Application of small molecule compound C2 in preparation of preparation or medicine for treating malignant tumors

Abstract

The invention discloses an application of a small molecule compound C2 in preparation of a preparation or a medicine for treating malignant tumors. The molecular formula of the small molecule compound C2 is C25H27N3O4S, and the small molecule compound C2 can significantly promote degradation of PD-L1 in triple negative breast cancer cells and enhance tumor immune response. The research on a triple negative breast cancer mouse transplantation tumor model proves that the small molecule compound C2 can obviously enhance the infiltration of immune cells in a tumor microenvironment and inhibit tumor growth, and shows a good treatment effect on malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a small molecule compound C2 in the preparation of a preparation or medicine for treating malignant tumors. Background Art

[0002] Malignant tumors are one of the major diseases that threaten human life and health. In the field of tumor immunotherapy, the emergence of immune checkpoint inhibitors has changed the treatment pattern of malignant tumors. Among them, programmed death ligand 1 (PD-L1) plays a key role as an important immune checkpoint molecule on the surface of tumor cells. PD-L1 inhibits the activation and proliferation of T cells by binding to programmed death receptor 1 (PD-1) on the surface of T cells, causing tumor cells to escape host immune surveillance. This mechanism makes PD-L1 an important target in cancer treatment.

[0003] Studies have shown that PD-L1 is highly expressed in a variety of malignant tumors (such as lung cancer, melanoma, triple-negative breast cancer, etc.), and is closely related to tumor progression and patient prognosis. Although existing anti-PD-L1 monoclonal antibodies (such as atezolizumab) have shown good therapeutic effects in some patients, there are still problems such as large side effects and drug resistance. Therefore, it is urgent to develop new small molecule drugs to overcome these limitations.

[0004] In recent years, the research on the degradation mechanism of PD-L1 has gradually increased. Studies have shown that certain small molecule compounds can significantly enhance the immune response of T cells by promoting the degradation of PD-L1. For example, the activation of E3 ubiquitin ligase can mediate the ubiquitination of PD-L1 and promote its degradation, thereby restoring the activity of T cells. These studies provide new ideas for the application of small molecule compounds in tumor immunotherapy. However, there is currently a lack of effective small molecule compounds targeting PD-L1. Summary of the invention

[0005] The present invention provides the use of a small molecule compound C2 in the preparation of a preparation or a drug for treating malignant tumors. The small molecule compound C2 significantly degrades PD-L1 protein and regulates the tumor immune microenvironment, thereby effectively inhibiting tumor growth and progression.

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

[0007] The present invention provides the use of a small molecule compound C2 in preparing a preparation or a medicine for treating malignant tumors.

[0008] Furthermore, the structural formula of the small molecule compound C2 is:

[0009]

[0010] Furthermore, the malignant tumor is triple-negative breast cancer.

[0011] Furthermore, the dosage of the small molecule compound C2 is 10 mg-25 mg per kilogram of body weight.

[0012] Furthermore, the preparation or medicine is an injection.

[0013] Furthermore, the administration methods of the preparation or drug include intraperitoneal injection, intramuscular injection, intravenous injection, and subcutaneous injection.

[0014] Furthermore, the small molecule compound C2 can promote the degradation of PD-L1 protein in malignant tumor cells and increase the expression of CD8 + The infiltration of T cells significantly enhances the tumor immune response and improves the immune microenvironment, thereby achieving the effect of treating malignant tumors.

[0015] The present invention also provides the use of the small molecule compound C2 in the preparation of a malignant tumor PD-L1 protein degrader.

[0016] Furthermore, the concentration of the small molecule compound C2 contained in the PD-L1 protein inhibitor is not less than 10 μM.

[0017] The present invention also provides a drug for treating triple-negative breast cancer, wherein the active ingredient is the small molecule compound C2 or its stereoisomers and pharmaceutically acceptable salts.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] 1. The present invention has been verified by in vitro experiments that C2, as a small molecule drug, can significantly degrade PD-L1 protein in tumor cells;

[0020] 2. C2 has been verified through in vivo experiments to have a significant tumor-suppressing effect on triple-negative breast cancer;

[0021] 3. C2 can enhance tumor immune response and promote immune cells such as CD8 + Infiltration of T cells.

[0022] In summary, C2 regulates the immune microenvironment, enhances the anti-tumor activity of immune cells, achieves multi-target synergy, inhibits tumor growth, and shows good therapeutic effects on malignant tumors. C2 has no obvious toxic side effects and is safe to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the chemical structural formula of small molecule compound C2.

[0024] Figure 2C2 can significantly degrade PD-L1 protein in tumor cells.

[0025] Figure 3 C2 can significantly inhibit tumor growth, enhance tumor immune response, and promote immune cells such as CD8 + T cell infiltration; A is a picture taken after the tumor is removed from the body, B is a picture of tumor volume distribution, C is a picture of tumor weight, and D is a picture of CD8 counted by flow cytometry of tumor tissue + T cell infiltration. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, each embodiment of the present invention will be described in detail below in conjunction with the embodiments. However, it will be appreciated by those skilled in the art that in each embodiment of the present invention, many technical details are proposed in order to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented.

[0027] Example 1: Chemical structure of C2

[0028] 1. Purpose of the experiment

[0029] The chemical structure of the small molecule compound C2 was elucidated to provide a basis for subsequent biological activity verification.

[0030] 2. Results description

[0031] The chemical structure of the small molecule compound C2 (purchased from www.chemdiv, com, catalog number: 8005-0050) is as follows Figure 1 As shown, its molecular formula is C 25 H 27 N3O4S, including specific functional groups and skeletons, has the potential to target and degrade PD-L1.

[0032] Example 2: Effect of C2 on PD-L1 degradation in triple-negative breast cancer cells

[0033] 1. Purpose of the experiment

[0034] To study the degradation effect of C2 on the expression of PD-L1 protein, an immune checkpoint molecule, in triple-negative breast cancer cells 4T1 and to clarify its target effect.

[0035] 2. Experimental Materials and Equipment

[0036] 2.1 Cell lines

[0037] The triple-negative breast cancer cell line 4T1.

[0038] 2.2 Reagents

[0039] RPMI-1640 culture medium, fetal bovine serum (FBS, 10%), trypsin (0.25%), C2 compound (dissolved in DMSO, stock solution 10 mM), anti-PD-L1 antibody (Proteintech, 66248-1-Ig), anti-β-actin antibody (Hua'an Biotechnology, M1210-2).

[0040] 2.3 Equipment

[0041] Carbon dioxide incubator (37°C, 5% CO2), clean bench, SDS-PAGE electrophoresis equipment, PVDF membrane, electrotransfer instrument, ECL chemiluminescence detection system.

[0042] 3. Experimental Methods

[0043] 3.1 Cell culture

[0044] 4T1 cells were seeded in six-well plates at 4 × 10 5 cells, add 2 mL of RPMI-1640 medium containing 10% FBS, and culture in a 37°C, 5% CO2 incubator for 24 hours until the cells grow to 70%-80% confluence.

[0045] 3.2 Compound treatment

[0046] After replacing the culture medium, C2 compound (concentration 10 μM) was added 3, 6, 12, 24, and 36 hours before sampling, and an equal volume of DMSO was added to the control group.

[0047] 3.3 Protein extraction and detection

[0048] 1. Aspirate the culture medium and wash twice with pre-cooled PBS;

[0049] 2. Add 100 μL RIPA lysis buffer to lyse the cells and collect the lysate after ice bathing for 10 minutes;

[0050] 3. Centrifuge (12,000 rpm, 4°C, 5 minutes), take the supernatant and detect the protein concentration;

[0051] 4. Take an equal amount of protein sample (40 μg), add 5× protein loading buffer, denature at 95°C for 5 minutes and then load the sample;

[0052] 5. Perform SDS-PAGE electrophoresis (12% separation gel) and transfer to PVDF membrane;

[0053] 6. Block the membrane with 5% skim milk for 1 hour, incubate with anti-PD-L1 and anti-β-actin antibodies in sequence (4°C, overnight), wash the membrane with TBST and incubate with secondary antibodies for 1 hour;

[0054] 7. Chemiluminescence detection of PD-L1 and β-actin expression.

[0055] 4. Experimental Results

[0056] Experimental results ( Figure 2 ) showed that C2 could significantly promote the degradation of PD-L1. Compared with the control group, the PD-L1 expression in the C2 compound 10μM treatment group was significantly reduced.

[0057] 5. Conclusion

[0058] C2 can significantly promote the degradation of PD-L1, showing its potential in the regulation of immune checkpoint molecules.

[0059] Example 3: Immunoenhancing effect of C2 in triple-negative breast cancer mouse transplant model

[0060] 1. Purpose of the experiment

[0061] To investigate the effect of C2 on the tumor immune microenvironment in the triple-negative breast cancer xenograft model and analyze its regulatory effect on immune cell infiltration.

[0062] 2. Experimental Materials and Equipment

[0063] 2.1 Experimental animals

[0064] Female BALB / c mice aged 4-6 weeks (purchased from a regular animal experimental center and kept in an SPF environment).

[0065] 2.2 Cell lines

[0066] Triple negative breast cancer cell line 4T1.

[0067] 2.3 Reagents

[0068] C2 compound (15 mg / kg, dissolved in preparation solvent: 5% DMSO, 25% PEG400, 5% Tween80, 65% PBS), FITC-CD8 antibody (Biolegend, 100706), flow cytometer staining buffer.

[0069] 2.4 Equipment

[0070] Carbon dioxide incubator, animal dissection tools, flow cytometer.

[0071] 3. Experimental Methods

[0072] 3.1 Preparation of cells

[0073] 4T1 mouse triple-negative breast cancer cells were revived one week in advance. When the cells proliferated to the third generation, the cells were routinely digested with 0.25% trypsin, washed twice with 1× PBS, and the cells were counted. 4×10 cells were added to each 50 μL of 1× PBS. 5 Resuspend the cells at a density of 100 cells / mL.

[0074] 3.2 Inoculation

[0075] Add the same volume of Matrigel melted at 4°C as the cell suspension, mix thoroughly with a pipette and place on ice for later use. After the mice are anesthetized, use a shaver to remove the hair on the left mammary pad of the mice, and inject 100 μL of the cell suspension prepared in the above step subcutaneously into each mouse.

[0076] 3.3 Medication treatment and records

[0077] When the tumor volume of mice grew to about 75mm 3 At the time of treatment, the mice were randomly divided into 4 groups (solvent control group, C2 treatment group), 15 mice in each group. The injection dose was 5 mg / kg, once a day by intraperitoneal injection. The solvent formula was: 5% DMSO, 25% PEG400, 5% Tween80 and 65% PBS. The weight of the mice and the long and short diameters of the tumor were recorded every other day, and the tumor volume was calculated using the following formula:

[0078] Tumor volume = 1 / 2 × long diameter × short diameter 2

[0079] When the tumor volume of mice in the solvent control group was close to 1000mm 3 At 4 pm, the mice were killed by cervical dislocation, and the tumor tissues were removed, photographed and weighed.

[0080] 3.4 Digestive Tumors

[0081] 3.4.1 Tissue digestion

[0082] After weighing, photographing and recording, 150 mg of tumor tissue was placed in a 1.5 mL centrifuge tube and the tumor tissue was minced with sterile scissors until there were no obvious lumps.

[0083] 3.4.2 Preparation of single cell suspension

[0084] 1. Add 1 mL of serum-free medium to each tube to resuspend the tumor tissue and transfer to a 6-well plate;

[0085] 2. Add 1 mL of culture medium to each well, add 200 μL of 10× type I collagenase (Gibco), shake well and place in a 37°C incubator for digestion for 30 minutes;

[0086] 3. Add 1 mL of culture medium containing 10% FBS to terminate digestion, and pass the mixture through a 40 μm cell sieve to collect the single cell suspension.

[0087] 3.4.3 Centrifugation and washing

[0088] 1. Centrifuge at 3,000 rpm for 3 minutes, discard the supernatant, resuspend the cells in 1 mL 1× PBS and centrifuge again;

[0089] 2. Finally, add 1 mL of 1×PBS to each tube to resuspend the cells and set aside.

[0090] 3.5 Flow Cytometry

[0091] 3.5.1 Zombie Violet Dyeing

[0092] Add 100 μL Zombie Violet working solution (BioLegend, 1:100 PBS dilution) to the cell pellet, resuspend the cell pellet, and stain at room temperature in the dark for 30 minutes. Add 1 mL PBS containing 0.5% BSA to homogenize the cells, centrifuge at 3,000 rpm for 3 minutes, and discard the supernatant.

[0093] 3.5.3 CD8 + T cell staining

[0094] After Zombie Violet staining, add 100 μL of T cell staining mixture to each tube: PerCP / Cyanine5.5anti-mouse CD45 (103132, BioLegend), FITC anti-mouse CD8 (100706, BioLegend), dilution ratio 1: 100. Resuspend the cells and stain for 30 minutes at room temperature in the dark.

[0095] 3.5.4 Washing and washing

[0096] Add 1 mL 1× PBS, centrifuge at 3,000 rpm for 3 minutes, discard the supernatant, and resuspend the cells in 1 mL fresh 1× PBS. Pass the cell suspension through a 40 μm cell sieve for flow cytometry detection.

[0097] 4. Experimental Results

[0098] like Figure 3 As shown in A, 3B, and 3C, intraperitoneal injection of C2 significantly reduced tumor volume; Figure 3 As shown in D, intraperitoneal injection of C2 increased the expression of CD8 + Infiltration of T cells.

[0099] 5. Discussion

[0100] Based on the above experimental results, C2 can degrade PD-L1, inhibit the growth of malignant tumors, improve the immune microenvironment, and has good potential for the treatment of malignant tumors.

[0101] The above embodiments are only intended to illustrate the principles and effects of the present invention and are not intended to limit the scope of the present invention. Any person skilled in the art may make various modifications, substitutions or changes to the above embodiments without violating the spirit and scope of the present invention. These modifications or changes, if they do not depart from the technical ideas and spirits disclosed by the present invention, shall be deemed to belong to the protection scope of the present invention and shall be covered and protected by the claims of the present invention.

Claims

1. Application of small molecule compound C2 in the preparation of preparations or drugs for treating malignant tumors.

2. The use according to claim 1, characterized in that: The structural formula of the small molecule compound C2 is:

3. The use according to claim 1, characterized in that: The malignant tumor is triple-negative breast cancer.

4. The use according to claim 1, characterized in that: The dosage of the small molecule compound C2 is 10 mg to 25 mg per kilogram of body weight.

5. The use according to claim 1, characterized in that: The preparation or medicine is an injection.

6. The use according to claim 5, characterized in that: The administration methods of the preparation or drug include intraperitoneal injection, intramuscular injection, intravenous injection, and subcutaneous injection.

7. The use according to claim 1, characterized in that: The small molecule compound C2 can promote the degradation of PD-L1 protein in malignant tumor cells, increase the expression of CD8 + The infiltration of T cells significantly enhances the tumor immune response and improves the immune microenvironment, thereby achieving the effect of treating malignant tumors.

8. Application of small molecule compound C2 in the preparation of PD-L1 protein inhibitors for malignant tumors.

9. The use according to claim 8, characterized in that: The concentration of the small molecule compound C2 contained in the PD-L1 protein inhibitor is not less than 10 μM.

10. A drug for treating triple-negative breast cancer, characterized in that: The active ingredient in the drug is the small molecule compound C2 or its stereoisomers and pharmaceutically acceptable salts as described in claim 1.

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