Use of the small molecule compound C2 in the preparation of a formulation or medicament for the treatment of a malignant tumor

Abstract

The application discloses application of a small molecule compound C2 in preparation of a preparation or a medicine for treating malignant tumors. 25 H 27 N3O4S, which can significantly promote degradation of PD-L1 in triple-negative breast cancer cells, enhance a tumor immune response; the application also proves through research on a triple-negative breast cancer mouse transplanted tumor model that the small molecule compound C2 can significantly enhance infiltration of immune cells in a tumor microenvironment, and inhibit tumor growth, and good treatment effects on malignant tumors are shown.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of a small molecule compound C2 in preparation of a preparation or a medicine for treating malignant tumors. BACKGROUND

[0002] Malignant tumor is one of major diseases threatening human life and health, and 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) as an important immune checkpoint molecule on the surface of tumor cells plays a key role. 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, leading to tumor cells escaping from 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 various malignant tumors (such as lung cancer, melanoma, triple-negative breast cancer, etc.), and is closely related to the progression of tumors and the prognosis of patients. Although existing anti-PD-L1 monoclonal antibodies (such as atezolizumab) show good therapeutic effect 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, 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, promote its degradation, and thus restore the activity of T cells. These studies provide a new idea for the application of small molecule compounds in tumor immunotherapy. However, there is still a lack of effective small molecule compounds targeting PD-L1. SUMMARY

[0005] The present application provides application of a small molecule compound C2 in preparation of a preparation or a medicine for treating malignant tumors. The small molecule compound C2 significantly degrades PD-L1 protein, regulates tumor immune microenvironment, and thus effectively inhibits tumor growth and progression.

[0006] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

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

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

[0009] Further, the structural formula of the small molecule compound C2 is:

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

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

[0012] Further, the preparation or drug is an injection.

[0013] Further, the administration mode of the preparation or drug includes intraperitoneal injection, intramuscular injection, intravenous injection, and subcutaneous injection.

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

[0015] The application also provides a use of the small molecule compound C2 in the preparation of a PD-L1 protein degradation agent for malignant tumors.

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

[0017] The application also provides a drug for treating triple-negative breast cancer, wherein the active ingredient is the small molecule compound C2 or a stereoisomer or a pharmaceutically acceptable salt thereof.

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

[0019] 1. The application is verified by in vitro experiments, and C2 as a small molecule drug can significantly degrade PD-L1 protein in tumor cells;

[0020] 2. C2 is verified by in vivo experiments to have a significant tumor inhibition effect on triple-negative breast cancer;

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

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

[0023] Figure 1 The chemical structural formula of the 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 tumor ex vivo, 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] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, each embodiment of the present invention will be described in detail below with reference to the embodiments. However, it will be understood by those skilled in the art that in each embodiment of the present invention, many technical details are provided to help readers better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical solutions claimed in the present application can still 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

[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 the immune checkpoint molecule PD-L1 protein in triple-negative breast cancer cells 4T1 and clarify its target effect.

[0035] 2. Experimental Materials and Equipment

[0036] 2.1 Cell lines

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

[0038] 2.2 Reagents

[0039] RPMI-1640 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 (Huanyaibio, M1210-2).

[0040] 2.3 Equipment

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

[0042] 3. Experimental method

[0043] 3.1 Cell culture

[0044] 4T1 cells were inoculated in a six-well plate at 4×10 5 cells per well, 2 mL of RPMI-1640 medium containing 10% FBS was added, and the plate was incubated in a 37℃, 5% CO2 incubator for 24 hours. When the cells grew to 70%-80% confluence, the medium was replaced with fresh RPMI-1640 medium containing 10% FBS.

[0045] 3.2 Compound treatment

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

[0047] 3.3 Protein extraction and detection

[0048] 1. Remove the culture solution 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 bath for 10 minutes;

[0050] 3. Centrifuge (12,000 rpm, 4℃, 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℃ for 5 minutes, and then load;

[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 (4℃, overnight), wash the membrane with TBST, and then add secondary antibody and incubate for 1 hour;

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

[0055] 4. Experimental results

[0056] The experimental results (Fig. 2) show that C2 can significantly promote the degradation of PD-L1. Compared with the control group, the PD-L1 expression of the 10 μM C2 compound treatment group was significantly reduced. Figure 2

[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: Immune-enhancing effect of C2 in a triple-negative breast cancer mouse xenograft model

[0060] 1. Purpose of the experiment

[0061] To explore the effect of C2 on the tumor immune microenvironment in a 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] 4-6 week old female BALB / c mice (purchased from a regular animal experiment center and kept in a SPF level environment for feeding).

[0065] 2.2 Cell lines

[0066] Triple-negative breast cancer cells 4T1.

[0067] 2.3 Reagents

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

[0069] 2.4 Equipment

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

[0071] 3. Experimental methods

[0072] 3.1 Prepare cells

[0073] ​The 4T1 mouse triple negative breast cancer cells were recovered one week in advance. When the cells proliferated to the 3rd generation, the cells were routinely digested with 0.25% trypsin, washed twice with 1xPBS, and counted. The cells were resuspended at a density of 4x10 5 cells per 50 μL of 1xPBS.

[0074] 3.2 Inoculation

[0075] An equal volume of 4°C melted Matrigel was added to the cell suspension, and the mixture was thoroughly mixed using a pipette gun and then placed on ice for standby. After the mice were anesthetized, the hair on the left mammary pad of each mouse was removed using a hair clipper, and 100 μL of the cell suspension prepared in the above step was subcutaneously injected into each mouse.

[0076] 3.3 Drug treatment and recording

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

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

[0079] When the tumor volume of the mice in the solvent control group approached 1000 mm 3 , the mice were sacrificed by decapitation, and the tumor tissue was peeled off and photographed and weighed.

[0080] 3.4 Digestion of tumor

[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 cut into small pieces 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 medium to each well, add 200 μL of 10x collagenase type I (Gibco), mix well, and then place in a 37°C incubator for 30 minutes for digestion;

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

[0087] 3.4.3 Centrifugation and washing

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

[0089] 2. Finally, resuspend the cells with 1 mL 1xPBS per tube, and keep for use.

[0090] 3.5 Flow cytometry staining

[0091] 3.5.1 Zombie Violet staining

[0092] Add 100 μL Zombie Violet working solution (BioLegend, 1:100 dilution in PBS) to the cell pellet, resuspend the cell pellet, and stain at room temperature for 30 minutes in the dark. Blow the cells evenly with 1 mL PBS containing 0.5% BSA, 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 T cell staining mixture to each tube: PerCP / Cyanine5.5 anti-mouse CD45 (103132, BioLegend), FITC anti-mouse CD8 (100706, BioLegend), dilution ratio 1:100. Resuspend the cells, and stain at room temperature for 30 minutes in the dark.

[0095] 3.5.4 Washing and machine

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

[0097] 4. Experimental results

[0098] As shown in Figure 3 A, 3B and 3C, C2 intraperitoneal administration significantly reduced tumor volume; as shown in Figure 3 D, C2 intraperitoneal administration increased the infiltration of CD8 + T cells in tumor tissue.

[0099] 5. Discussion

[0100] In summary, 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 merely illustrative of the principles and effects of the present application, and are not intended to limit the scope of the present application. Any modification, substitution or change made by those skilled in the art without departing from the spirit and scope of the present application should be considered as falling within the scope of the present application, and should be covered by the claims of the present application.

Claims

1. Use of a small molecule compound C2 or a pharmaceutically acceptable salt thereof in the preparation of a preparation or a drug for treating malignant tumors, wherein: The structural formula of the small molecule compound C2 is: The malignant tumor is triple-negative breast cancer.

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

3. The use according to claim 2, characterized in that The preparation or medicine is administered by intraperitoneal injection, intramuscular injection, intravenous injection or subcutaneous injection.

4. Use of the small molecule compound C2 or a pharmaceutically acceptable salt thereof in the preparation of a PD-L1 protein inhibitor for malignant tumors, wherein: The structural formula of the small molecule compound C2 is: The malignant tumor is triple-negative breast cancer, and the concentration of the small molecule compound C2 contained in the PD-L1 protein inhibitor is not less than 10 μM.

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