Small molecule polypeptide for p53 mutant targeted degradation based on p53c176 palmitoylation site and use thereof

Abstract

Provided are a small molecule polypeptide XY-0728 for p53 mutant targeted degradation based on a p53C176 palmitoylation site and a use thereof. The sequence of the small molecule polypeptide XY-0728 is R8-TEVVRRCPHHERC.

Description

Small molecule polypeptide targeting degradation of p53 mutant based on p53 C176 palmitic acid modification site and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a small molecule polypeptide targeting degradation of p53 mutant based on p53 C176 palmitic acid modification site and application thereof. BACKGROUND

[0002] TP53 is a tumor suppressor gene, known as the "guardian of the genome", located on human chromosome 17p13.1, and encodes 393 amino acids. It is a regulator in the cell cycle and is related to cell cycle regulation, DNA repair, cell differentiation, and apoptosis. The p53 protein is a 53kDa protein encoded by the TP53 gene, which is a powerful tumor suppressor that can inhibit tumor growth through multiple ways. As a transcription factor, p53 regulates the expression of target genes, promotes cell cycle arrest, apoptosis, DNA repair, etc. In addition, p53 can also exert anti-proliferative effects through mechanisms independent of the transcription machinery. It has been reported that p53 affects almost all cellular compartments and organelles, including mitochondria, lysosomes, endoplasmic reticulum, etc. In normal cells without stress, the level of p53 is maintained at a low level by the proteasome degradation process mediated by the E3 ubiquitin ligase MDM2.

[0003] When the p53 protein is mutated, its tumor suppressor activity is impaired. Unlike other tumor suppressor genes, cancer-related p53 mutations are mainly missense mutations, resulting in the substitution of a single amino acid. Usually, the resulting mutant p53 accumulates in cancer cells. In addition to losing the ability to bind DNA and regulate transcription, which ultimately leads to the loss of tumor suppressor function, some mutant p53 proteins also acquire new functions, further promoting the occurrence and development of cancer. Studies have shown that about 50% of human cancers have p53 mutations, and inhibitors targeting mutant p53 proteins are a hot spot for the treatment of cancer. However, due to the lack of typical drug target characteristics as a nuclear transcription factor, p53 has been considered "undruggable" for a long time. However, in recent years, there have been many promising approaches based on p53 therapy, most of which are represented by inhibitors that stabilize wild-type p53 or restore the wild-type conformation of mutant p53 protein and inhibit the interaction between MDM2 and p53 protein. Although various treatment methods targeting p53 protein have emerged in recent years, no ideal drug has been developed so far. Therefore, the development of polypeptide inhibitors targeting degradation of p53 mutant will provide an important reference for the clinical treatment of p53 mutant cancer patients.

[0004] The complex functions of p53 protein are closely related to its various post-translational modifications, including phosphorylation, acetylation, ubiquitination and other modifications. Among them, palmitic acid modification is a modification form of covalently connecting saturated fatty acids (palmitic acid) to proteins, which can affect the subcellular localization, stability and function of proteins. Our team first revealed the palmitic acid modification of p53 protein, which occurred at p53C135, p53C176 and p53C275, and found that this modification played an important role in regulating the subcellular nuclear localization of wild-type p53 (Tang J, et al. Oncogene. 2021 Sep;40(35):5416-5426.). TECHNICAL PROBLEM

[0005] Therefore, the purpose of the present application is to provide a small molecule polypeptide targeting degradation of p53 mutant based on p53C176 palmitic acid modification site and its application, which has effective killing effect on p53 mutant tumors and can only kill tumor cells under the same action, has no toxic side effects on normal cells, has the advantages of high selectivity, weak toxic side effects, etc., and has the prospect of developing into a therapeutic drug for p53 mutant tumors. TECHNICAL SOLUTION

[0006] In order to achieve the above purpose, the present application provides the following technical scheme:

[0007] The present application provides a small molecule polypeptide XY-0728 targeting degradation of p53 mutant based on p53C176 palmitic acid modification site, and the sequence of the polypeptide XY-0728 is R8-TEVVRRCPHHERC.

[0008] The present application also provides the use of the small molecule polypeptide XY-0728 targeting degradation of p53 mutant in the preparation of a drug for treating p53 mutant tumors.

[0009] Further, the p53 mutant tumor is breast cancer, esophageal cancer, lung cancer, pancreatic cancer, liver cancer, colon cancer.

[0010] The present application also provides a drug for treating p53 mutant tumors, which contains the small molecule polypeptide XY-0728 targeting degradation of p53 mutant.

[0011] Further, the p53 mutant tumor is breast cancer, esophageal cancer, lung cancer, pancreatic cancer, liver cancer, colon cancer. ADVANTAGEOUS EFFECTS

[0012] The present application has the beneficial effect that the present application provides a small molecule polypeptide XY-0728 based on a p53C176 palmitic acid modification site for targeted degradation of p53 mutants and an application thereof, the sequence of the small molecule polypeptide XY-0728 is R8-TEVVRRCPHHERC, which exhibits an effective killing effect on p53 mutant tumors, and under the same action, only tumor cells can be killed, and normal cells are basically non-toxic and have no side effects, has the advantages of high selectivity, weak side effects, etc., has the prospect of developing into a therapeutic drug for p53 mutant tumors, and provides a new idea for realizing the clinical treatment of p53 mutant tumors. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a dynamic continuous monitoring experiment of living cells, which proves that XY-0728 can quickly enter tumor cells, but it is difficult to enter normal immortalized epithelial cells 293T.

[0014] Figure 2 is an immunofluorescence experiment, which proves that XY-0728 can target p53 and block p53 from entering the nucleus.

[0015] Figure 3 is an immunofluorescence experiment, which proves that XY-0728 can degrade mutant p53 in cells and the nucleus.

[0016] Figure 4 is the CCK8 proliferation toxicity result of polypeptide XY-0728 (µM) on different p53 mutant tumor cells.

[0017] Figure 5 is the CCK8 proliferation toxicity of polypeptide XY-0728 (µM) on BEAS-2B cells and 293T cells.

[0018] Figure 6 is the influence of polypeptide XY-0728 on the clonogenic ability of MB231 cells and KYSE150 cells.

[0019] Figure 7 is the influence of polypeptide XY-0728 on the clonogenic ability of H2122 cells and HT29 cells.

[0020] Figure 8 is a mouse tumor photo, wherein the sample is taken on the 15th day after administration.

[0021] Figure 9 is the analysis result of mouse tumor volume, wherein the sample is taken on the 15th day after administration.

[0022] Figure 10 is a line graph of the influence of polypeptide XY-0728 on mouse tumor volume.

[0023] Figure 11 is a line graph of the influence of polypeptide XY-0728 on mouse body weight. Embodiment of the present application

[0024] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only illustrative and explanatory of the present application and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above description of the present application is covered by the scope of protection intended by the present application. It should be noted that the experimental materials not mentioned in the experimental materials source in the embodiments of the present application are commercially available, and the experimental methods not mentioned in the specific conditions in the embodiments of the present application are usually carried out according to the conventional experimental methods or according to the experimental material manufacturer's recommended method. It should be noted that the p53 mutant tumor cells involved in the present application are PC9 cells (p53 mutant lung cancer cell line), PC9 / GR cells (PC9 drug-resistant cell line), H2122 cells (p53 mutant lung adenocarcinoma cell line), H1299 cells (p53 mutant non-small cell lung cancer cell line), H1975 cells (p53R273H mutant non-small cell lung cancer cell line), MB231 cells (p53R280K mutant breast cancer cell line), SK-BR-3 cells (p53R175H mutant breast cancer cell line), BT549 cells (p53R249S mutant breast cancer cell line), KYSE150 cells (p53R248Q mutant esophageal squamous cell carcinoma cell line), and HT29 cells (p53R273H mutant colon cancer cell line). Example 1 Dynamic continuous monitoring of living cells confirmed that XY-0728 can quickly enter tumor cells, but it is difficult to enter normal immortalized epithelial cells 293T

[0025] PC9 lung cancer cells expressing p53 mutations were selected, and human embryonic kidney cell line 293T was used as a control cell. After treatment with FITC-labeled XY-0728 (10 μM), continuous live cell detection was performed using a Leica STELLARIS 5 & STED laser confocal microscopy system. Green represents the FITC-labeled XY-0728 tracking polypeptide inhibitor, red represents PI tracking cell death, blue represents DAPI staining representing the cell nucleus, and bright field is added for real-time monitoring. Photographs were taken every 5 minutes, and the images were selected according to the time points to form a group of images. The results are shown in Figure 1, which confirmed that XY-0728 can quickly enter tumor cells, but it is difficult to enter normal immortalized epithelial cells 293T. Example 2 Immunofluorescence experiment confirmed that XY-0728 can target p53 and block p53 into the nucleus

[0026] H1299 cells which do not express p53 by themselves were selected, and wild-type p53 (p53WT) was transfected, and the effect of FITC-labeled XY-0728 (10 μM) on the localization of cancer cells after 6 hours of treatment was studied by immunofluorescence method. Briefly, the treated cells on the coverslips were washed with PBS for 3 times, 5 min each time. 4% paraformaldehyde was used for room temperature fixation for 30 min, and the coverslips were washed with PBS for 3 times, 5 min each time. An appropriate amount of 0.5% Triton-100 was added, and the mixture was incubated at room temperature for 10 min, and the coverslips were washed with PBS for 3 times. Immunostaining blocking solution was used for blocking at room temperature for 1 h. The primary antibody (anti-p53, D01, sc-126, 1:200) was incubated at 4°C overnight. The mixture was incubated at room temperature for 1 h. The coverslips were washed with PBS for 3 times, 5 min each time. The secondary antibody (anti-Mouse IgG 594, Jackson Immuno Research, 111-585-003, 1:200) was incubated at room temperature for 2 h (avoiding light operation). The coverslips were washed with PBS for 3 times, 5 min each time. An appropriate amount of DAPI was added directly, and the mixture was incubated at room temperature for 5 min. The DAPI was removed, and the coverslips were washed with PBS for 3 times. The coverslips were mounted with antifade reagent, and observed under a confocal microscope. The images were collected, and the results are shown in FIG. 2, which confirmed that XY-0728 can target p53 and block the entry of p53 into the nucleus. Example 3 Immunofluorescence experiment confirmed that XY-0728 can degrade mutant p53 in cells and nucleus

[0027] Various p53 mutant cancer cells were selected, and 293T cells were used as control cells. The effect of FITC-labeled XY-0728 (10 μM) on cancer cells after 6 hours of treatment was studied by immunofluorescence method. The method was the same as that in Example 2, and the results are shown in FIG. 3, which confirmed that XY-0728 can degrade mutant p53 in cells and nucleus: MB231 (p53R280K), NCIH1975 (p53R273H), KYSE150 (p53R248Q), Huh7 (p53Y220H), SK-BR-3 (p53R175H), BT549 (p53R249S), and KYSE410 (p53R337C) cells. CCK8 proliferation toxicity analysis of polypeptide XY-0728 on different P53 mutant tumor cells and normal cells

[0028] CCK8 proliferation effect of polypeptide XY-0728 on p53 mutant tumor cells: PC9 cells, PC9 / GR cells, H2122 cells, H1299 cells, H1975 cells, MB231 cells, SK-BR-3 cells, BT549 cells, KYSE150 cells, HT29 cells and normal cells BEAS-2B cells, 293T cells was detected at the cellular level. The logarithmic growth phase of each cell was taken, and after discarding the culture medium, the cells were washed twice with PBS, trypsinized, centrifuged to obtain cell pellets and counted, and then plated in 96-well plates. After the cells adhered, 2.5, 5, 10 and 20 μM of polypeptide XY-0728 were added to each cell, respectively, and the culture medium was used as a control. CCK8 reagent was used to measure OD450nm at 0, 24, 48 and 72 h. The experimental results are shown in Figures 4-5. The experimental results show that polypeptide XY-0728 has no effect on the growth of human embryonic kidney cells 293T and normal lung epithelial cells BEAS-2B (Figure 4), and with increasing concentration of polypeptide XY-0728, the cell activity of p53 mutant tumor cells: PC9 cells, PC9 / GR cells, H2122 cells, H1299 cells, H1975 cells, MB231 cells, SK-BR-3 cells, BT549 cells, KYSE150 cells, HT29 cells decreased significantly (Figure 5). It is shown that XY-0728 has no effect on the growth of normal cells, can significantly inhibit p53 mutant tumor cells: PC9 cells, PC9 / GR cells, H2122 cells, H1299 cells, H1975 cells, MB231 cells, SK-BR-3 cells, BT549 cells, KYSE150 cells, HT29 cells, and the inhibition ability increases with increasing concentration of polypeptide XY-0728. Example 5 Analysis of the clonogenic ability of polypeptide XY-0728 on different p53 mutant tumor cells

[0029] The effect of polypeptide XY-0728 on the clone formation ability of different p53 mutant tumor cells: MB231 cells, KYSE150 cells, H2122 cells, and HT29 cells was detected at the cell level. The logarithmic growth phase cells were taken, washed twice with PBS after discarding the culture medium, trypsinized, centrifuged to obtain cell pellets, counted, and then plated in a 24-well plate. After the cells adhered, 2.5, 5, and 10 μM of polypeptide XY-0728 was added to each cell, respectively, and the culture medium was used as a control. When the cells grew into obvious clone groups, the culture medium was discarded, washed with PBS, fixed with 4% paraformaldehyde for 30 min, washed with PBS after discarding the fixing solution, stained with crystal violet solution, dried, and then photographed. The experimental results are shown in FIGS. 6-7, and the results show that as the concentration of polypeptide XY-0728 increases, the clone formation ability of p53 mutant tumor cells: MB231 cells, KYSE150 cells, H2122 cells, and HT29 cells significantly decreases, indicating that polypeptide XY-0728 can significantly inhibit the clone formation ability of p53 mutant tumor cells: MB231 cells, KYSE150 cells, H2122 cells, and HT29, and the inhibition ability increases as the concentration of polypeptide XY-0728 increases. Example 6 Analysis of the Tumor Inhibition Effect and Body Weight Impact of Polypeptide XY-0728 on MB231 Cell and KYSE150 Cell Tumor-bearing Mice

[0030] Establishment of breast cancer and esophageal cancer mouse models: A large number of MB231 and KYSE150 cells were cultured, and the logarithmic growth phase cells were collected, washed twice with PBS, and counted. The cells were collected and counted. 0.1 ml of about 2 x 10 6 The cells were subcutaneously injected into nude mice, and the subcutaneous tumor formation was observed regularly. After confirming that the mice had tumors of 5 mm 2 The control group was injected with solvent DMSO (50 mg / kg) around the tumor, and the experimental group was injected with polypeptide XY-0728 (50 mg / kg) around the tumor. Within 15 days, the experimental group was administered a total of 3 times, on the first day, the third day, and the fifth day. Fifteen days after administration, the experimental subjects were sacrificed, the tumor was observed by taking pictures, and the tumor volume was measured.

[0031] The mouse tumor photos are shown in Figure 8, and each group has 9 mice. The samples are taken from the 15th day after administration. Compared with the tumor of the control group, the tumor of the mice in the experimental group is significantly reduced. The mouse tumor volume analysis is shown in Figure 9, and each group has 9 mice. In KYSE150 tumor-bearing mice, compared with the baseline tumor volume, the tumor volume of 9 mice in the control group gradually increased, and in the experimental group, except for 1 mouse, the tumor volume of the remaining mice was significantly reduced compared with the baseline volume. In MB231 tumor-bearing mice, compared with the baseline, the tumor volume of the mice in the control group increased during the experiment, and in the experimental group, only 1 mouse had an increased tumor volume compared with the baseline, and the tumor volume of the remaining mice decreased.

[0032] The mouse tumor volume was measured regularly during the experiment, and the line chart is shown in Figure 10. Compared with the control group, the tumor volume of the mice in the experimental group was significantly reduced.

[0033] The mouse weight was measured regularly during the experiment, and the experimental results are shown in Figure 11. The results show that there is no significant difference in the weight of the mice in the experimental and control groups, indicating that the polypeptide XY-0728 has little systemic toxicity to the mice and has no effect on the weight of the growing mice.

[0034] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A small molecule polypeptide XY-0728 that targets and degrades p53 mutants based on the p53C176 palmitic acid modification site, characterized in that, The sequence of the polypeptide XY-0728 is: R8-TEVVRRCPHHERC, where R8 represents RRRRRRRRR.

2. The use of the small molecule polypeptide XY-0728, which targets and degrades p53 mutants, as described in claim 1, in the preparation of a drug for treating p53-mutant tumors, wherein, The p53-mutated tumors include breast cancer, esophageal cancer, lung cancer, pancreatic cancer, liver cancer, and colon cancer.

3. A drug for treating p53-mutant tumors, characterized in that, The drug contains the small molecule polypeptide XY-0728 that targets and degrades p53 mutants as described in claim 1, wherein the p53 mutant tumor is breast cancer, esophageal cancer, lung cancer, pancreatic cancer, liver cancer, or colon cancer.

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