Application of small molecule compound LZ-A4 in preparation of preparation or medicine for treating malignant tumors

Abstract

The invention discloses an application of a small molecular compound LZ-A4 in preparation of a preparation or a medicine for treating malignant tumors. The chemical formula of the small molecule compound LZ-A4 is C19H11Cl2N3O3S, and the small molecule compound LZ-A4 can promote degradation of LGMN in malignant tumor cells, inhibit proliferation of tumor cells and inhibit polarization of macrophages to M2 type. The research on a colorectal cancer mouse transplantation tumor model proves that the development of the colorectal cancer can be effectively inhibited by single treatment of the LZ-A4, and the volume of the tumor can be further reduced after the LZ-A4 is combined with a clinical first-line chemotherapeutic drug Oxaliplatin for the colorectal cancer. Meanwhile, the LZ-A4 can inhibit M2 type polarization of macrophages in a tumor microenvironment in vivo, promote infiltration of cytotoxic T cells, remodel the tumor microenvironment and activate anti-tumor immunity. In addition, the A4 has good toxic and side effects, and does not cause further reduction of the body weight of the mouse after being combined with chemotherapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a small molecule compound LZ-A4 in preparing a preparation or medicine for treating malignant tumors. Background Art

[0002] Malignant tumors are one of the major diseases that seriously threaten human health. Colorectal cancer is a common malignant tumor with high morbidity and mortality. LGMN (Legumain) is a cysteine ​​protease that is widely present in a variety of malignant tumors and is involved in the proliferation, migration and invasion of tumor cells. Studies have shown that high expression of LGMN is closely related to the malignant behavior of tumor cells, and can promote the polarization of tumor-associated macrophages to M2 type, thereby inhibiting anti-tumor immune response.

[0003] However, there is currently no effective small molecule compound that can significantly degrade LGMN, inhibit tumor cell proliferation and regulate the tumor immune microenvironment. Therefore, the development of a small molecule compound that can degrade LGMN, inhibit tumor cell proliferation and prevent macrophages from polarizing to M2 type is of great clinical significance for the treatment of malignant tumors. Summary of the invention

[0004] The purpose of the present invention is to provide the use of a small molecule compound LZ-A4 in the preparation of a preparation or a drug for treating malignant tumors. The small molecule compound LZ-A4 can significantly degrade LGMN, inhibit tumor cell proliferation, and inhibit macrophage polarization to M2 type. Through experiments on a colorectal cancer mouse transplant tumor model, LZ-A4 showed a significant inhibitory effect on tumor cell proliferation and immune regulation of the tumor microenvironment, and can effectively delay the progression of colorectal cancer.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] The present invention provides application of a small molecule compound LZ-A4 in preparing a preparation or medicine for treating malignant tumors.

[0007] Furthermore, the chemical structural formula of the small molecule compound LZ-A4 is

[0008]

[0009] Furthermore, the malignant tumors include colorectal cancer and cervical cancer.

[0010] Furthermore, the dosage of the preparation or drug is 5 mg-15 mg per kilogram of body weight.

[0011] Furthermore, the administration of the preparation or drug includes oral administration, sublingual administration, rectal administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, absorption through the lungs, or absorption through the conjunctiva, nasopharynx, mouth, rectum, urethra or bladder, and external use.

[0012] Furthermore, the small molecule compound LZ-A4 degrades LGMN protein in malignant tumor cells, inhibits the polarization of malignant tumor-associated macrophages to M2 type, thereby inhibiting the proliferation of malignant tumor cells and improving the tumor immune microenvironment. At the same time, it also synergistically enhances the anti-tumor immune response and promotes the infiltration of immune cells, thereby achieving the effect of treating malignant tumors.

[0013] Furthermore, the small molecule compound LZ-A4 can be used alone or in combination with chemotherapeutic drugs.

[0014] Furthermore, the chemotherapy drug is Oxaliplatin.

[0015] The present invention also provides the use of the small molecule compound LZ-A4 in the preparation of a malignant tumor LGMN protein degradation agent.

[0016] Furthermore, the concentration of the small molecule compound LZ-A4 contained in the LGMN protein degrading agent is not less than 10 μM.

[0017] The present invention also provides a drug for treating colorectal cancer, wherein the active ingredient is the small molecule compound LZ-A4 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 experimentally confirmed to have a significant effect in degrading LGMN by the small molecule compound LZ-A4, filling the gap of the current lack of a degrader for this target;

[0020] 2. The small molecule compound LZ-A4 of the present invention regulates the tumor microenvironment and inhibits tumor proliferation, inhibits the polarization of macrophages in the tumor microenvironment to M2 type, enhances anti-tumor immunity, and achieves anti-tumor synergistic effects, thereby effectively treating colorectal cancer;

[0021] 3. The effective dose of the small molecule compound LZ-A4 of the present invention is low, and its effect is significant when used in combination with chemotherapeutic drugs. Experimental verification shows that it has no obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the chemical structural formula of the small molecule compound LZ-A4.

[0023] Figure 2This is the effect of the small molecule compound LZ-A4 on the LGMN protein level in human cervical cancer cells HeLa.

[0024] Figure 3 This is the effect of the small molecule compound LZ-A4 on the in vitro proliferation of mouse colorectal cancer cell line CT26.

[0025] Figure 4 The small molecule compound LZ-A4 inhibits tumors in the mouse CT26 colorectal cancer model; A is the experimental flow chart of the solvent control, LZ-A4 (5 mg / kg, intraperitoneal injection, once a day), Oxaliplatin (5 mg / kg, intraperitoneal injection, once a day) and Oxaliplatin combined with LZ-A4 (5 mg / kg + 5 mg / kg, intraperitoneal injection, once a day) treatment of the mouse CT26 colorectal tumor model; B is the photograph of the tumors in each group after the tumors were removed at the end of the experiment; C is the growth of the tumor volume of mice in each group; D is the tumor weight of mice in each group; E is the record of the weight change of mice in each group; F is the protein immunoblot detection of LGMN levels in tumor tissues of the solvent control group and LZ-A4 group; G is the flow cytometry detection of CD206 in tumors of the solvent control group, LZ-A4 group, and LZ-A4 combined with Oxaliplatin group + F4 / 80 + M2 macrophages have CD45 + CD1lb + H is the percentage of CD8 in tumors detected by flow cytometry in solvent control group, LZ-A4 group, and LZ-A4 combined with Oxaliplatin group + T cells on CD45 + CD3 + The percentage of cells.

[0026] In the figure, LGMN is Legumain, Tub is Tubulin, IC 50 is the half-inhibitory concentration of the drug on cells, Oxaliplatin is the chemotherapy drug oxaliplatin, ip is intraperitoneal injection, Injection is injection, Termination is the experimental endpoint, + indicates combined use, and DAPI is a nuclear dye. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0028] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0029] Example 1: Western blotting to determine the degradation efficiency of LZ-A4 on Hela cell LGMN

[0030] 1 Experimental subject: Human cervical cancer cells Hela

[0031] 2 Experimental drug: LZ-A4, full name N-{4-[(2,1,3-benzothiadiazole-5-yl)oxy]-3-chlorophenyl}-5-chloro-2-hydroxybenzamide, chemical formula: C 19 H 11 Cl2N3O3S, chemical structure is as follows Figure 1 As shown, the compound numbered 0845-0932 was purchased from the ChemDiv compound library.

[0032] 3 Experimental methods:

[0033] 3.1 Cell plating: HeLa cells were seeded in six-well plates, with 4×10 cells per well. 5 cells, add 2 ml of DMEM medium containing 10% FBS, and culture in a 37°C, 5% CO2 incubator for 24 hours until the cells grow to 60%-70% confluence.

[0034] 3.2 Compound treatment: The cells were treated with LZ-A4 (final concentration 10 μM) for 1, 2, 4, 8, and 12 hours, respectively, and an equal volume of DMSO was added to the 0-hour group.

[0035] 3.3 Protein extraction and detection: Aspirate the culture medium and wash twice with pre-cooled PBS. Add 200 μL RIPA lysis buffer to each well to lyse the cells and collect the lysate. Centrifuge (12,000 rpm, 4°C, 10 minutes), collect the supernatant, detect the protein concentration of each sample by BCA method, and adjust each sample to the same concentration with RIPA lysis buffer. Add the corresponding volume of 5× protein loading buffer to each sample, denature at 95°C for 5 minutes and then load. Perform SDS-PAGE electrophoresis (12% separation gel), and transfer the protein to a PVDF membrane after electrophoresis. After transfer, block the membrane with 5% skim milk for 1 hour, and incubate the corresponding proteins with LGMN antibody (A23776, Abclonal), Tubulin antibody (M1305-2, HABIO) and β-actin antibody (AC026, Abclonal) (4°C, overnight). After washing the membrane with TBST (10 min each time, 3 times), the corresponding secondary antibody was added and incubated at room temperature for 1 hour. After washing the membrane (10 min each time, 3 times), the protein levels of LGMN and Tubulin were detected by chemiluminescence.

[0036] 4 Experimental results:

[0037] like Figure 2The results showed that LZ-A4 can significantly promote the degradation of LGMN in Hela cells. Compared with the control group (0 hours), the LGMN protein expression level in the LZ-A4 compound treatment group was significantly reduced. The LGMN protein level in the 4-hour treatment group decreased by about 40%, the 8-hour treatment group decreased by about 80%, and the 12-hour treatment group decreased by about 90%.

[0038] 5 Conclusion:

[0039] A4 can significantly promote the degradation of LGMN in HeLa cells, indicating that it has a good effect in targeted degradation of LGMN, further verifying its potential as an anti-tumor drug candidate.

[0040] Example 2: CCK-8 assay for the effect of LZ-A4 on the proliferation of CT26 cells in vitro

[0041] 1 Experimental subject: Mouse colorectal cancer cells Hela

[0042] 2 Experimental drug: LZ-A4

[0043] 3 Experimental methods:

[0044] 3.1 Cell plating: CT26 cells were seeded in 96-well plates, with 1×10 5 cells, add 100 μL of 1640 culture medium containing 10% FBS, and set up a culture medium control group and culture in a 37° C., 5% CO 2 incubator for 24 hours until the cells grow to 50%-60% confluence.

[0045] 3.2 Compound treatment: The cells were treated with different concentrations of LZ-A4 (100 nM, 330 nM, 1000 nM, 3300 nM, 10000 nM, 33000 nM) for 36 hours. The control group was added with the same volume of DMSO as the 33000 nM concentration group. Three replicates were set for each group.

[0046] 3.3 CCK-8 measurement of cell viability: After the drug treatment time, 10 μL CCK-8 detection solution (CK001, Langbolide) was added to each well, and the wells were gently mixed by swiping the edge of the well plate. The wells were placed in a 37°C, 5% CO2 incubator for 30 minutes, and the OD was measured by a microplate reader. 450 .

[0047] 3.4 Calculation:

[0048] Relative cell viability = (OD 450 -Blank group OD 450 ) / (control group OD 450 -Blank group OD 450 )×100%.

[0049] 4 Experimental results:

[0050] like Figure 3 As shown in Figure 2, LZ-A4 can significantly reduce the viability of CT26 cells. A concentration of 10 μM can reduce the viability by 80%, and a concentration of 33 μM can reduce the viability by 95%. The half-inhibitory concentration of LZ-A4 on CT26 cells is IC 50 =3.24×10 -6 mol / L.

[0051] 5 Conclusion:

[0052] LZ-A4 has a significant inhibitory effect on the proliferation of colorectal cancer cells in vitro, providing support for its use as an anti-tumor drug.

[0053] Example 3: Construction of a mouse CT26 colorectal tumor model to detect the anti-tumor effect of LZ-A4 in vivo

[0054] 1 Experimental subjects: 6-week-old female BALB / c mice inoculated with CT26 cells

[0055] 2 Experimental drugs: LZ-A4, Oxaliplatin

[0056] 3 Experimental methods:

[0057] 3.1 Cell preparation: CT26 mouse colorectal cancer cells were revived one week in advance. When the cells proliferated to the third generation, the cells were routinely digested, 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.

[0058] 3.2 Inoculation: Add the same volume of cells as the matrix gel melted at 4°C in advance, mix thoroughly with a pipette and place on ice for later use. After the mice are anesthetized, use a shaver to shave the hair under the left armpit of the mice, and inject 100 μL of the cell suspension prepared in the above step subcutaneously into each mouse.

[0059] 3.3 Medication treatment and records: Figure 4 A shows that when the tumor volume of mice grows to about 75mm 3 At the same time, the mice were evenly divided into 4 groups (solvent control group, LZ-A4 treatment group, Oxaliplatin treatment group, LZ-A4 and Oxaliplatin combination group), 8 mice in each group, the injection dose was 5 mg / kg, once a day by intraperitoneal injection. The solvent formula is: 5% DMSO, 25% PEG400, 5% Tween-80 and 65% PBS. The weight of the mice, as well as the long and short diameters of the tumor were recorded every other day to calculate the tumor volume. The calculation formula is: tumor volume = 1 / 2 × long diameter × short diameter 2. When the tumor diameter of the solvent control mouse reaches 1000mm 3At about 12 h, the mice were killed by cervical dislocation, and the tumors were removed, photographed and weighed.

[0060] 3.4 Digestion of tumor: After weighing, taking photos and recording, take 150mg of the mouse tumor parenchyma and put it into a clean 1.5mL centrifuge tube. Use scissors to cut the tumor tissue in the centrifuge tube until there are no obvious clumps. Add 1mL of serum-free cell culture medium to each centrifuge tube, resuspend the tissue fragments and transfer them to a 6-well plate, and add 1mL of culture medium to the 6-well plate. Add 200μL of 10× type I collagenase (17100017, Gibco) to each well, shake the well plate to mix it with the sample thoroughly, and put it in a 37℃ incubator for digestion for 30 minutes. Then add 1mL of culture medium containing 10% FBS to each well, mix evenly to terminate digestion. Pass the cell suspension through a 40μm cell sieve, collect the filtered single cell suspension, centrifuge at 3,000rpm for 3 minutes, and discard the supernatant. Add 1mL of 1×PBS to each sample to resuspend the cells and centrifuge at 3,000rpm for 3 minutes.

[0061] 3.5 Flow staining: Add 100 μL of Zombie Violet dye working solution (423114, BioLegend, 1:100 PBS dilution) to the cell pellet in the above step to resuspend the cell pellet and stain at room temperature in the dark for 30 minutes. Add 1 mL of PBS containing 0.5% BSA to each tube, blow evenly, centrifuge at 3,000 rpm for 3 minutes, and discard the supernatant. For M2 macrophage staining, add 100 μL of M2 macrophage staining mixture to each tube of cell pellet: PerCP / Cyanine5.5anti-mouse CD45 (103132, BioLegend), FITC anti-mouse / human CD11b (101206, BioLegend), PE / Cyanine7anti-mouse CD206 (141720, BioLegend), APC anti-mouse F4 / 80 (157305, BioLegend), dilute at a ratio of 1:100, resuspend the cells, and stain at room temperature in the dark for 30 minutes. +T cell staining, Zombie Violet staining After each tube of cell pellet, add 100μL of T cell staining mixture: PerCP / Cyanine5.5anti-mouse CD45 (103132, BioLegend), PE / Cyanine7 anti-mouse CD3 (100320, BioLegen), FITC anti-mouse CD8 (100706, BioLegend), dilute at a ratio of 1:100, resuspend the cells, and stain at room temperature in the dark for 30 minutes. After staining, add 1mL 1×PBS, centrifuge at 3,000rpm for 3 minutes, discard the supernatant, resuspend the cells with fresh 1mL 1×PBS, pass through the cell screen, and wait for flow cytometry.

[0062] 4 Experimental results:

[0063] like Figure 4 As shown in BD, LZ-A4 alone significantly inhibited tumor growth, and the tumor volume and weight were reduced by more than 60% compared with the solvent control group. The combination of LZ-A4 and Oxaliplatin showed an additive tumor inhibition effect, which could reduce the tumor volume by more than 90%. Compared with the Oxaliplatin alone treatment group, the tumor volume of the combination group was further reduced by 50%. Figure 4 As shown in Figure E, in terms of safety, LZ-A4 also showed less toxic side effects. Even when used in combination with the chemotherapy drug Oxaliplatin, the weight of mice did not decrease further. Figure 4 As shown in F, Western blotting analysis of mouse tumor tissues showed that the LGMN protein level in the tumor tissues of mice treated with LZ-A4 was significantly decreased. Figure 4 As shown in GH, LZ-A4 can significantly reduce the expression of CD206 in tumor tissues. + F4 / 80 + Macrophage numbers and CD8 + Infiltration of T cells.

[0064] 5 Discussion:

[0065] The above experiments prove that LZ-A4 also exhibits efficient LGMN degradation ability in vivo, significantly inhibiting the growth of colorectal cancer tumors in mice. In combination with the chemotherapy drug Oxaliplatin, the tumor volume can be further reduced on the basis of Oxaliplatin, showing an excellent chemotherapy sensitization effect. In addition, LZ-A4 can reshape the tumor immune microenvironment by reducing the proportion of M2 macrophages in tumor tissues and promoting the infiltration of cytotoxic T cells. Therefore, LZ-A4 has the dual efficacy of inhibiting tumor cell proliferation and promoting anti-tumor immunity. At the same time, the expression of LGMN, the target of LZ-A4 degradation, is tissue-specific, that is, LGMN is highly expressed in a variety of tumor cells and immunosuppressive cells, but lowly expressed or not expressed in normal cells. This characteristic allows LZ-A4 to degrade LGMN in tumor cells, etc., while minimizing unnecessary damage to normal tissues, ensuring the safety of treatment.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. Application of the small molecule compound LZ-A4 in the preparation of preparations or drugs for treating malignant tumors.

2. The use according to claim 1, characterized in that: The chemical structural formula of the small molecule compound LZ-A4 is 3. The use according to claim 1, characterized in that: The malignant tumors include colorectal cancer and cervical cancer.

4. The use according to claim 1, characterized in that: The dosage of the preparation or drug is 5 mg to 15 mg per kilogram of body weight.

5. The use according to claim 1, characterized in that: The administration methods of the preparation or drug include oral administration, sublingual administration, rectal administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, absorption through the lungs, or absorption through the conjunctiva, nasopharynx, oral cavity, rectum, urethra or bladder, and external use.

6. The use according to claim 1, characterized in that: The small molecule compound LZ-A4 degrades LGMN protein in malignant tumor cells, inhibits the polarization of malignant tumor-associated macrophages to M2 type, thereby inhibiting the proliferation of malignant tumor cells and improving the tumor immune microenvironment. It also synergistically enhances the anti-tumor immune response and promotes the infiltration of immune cells, thereby achieving the effect of treating malignant tumors.

7. The use according to claim 1, characterized in that: The small molecule compound LZ-A4 can be used alone or in combination with a chemotherapeutic drug.

8. Use of the small molecule compound LZ-A4 according to claim 1 in the preparation of a degrading agent for malignant tumor LGMN protein.

9. The use according to claim 8, characterized in that: The concentration of the small molecule compound LZ-A4 contained in the LGMN protein degrading agent is not less than 10 μM.

10. A drug for treating colorectal cancer, characterized in that: The active ingredient in the drug is the small molecule compound LZ-A4 or its stereoisomers and pharmaceutically acceptable salts as described in claim 1.

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