Small molecule peptides targeting degradation of p53 mutants based on p53C176 palmitic acid modification site and their applications

Abstract

The present invention provides a small molecule polypeptide XY-0728 for targeted degradation of p53 mutants based on the p53C176 palmitic acid modification site 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 can only kill tumor cells under the same action, and has basically no toxic side effects on normal cells. It has the advantages of high selectivity, weak toxic side effects, etc., and has the prospect of being developed into a therapeutic drug for p53 mutant tumors, providing a new idea for realizing the clinical treatment of p53 mutant tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule polypeptide for targeted degradation of p53 mutants based on a p53C176 palmitic acid modification site and an application thereof. Background Art

[0002] TP53 is a tumor suppressor gene, known as the "guardian of the genome". It is located on human chromosome 17p13.1 and encodes a total of 393 amino acids. It is a regulatory factor in the cell growth cycle and is related to cell cycle regulation, DNA repair, cell differentiation, apoptosis and other pathways. The p53 protein is a 53kDa protein encoded by the TP53 gene. It is a powerful tumor suppressor that can inhibit tumor growth in many ways. As a transcription factor, p53 regulates the expression of target genes and promotes cell cycle arrest, apoptosis, DNA repair, etc. In addition, p53 can also exert anti-proliferative effects through other mechanisms independent of the transcriptional mechanism. It is reported that p53 affects almost all cell compartments and organelles, including mitochondria, lysosomes, endoplasmic reticulum, etc. In normal cells that are not under stress, the level of p53 is maintained at a low level by the protease degradation process mediated by the E3 ubiquitin ligase MDM2 in the body.

[0003] When the p53 protein mutates, its tumor suppressor activity is impaired. Unlike other tumor suppressor genes, cancer-related p53 mutations are mainly missense mutations, resulting in the replacement of a single amino acid. Usually, the resulting mutant p53 accumulates in large quantities in cancer cells. In addition to losing the ability to bind to 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 confirmed that about 50% of human cancers have p53 protein mutations, and inhibitors targeting mutant p53 proteins are a hot spot for cancer treatment. However, as p53 is a nuclear transcription factor, it lacks typical drug target characteristics and has long been considered "undruggable". Despite this, many promising methods based on p53 therapy have emerged in recent years, most of which are represented by inhibitors that stabilize wild-type p53 or restore the wild-type conformation of mutant p53 proteins and inhibit the interaction between MDM2 and p53 proteins. Although various treatments for p53 proteins have emerged in recent years, a drug with ideal efficacy has not yet been developed. Therefore, research and development of peptide inhibitors that target and degrade p53 mutants will provide important references for the clinical treatment of cancer patients with p53 mutations.

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

[0005] In view of this, the purpose of the present invention is to provide a small molecule polypeptide for targeted degradation of p53 mutants based on the p53C176 palmitic acid modification site and its application. The small molecule polypeptide has an effective killing effect on p53 mutant tumors, and can only kill tumor cells under the same action, and has basically no toxic side effects on normal cells. It has the advantages of high selectivity and weak toxic side effects, and has the prospect of being developed into a therapeutic drug for p53 mutant tumors.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a small molecule polypeptide XY-0728 for targeted degradation of p53 mutants based on the p53C176 palmitic acid modification site. The sequence of the polypeptide XY-0728 is: R8-TEVVRRCPHHERC.

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

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

[0010] The present invention also provides a medicine for treating p53 mutant tumors, wherein the medicine contains the above-mentioned small molecule polypeptide XY-0728 for targeted degradation of p53 mutants.

[0011] Furthermore, the p53 mutant tumor is breast cancer, esophageal cancer, lung cancer, pancreatic cancer, liver cancer, or colon cancer.

[0012] The beneficial effects of the present invention are as follows: the present invention provides a small molecule polypeptide XY-0728 for targeted degradation of p53 mutants based on the p53C176 palmitic acid modification site and its application, the sequence of the small molecule polypeptide XY-0728 is R8-TEVVRRCPHHERC, it exhibits an effective killing effect on p53 mutant tumors, and under the same action can only kill tumor cells, and has basically no toxic side effects on normal cells, has the advantages of high selectivity, weak toxic side effects, etc., has the prospect of being developed 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 THE DRAWINGS

[0013] Figure 1 The dynamic continuous monitoring experiment of living cells confirmed that XY-0728 can quickly enter tumor cells, but has difficulty entering normal immortalized epithelial cells 293T.

[0014] Figure 2 Immunofluorescence experiments confirmed that XY-0728 could target p53 and block p53 from entering the cell nucleus.

[0015] Figure 3 Immunofluorescence experiments confirmed that XY-0728 can degrade mutant p53 and cell nuclei in cells.

[0016] Figure 4 The results of the CCK8 proliferation toxicity of polypeptide XY-0728 (μM) on tumor cells with different p53 mutations.

[0017] Figure 5 The results of the proliferation toxicity of polypeptide XY-072 (μM) 8 to CCK8 in BEAS-2B cells and 293T cells.

[0018] Figure 6 The results show the effect of polypeptide XY-0728 on the clone-forming ability of MB231 cells and KYSE150 cells.

[0019] Figure 7 The results show the effect of polypeptide XY-0728 on the clone-forming ability of H2122 cells and HT29 cells.

[0020] Figure 8 These are photos of mouse tumors, where samples were taken on the 15th day after administration.

[0021] Fig. 9 The results of mouse tumor volume analysis are shown in Figure 2, where samples were taken on the 15th day after administration.

[0022] Fig.10 This is a line graph showing the effect of polypeptide XY-0728 on mouse tumor volume.

[0023] Fig.11 It is a line graph showing the effect of polypeptide XY-0728 on the body weight of mice. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations 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 that the present invention is intended to protect. It should be noted that the experimental materials whose sources are not indicated in the embodiments of the present invention can all be obtained commercially, and the experimental methods in the embodiments of the present invention whose specific conditions are not indicated are usually carried out according to conventional experimental methods or according to the methods recommended by the experimental material manufacturers. It should be noted that the p53 mutant tumor cells involved in the present invention are PC9 cells (p53 mutant lung cancer cell line), PC9 / GR cells (PC9 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).

[0025] Example 1 Dynamic and continuous monitoring of living cells confirmed that XY-0728 can quickly enter tumor cells, but has difficulty entering normal immortalized epithelial cells 293T

[0026] PC9 lung cancer cells expressing p53 mutation and human embryonic kidney cell line 293T were selected as control cells. After treatment with FITC-labeled XY-0728 (10 μM), continuous live cell detection was performed using the Leica STELLARIS 5 & STED laser confocal microscopy system. Green is FITC-labeled XY-0728 that tracks peptide inhibitors, red is PI that tracks cell death, and blue is DAPI staining that represents the cell nucleus. In addition, bright field and real-time monitoring were performed. Photos were taken every 5 minutes, and pictures were selected according to time points. The results are shown in the figure. Figure 1 As shown, the results confirmed that XY-0728 can quickly enter tumor cells, but has difficulty entering normal immortalized epithelial cells 293T.

[0027] Example 2 Immunofluorescence experiments confirmed that XY-0728 can target p53 and block p53 from entering the cell nucleus

[0028] H1299 cells that do not express p53 were selected and transfected with wild-type p53 (p53WT). Immunofluorescence was used to study the effect of FITC-labeled XY-0728 (10μM) on the localization of cancer cells after 6 hours of treatment. Simple method: First, wash the treated slide cells with PBS 3 times, 5 minutes each time. Fix with 4% paraformaldehyde at room temperature for 30 minutes, wash with PBS 3 times, 5 minutes each time. Add appropriate amount of 0.5% Triton-100, room temperature for 10 minutes, and wash the slides with PBS 3 times. Immunostaining blocking solution, block at room temperature for 1 hour. Incubate with primary antibody (anti-p53, D01, sc-126, 1:200) at 4°C overnight. Rewarm at room temperature for 1 hour. Wash with PBS 3 times, 5 min each time; incubate with fluorescent secondary antibody (anti-Mouse IgG 594, Jackson Immuno Research, 111-585-003, 1:200) at room temperature for 2 h (avoid light). Wash with PBS 3 times, 5 min each time. Directly add appropriate amount of DAPI for staining at room temperature for 5 min. Aspirate and discard DAPI, wash with PBS 3 times. Seal the slide with antifade agent, observe under confocal microscope, and collect images. The results are shown in the figure. Figure 2 As shown, the results confirmed that XY-0728 could target p53 and block p53 from entering the cell nucleus.

[0029] Example 3 Immunofluorescence experiments confirmed that XY-0728 can degrade mutant p53 and cell nuclei in cells

[0030] Various p53 mutant cancer cells were selected, 293T cells were used as control cells, and 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 in Example 2, and the results were as follows: Figure 3 As shown, the results confirmed that XY-0728 could degrade mutant p53 and cell nuclei in cells: such as MB231 (p53R280K), NCIH1975 (p53R273H), KYSE150 (p53R248Q), Huh7 (p53Y220H), SK-BR-3 (p53R175H), BT549 (p53R249S) and KYSE410 (p53R337C).

[0031] Example 4 Analysis of the proliferative toxicity of polypeptide XY-0728 to CCK8 of different P53 mutant tumor cells and normal cells

[0032] At the cellular level, the effect of polypeptide XY-0728 on the proliferation 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 and normal cells BEAS-2B cells, 293T cells were detected by CCK8. Take each cell in the logarithmic growth phase, discard the culture medium and wash twice with PBS, digest with trypsin and centrifuge to obtain cell pellets, count and plate them in 96-well plates. After the cells adhered, add 2.5, 5, 10, and 20 μM of polypeptide XY-0728 to each cell, respectively. The culture medium was used as a control. CCK8 reagent was used to measure OD450nm at 0, 24, 48, and 72 hours. The experimental results are as follows Figure 4-5 As shown in the results, the peptide XY-0728 had little effect on the growth of human embryonic kidney cells 293T cells and normal lung epithelial cells BEAS-2B ( Figure 4 ), with the increase of peptide XY-0728 concentration, 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 shows that XY-0728 has no effect on the growth of normal cells, but can significantly inhibit the growth 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, and HT29 cells, and the inhibitory ability increases with the increase of the concentration of polypeptide XY-0728.

[0033] Example 5 Analysis of the clone-forming ability of polypeptide XY-0728 on tumor cells with different p53 mutations

[0034] At the cellular level, the effect of polypeptide XY-0728 on the cloning ability of different p53 mutant tumor cells: MB231 cells, KYSE150 cells, H2122 cells, and HT29 cells was detected. Take each cell in the logarithmic growth phase, discard the culture medium and wash twice with PBS, digest with trypsin, centrifuge to obtain the cell precipitate, count and spread it in a 24-well plate. After the cells adhere, add 2.5, 5, and 10 μM of polypeptide XY-0728 to each cell, respectively, and the culture medium is used as a control. When the cells grow to have obvious clones, discard the culture medium and wash with PBS, add 4% paraformaldehyde to fix for 30 minutes, discard the fixative and wash once with PBS, add crystal violet solution for staining, and take pictures after drying. The experimental results are as follows Figure 6-7As shown, the results showed that with the increase of the concentration of polypeptide XY-0728, the cloning ability of p53 mutant tumor cells: MB231 cells, KYSE150 cells, H2122 cells, and HT29 cells decreased significantly, indicating that polypeptide XY-0728 can significantly inhibit the cloning ability of p53 mutant tumor cells: MB231 cells, KYSE150 cells, H2122 cells, and HT29, and the inhibitory ability increases with the increase of polypeptide XY-0728 concentration.

[0035] Example 6 Analysis of the tumor inhibition effect and body weight effect of polypeptide XY-0728 on MB231 cell and KYSE150 cell-bearing mice

[0036] Establishment of breast cancer and esophageal cancer mouse models: MB231 and KYSE150 cells were cultured in large quantities, cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS. The cell pellets were collected and counted. 0.1 ml of about 2 × 10 6 The cells were then used to observe the subcutaneous tumor formation periodically. 2 Afterwards, the control group was injected with solvent DMSO (50 mg / kg) peritumorally, and the experimental group was injected with peptide XY-0728 (50 mg / kg) peritumorally. Within 15 days, the experimental group was given the drug three times in total, on the first day, the third day, and the fifth day. 15 days after the drug administration, the experimental subjects were killed, and the tumor conditions were observed and the tumor volume was measured by taking photos.

[0037] Mouse tumor photos Figure 8 As shown, there were 9 mice in each experimental group and control group. The samples were taken on the 15th day after administration. Compared with the tumors in the control group, the tumors in the experimental group were significantly smaller. Fig. 9 As shown, there were 9 mice in each experimental group and control group. In KYSE150 tumor-bearing mice, the tumor volume of 9 control mice gradually increased compared with the baseline tumor volume. In the experimental group, except for one mouse with an increase in tumor volume, the tumor volume of the remaining mice decreased significantly compared with the baseline volume. In MB231 tumor-bearing mice, during the experiment, the tumor volume of the control mice increased compared with the baseline. In the experimental group, only one mouse had an increase in tumor volume compared with the baseline, and the tumor volume of the remaining mice decreased.

[0038] During the experiment, the tumor volume of mice was measured regularly and a line graph was drawn. The experimental results are shown in Fig.10 As shown, the tumor volume of mice in the experimental group was significantly reduced compared with that in the control group.

[0039] The weight of mice was measured regularly during the experiment. Fig.11As shown, the results showed that there was no significant difference in the body weight of mice in the experimental group and the control group, indicating that the polypeptide XY-0728 had little systemic toxicity to mice and had basically no effect on the weight of mice.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A small molecule polypeptide XY-0728 for targeted degradation of p53 mutants based on the p53C176 palmitic acid modification site, characterized in that: The sequence of the polypeptide XY-0728 is: R8-TEVVRRCPHHERC, wherein R8 represents RRRRRRRR.

2. Use of the small molecule polypeptide XY-0728 for targeted degradation of p53 mutants according to claim 1 in the preparation of a drug for treating p53 mutant tumors, wherein: The p53 mutant tumors are breast cancer, esophageal cancer, lung cancer, and colon cancer.

3. A drug for treating p53 mutant tumors, characterized in that: The drug contains the small molecule polypeptide XY-0728 for targeted degradation of p53 mutants as claimed in claim 1, wherein the p53 mutant tumor is breast cancer, esophageal cancer, lung cancer, and colon cancer.

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