53 protein levels and the binding ability of p53 to palmitate transferase ZDHHC1 in AsPC-1 tumor cells were analyzed. Cells in the logarithmic growth phase were washed twice with PBS after discarding the culture medium, digested with trypsin, centrifuged to obtain cell pellets, and counted. The pellets were then seeded into 6-well plates, 10cm dishes, and laser confocal dishes, respectively. After cell attachment, 0 and 10 μM peptides were added to the cells, respectively. After 24 h of treatment, cells were collected for Western blot, Co-IP, and Fret experiments. The results showed that XY-0913 significantly inhibited p53 protein levels and suppressed the binding of p53 to ZDHHC1. (See attached figures). Figure 12 .

[0030] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A polypeptide that selectively inhibits the interaction between ZDHHC1 and p53C135, characterized in that, The polypeptide contains the following core sequence: SEQ ID No.1: NKMFCQLAK.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is one of the following: (1) SEQ ID No.2: RRRRRRRR-NKMFCQLAK-NH2; (2) SEQ ID No.3: Ac-NKMFCQLAK-Ahx-RRRRRRRR-NH2, where Ac is an N-terminal acetyl group, Ahx is a 6-aminohexanoic acid linker, and -NH2 is a C-terminal amino group.

3. The use of the polypeptide according to any one of claims 1 to 2 in the preparation of a tumor therapeutic drug, characterized in that, The tumor is a p53 mutation-related tumor.

4. The application according to claim 3, characterized in that, The p53 mutation-related tumors include, but are not limited to, human metastatic pancreatic adenocarcinoma, human colorectal adenocarcinoma, human esophageal squamous cell carcinoma, human breast adenocarcinoma, human pancreatic ductal carcinoma, human renal clear cell adenocarcinoma, human non-small cell lung cancer, and human hepatocellular carcinoma.

5. The application according to claim 3, characterized in that, The drug inhibits the interaction between ZDHHC1 and p53C135, promotes the degradation of mutant p53, thereby inhibiting tumor cell proliferation, inducing tumor cell apoptosis, arresting the tumor cell cycle, and reducing the migration and invasion capabilities of tumor cells.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective dose of the polypeptide of claim 1 or 2.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage forms of the pharmaceutical composition include, but are not limited to, injections, oral preparations, or topical preparations. The injections include, but are not limited to, intravenous injections and subcutaneous injections. The oral preparations include, but are not limited to, tablets, capsules, and oral liquids. The topical preparations include, but are not limited to, ointments and gels.

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