Use of small molecule compound LZ-A4 in preparation of a preparation or medicament for treating malignant tumor

Abstract

The application discloses application of a small-molecule compound LZ-A4 in preparation of a preparation or a medicine for treating malignant tumors. 19 H 11 Cl2N3O3S, which can promote LGMN degradation in malignant tumor cells, inhibit tumor cell proliferation, and inhibit macrophage polarization to M2 type. The application proves through research on a colorectal cancer mouse transplanted tumor model that LZ-A4 alone can effectively inhibit the progression of colorectal cancer, and after being combined with a colorectal cancer first-line chemotherapy medicine oxaliplatin, the tumor volume can be further reduced. Meanwhile, LZ-A4 can inhibit M2 type polarization of macrophages in a tumor microenvironment in vivo, promote infiltration of cytotoxic T cells, reshape the tumor microenvironment, and activate anti-tumor immunity. In addition, A4 performs well in terms of toxic side effects, and after being combined with the chemotherapy medicine, the mouse weight is not further reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of small molecule compound LZ-A4 in preparation of preparation or medicine for treating malignant tumor. BACKGROUND

[0002] Malignant tumor is one of the major diseases that seriously threaten human health, and colorectal cancer is a common malignant tumor with high incidence and mortality. LGMN (Legumain) is a cysteine protease that widely exists in various 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 the 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 tumor immune microenvironment. Therefore, developing a new type of drug that can degrade LGMN, inhibit tumor cell proliferation and prevent the polarization of macrophages to M2 type has important clinical significance for treating malignant tumor. SUMMARY

[0004] The purpose of the present application is to provide application of small molecule compound LZ-A4 in preparation of preparation or medicine for treating malignant tumor. Small molecule compound LZ-A4 can significantly degrade LGMN, inhibit tumor cell proliferation and inhibit the polarization of macrophages to M2 type. Through experiments on colorectal cancer mouse xenograft model, LZ-A4 shows significant inhibitory effect on tumor cell proliferation and immune regulation of tumor microenvironment, and can effectively delay the progression of colorectal cancer.

[0005] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

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

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

[0008]

[0009] Further, the malignant tumor includes colorectal cancer and cervical cancer.

[0010] Further, the administration dose of the preparation or medicine is 5-15 mg per kilogram of body weight.

[0011] Further, the administration mode of the preparation or drug includes oral administration, sublingual administration, rectal administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, pulmonary absorption, or conjunctival, nasopharyngeal, oral, rectal, urethral or bladder absorption, external use.

[0012] Further, the small molecule compound LZ-A4 can inhibit the polarization of malignant tumor-associated macrophages to M2 type by degrading LGMN protein in malignant tumor cells, thereby inhibiting the proliferation of malignant tumor cells and improving the tumor immune microenvironment, and also synergistically enhancing the anti-tumor immune response and promoting the infiltration of immune cells, so as to achieve the effect of treating malignant tumors.

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

[0014] Further, the chemotherapeutic drug is Oxaliplatin.

[0015] The application further provides an application of the small molecule compound LZ-A4 in the preparation of a LGMN protein degrading agent for malignant tumors.

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

[0017] The application further provides a drug for treating colorectal cancer, wherein the active ingredient is the small molecule compound LZ-A4 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 small molecule compound LZ-A4 can significantly degrade LGMN, filling the gap of the target point without degrading agents at present.

[0020] 2. The small molecule compound LZ-A4 can regulate the tumor microenvironment and inhibit tumor proliferation, inhibit the polarization of macrophages in the tumor microenvironment to M2 type, enhance the anti-tumor immunity, realize the synergistic effect of anti-tumor, and effectively treat colorectal cancer.

[0021] 3. The effective dose of the small molecule compound LZ-A4 is low, and the effect of combination with a chemotherapeutic drug is remarkable, and no obvious toxic side effects are verified by experiments. DETAILED DESCRIPTION

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

[0023] Figure 2Effect of small molecule compound LZ-A4 on LGMN protein level of human cervical cancer cell HeLa.

[0024] Figure 3 Effect of small molecule compound LZ-A4 on proliferation of mouse colorectal cancer cell CT26 in vitro.

[0025] Figure 4 Inhibition effect of small molecule compound LZ-A4 on tumor in mouse CT26 colorectal cancer model; wherein: A is the experimental flow chart of 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 mouse CT26 colorectal tumor model; B is the photograph of tumor in each group after tumor was peeled off at the end of experiment; C is the tumor volume growth of mice in each group; D is the tumor weight of mice in each group; E is the weight change record of mice in each group; F is the detection of LGMN level in tumor tissue of solvent control group and LZ-A4 group by Western blotting; G is the detection of CD206 + F4 / 80 + M2 type macrophage in CD45 + CD1 lb + cells in each group by flow cytometry; H is the detection of CD8 + T cells in CD45 + CD3 + cells in each group by flow cytometry.

[0026] In the figure, LGMN is Legumain, Tub is Tubulin, IC 50 is the half inhibitory concentration of drug on cells, Oxaliplatin is a chemotherapy drug, i.p. is intraperitoneal injection, Injection is injection, Termination is the end of experiment, + indicates combination, DAPI is a nuclear stain. DETAILED DESCRIPTION

[0027] The technical solutions of the present application are further described in detail in combination with the following specific examples.

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

[0029] Example 1: Western blot assay of LZ-A4 on the degradation efficiency of LGMN in Hela cells

[0030] 1. Experimental object: human cervical cancer cell 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, as shown in the following chemical structure formula: Figure 1 0845-0932 in ChemDiv Compound Library.

[0032] 3. Experimental method:

[0033] 3.1 Cell plating: HeLa cells were seeded in a six-well plate at 4x10 5 cells per well, 2 ml of DMEM medium containing 10% FBS was added, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to 60%-70% confluence, the culture medium was removed, and the cells were washed twice with pre-cooled PBS.

[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 the same volume of DMSO was added to the 0 hour group.

[0035] 3.3 Protein extraction and detection: the culture medium was removed, and the cells were washed twice with pre-cooled PBS. Each well was added with 200 μL RIPA lysis buffer to lyse the cells, and the lysate was collected. Centrifugation (12,000 rpm, 4°C, 10 minutes), collection of supernatant, BCA method detection of protein concentration of each sample, and adjustment of each sample to the same concentration with RIPA lysis buffer. Each sample was added with a corresponding volume of 5x protein loading buffer, denatured at 95°C for 5 minutes, and then loaded. SDS-PAGE electrophoresis (12% separation gel) was performed, and the protein was transferred to a PVDF membrane after electrophoresis. After the transfer, the membrane was blocked with 5% skim milk for 1 hour, and the corresponding proteins were incubated 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 minutes each time, 3 times), the corresponding secondary antibody was added and incubated at room temperature for 1 hour. After washing the membrane (10 minutes each time, 3 times), the protein levels of LGMN and Tubulin were detected by chemiluminescence.

[0036] 4. Experimental results:

[0037] As Figure 2It is shown that LZ-A4 can significantly promote the degradation of LGMN in Hela cells. Compared with the control group (0 hours), the expression level of LGMN protein in the LZ-A4 compound treatment group is significantly reduced. Among them, 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 good effect in targeting the degradation of LGMN, and further verifying its potential as an anti-tumor candidate drug.

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

[0041] 1. Experimental object: mouse colorectal cancer cell Hela

[0042] 2. Experimental drug: LZ-A4

[0043] 3. Experimental method:

[0044] 3.1 Cell plating: CT26 cells were inoculated in a 96-well plate at 1×10 5 cells per well, 100 μL of 1640 culture medium containing 10% FBS was added, and the medium control group was placed in a 37°C, 5% CO2 incubator for 24 hours. The cells were grown to 50%-60% confluence.

[0045] 3.2 Compound treatment: treat cells 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, and each group was set up in triplicate.

[0046] 3.3 CCK-8 measurement of cell viability: after the drug treatment time, 10 μL of CCK-8 detection solution (CK001, Lambolide) was added to each well, the edge of the plate was mixed, and it was placed in a 37°C, 5% CO2 incubator for 30 minutes. The OD 450 .

[0047] 3.4 Calculation:

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

[0049] 4. Experimental results:

[0050] As shown in Figure 3 , LZ-A4 can significantly reduce the viability of CT26 cells. The concentration of 10 μΜ can reduce 80%, and the concentration of 33 μΜ can reduce 95%. The half-inhibitory concentration of LZ-A4 to CT26 cells is IC 50 = 3.24 x 10 -6 mol / L.

[0051] 5 Conclusion:

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

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

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

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

[0056] 3 Experimental method:

[0057] 3.1 Prepare cells: CT26 mouse colorectal cancer cells were recovered one week in advance, and when the cells proliferated to the 3rd generation, the cells were routinely digested and washed twice with 1 x PBS. The cells were resuspended at a density of 4 x 10 5 cells per 50 μL of 1 x PBS.

[0058] 3.2 Inoculation: Add the same volume of cell matrix gel melted in advance at 4°C, mix well with a pipette gun, and prepare on ice. After the mice were anesthetized, the mouse's left axillary hair was removed with a hair clipper, and each mouse was subcutaneously injected with 100 μL of the cell suspension prepared in the above step.

[0059] 3.3 Drug treatment and recording: as shown in Figure 4 A, when the tumor volume of the mice grew to about 75 mm 3 , the mice were evenly divided into 4 groups (solvent control group, LZ-A4 treatment group, Oxaliplatin treatment group, and LZ-A4 and Oxaliplatin combination group), 8 mice in each group, and the injection dose was 5 mg / kg, once a day intraperitoneally. The solvent formula is: 5% DMSO, 25% PEG400, 5% Tween-80 and 65% PBS. The body weight of the mice was recorded every other day, and the long diameter and short diameter of the tumor were recorded to calculate the tumor volume, the formula is: tumor volume = 1 / 2 x long diameter x short diameter2. When the tumor diameter of the solvent control mice reached 1000 mm 3At the right time, the mice were decapitated, the tumors were peeled off and photographed and weighed.

[0060] 3.4 Digestion of tumor: After weighing and photographing, 150 mg of tumor parenchyma of the mouse was taken into a clean 1.5 mL centrifuge tube, and the tumor tissue in the centrifuge tube was cut into pieces with scissors until there were no obvious lumps. 1 mL of serum-free cell culture medium was added to each centrifuge tube, the tissue fragments were resuspended and transferred to a 6-well plate, and 1 mL of culture medium was added to the 6-well plate. 200 μL of 10× collagenase type I (17100017, Gibco) was added to each well, the plate was shaken to mix well with the sample, and was placed in a 37°C incubator for 30 minutes. Then 1 mL of medium containing 10% FBS was added to each well, mixed well to stop the digestion. The cell suspension was passed through a 40 μm cell sieve, and the single cell suspension filtered was collected, centrifuged at 3,000 rpm for 3 minutes, and the supernatant was discarded. 1 mL of 1× PBS was added to each sample to resuspend the cells, and centrifuged at 3,000 rpm for 3 minutes.

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

[0062] 4. Experimental results:

[0063] As shown in Figure 4 B-D, 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, while the combination of LZ-A4 and Oxaliplatin showed an additive tumor inhibition effect, which could reduce the tumor volume by more than 90%, and the combination group further reduced the tumor volume by 50% compared with the Oxaliplatin alone treatment group. As shown in Figure 4 E, in terms of safety, LZ-A4 also showed smaller side effects, and even when combined with the chemotherapeutic drug Oxaliplatin, the body weight of the mice did not further decrease. As shown in Figure 4 F, by Western blot analysis of tumor tissues in mice, it can be seen that the LGMN protein level in the tumor tissues of LZ-A4 treated mice is significantly reduced. As shown in Figure 4 G-H, LZ-A4 can significantly reduce the number of CD206 + F4 / 80 + macrophages in tumor tissues, and promote the infiltration of CD8 + T cells.

[0064] 5. Discussion:

[0065] The above experiments prove that LZ-A4 also exhibits high-efficiency LGMN degradation ability in vivo, significantly inhibits the growth of colorectal cancer tumors in mice. And in the combined treatment with the chemotherapeutic drug Oxaliplatin, it can further reduce the tumor volume on the basis of Oxaliplatin, showing excellent chemosensitization effect. In addition, LZ-A4 can remodel the tumor immune microenvironment by reducing the proportion of M2-type macrophages in tumor tissues and promoting the infiltration of cytotoxic T cells. Therefore, LZ-A4 has a dual drug effect of inhibiting tumor cell proliferation and promoting anti-tumor immunity. At the same time, the expression of the target LGMN degraded by LZ-A4 has tissue specificity, that is, LGMN is highly expressed in various tumor cells and immunosuppressive cells, but is lowly expressed or not expressed in normal cells, which makes LZ-A4 degrade LGMN of tumor cells, etc. while it can maximize the avoidance of unnecessary damage to normal tissues, ensuring the safety of treatment.

[0066] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, for those skilled in the art, the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. The application of the small molecule compound LZ-A4 in the preparation of a drug for treating rectal cancer, wherein the chemical structural formula of the small molecule compound LZ-A4 is as follows:

2. The application according to claim 1, characterized in that, The dosage of the drug is 5 mg to 15 mg per kilogram of body weight.

3. The application according to claim 1, characterized in that, The drug can be administered orally, rectally, intravenously, subcutaneously, intramuscularly, intraperitoneally, or absorbed via the lungs, conjunctiva, nasopharynx, rectum, urethra, or bladder.

4. The application according to claim 1, characterized in that, The small molecule compound LZ-A4 inhibits the M2 polarization of malignant tumor-associated macrophages by degrading LGMN protein in malignant tumor cells, 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, thus achieving the therapeutic effect on malignant tumors.

5. The application according to claim 1, characterized in that, The small molecule compound LZ-A4 can be used alone or in combination with chemotherapy drugs.

6. The use of the small molecule compound LZ-A4 according to claim 1 in the preparation of an LGMN protein degrading agent for rectal cancer.

7. The application according to claim 6, characterized in that, The LGMN protein degrader contains a concentration of the small molecule compound LZ-A4 of not less than 10 μM.

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