Use of a small molecule inhibitor of stress granules in the preparation of a medicament for treating a tumor

Abstract

The application discloses an application of a small molecule inhibitor of stress granules in preparation of a tumor treatment drug, and relates to the technical field of biological medicines.The small molecule inhibitor of stress granules, i.e., compound UA-0007373, is obtained by high-throughput screening of "ChemDiv Protein-Protein Interaction (PPI) Library". The small molecule inhibitor can effectively inhibit the formation of cell stress granules caused by G3BP1, thereby reducing the drug resistance of tumor cells. The small molecule inhibitor can effectively inhibit the formation of stress granules induced by an anticancer drug through cell experiments. Therefore, the application provides a new auxiliary compound for the chemical drug treatment of cancer, and can provide a new technical thought for the research and development of cancer drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of a small molecule inhibitor of stress granules in preparation of a tumor treatment drug. BACKGROUND

[0002] Stress granules (SGs) are membraneless organelles formed by phase separation in response to external stress, which promote cell survival by pausing non-essential mRNA translation. Studies have found that SGs exist widely in various tumor cells, participate in the expression regulation of oncogenes, metabolic regulation of tumors and adaptation of tumor microenvironment, and directly affect tumor development and anticancer drug efficacy. Some tumor cells enhance the formation of SGs to resist translation stress induced by chemotherapy and radiotherapy, improve the drug resistance to anticancer drugs, and lead to poor clinical therapeutic effect of patients with cancer. Therefore, inhibiting the formation of SGs is a new tumor treatment strategy.

[0003] G3BP1 is a core component of SGs, which is up-regulated in various tumor cells, and the formation of SGs can be effectively inhibited by knocking out G3BP1 in cells. Studies have found that proteins such as USP10 and Caprin-1 can bind to the same site of the G3BP1 NTF2L domain and inhibit the formation of SGs. Therefore, targeting the G3BP1 NTF2L domain to screen inhibitors of SGs can provide a new idea for the research and development of cancer drugs. SUMMARY

[0004] The purpose of the present application is to provide application of a small molecule inhibitor of stress granules in preparation of a tumor treatment drug, so as to solve the problems existing in the prior art. The small molecule inhibitor can effectively inhibit the formation of cell stress granules caused by G3BP1 or anticancer drugs, thereby reducing the drug resistance of tumor cells.

[0005] To achieve the above purpose, the present application provides the following solutions.

[0006] The present application provides application of compound UA-0007373 in preparation of a small molecule inhibitor for inhibiting the formation of cell stress granules, and the structural formula of the compound UA-0007373 is as follows:

[0007]

[0008] Further, the cell stress granules are formed by G3BP1 or anticancer drugs.

[0009] The present application also provides a small molecule inhibitor of cell stress granules, and the active ingredient comprises compound UA-0007373; and the structural formula of the compound UA-0007373 is as follows:

[0010]

[0011] The present application also provides the use of the small molecule inhibitor in the preparation of a medicament for treating tumors.

[0012] Further, the tumor is breast cancer.

[0013] The present application also provides a pharmaceutical composition for treating tumors, wherein the active ingredients include an anticancer drug and the small molecule inhibitor.

[0014] Further, the anticancer drug is sorafenib.

[0015] The present application also provides the use of the pharmaceutical composition in the preparation of a medicament for treating tumors.

[0016] The present application also provides a medicament for treating tumors, wherein the active ingredients include the pharmaceutical composition.

[0017] The present application also provides the use of compound UA-0007373 in the preparation of an inhibitor of G3BP1, wherein the compound UA-0007373 has the following structural formula:

[0018]

[0019] The present application discloses the following technical effects:

[0020] The present application obtains a compound combined with G3BP1 NTF2L by high-throughput screening of "ChemDiv Protein-Protein Interaction (PPI) Library" Figure 2 ), which is (2-[bis(2-methylpropyl)amino]-5-(3-chlorobenzamide)benzoic acid) (named UA-0007373, CAS number: 1223830-25-8, structural formula see Figure 1 ). The present application finds that the compound UA-0007373 can be specifically combined with the G3BP1 NTF2L domain in cells, thereby inhibiting the stress granule formation mediated by G3BP1. In this way, the present application further finds that the compound UA-0007373 can effectively inhibit the stress granule formation caused by anticancer drugs, thereby reducing the drug resistance of tumor cells.

[0021] Cell experiments prove that the compound UA-0007373 can effectively inhibit the stress granule formation induced by sorafenib, and the use of the compound UA-0007373 in combination with sorafenib can significantly reduce the IC 50Therefore, the application provides a new auxiliary compound for chemical drug treatment of cancer, and provides a new technical idea for research and development of cancer drugs. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0023] Figure 1 is a structural formula of the compound UA-0007373;

[0024] Figure 2 is an experimental principle and flowchart for screening small molecule compounds according to the present application;

[0025] Figure 3 is a result of measuring the binding constant (KD) of the compound UA-0007373 and G3BP1 by surface plasmon resonance (SPR) experiment; wherein, A is a steady-state response value curve of different concentrations of UA-0007373; B is a fitting saturation curve with concentration as X axis and steady-state response value as Y axis;

[0026] Figure 4 is an immunofluorescence image of the compound UA-0007373 inhibiting stress granule formation induced by the chemotherapeutic drug sorafenib in human breast cancer cells MCF-7;

[0027] Figure 5 is a statistical column chart of the compound UA-0007373 inhibiting the number of stress granules induced by the chemotherapeutic drug sorafenib in human breast cancer cells MCF-7;

[0028] Figure 6 is an IC50 of the compound UA-0007373 combined with the chemotherapeutic drug sorafenib in human breast cancer cells MCF-7; 50 Detection results. DETAILED DESCRIPTION

[0029] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as a limitation of the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0030] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, amounts, and other

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not construed as an admission that it is prior art with respect to the present application.

[0032] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Additional implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. For example, the specification and examples give guidance regarding the use of the application in certain compositions and methods, but the application is not limited to these. Numerous specific details are set forth in order to provide a thorough understanding of the application. The implementations have been presented for the purpose of illustration and description. They are not intended to be exhaustive or to limit the application to the precise forms disclosed. Many other variations are possible given the wide number of possible embodiments.

[0033] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0034] Example 1 Surface Plasmon Resonance Assay for the Binding Constant (KD) of UA-0007373 to G3BP1

[0035] 1. Protein Coupling

[0036] (1) Take out the Biacore T200 maintenance chip and replace it with an NTA chip.

[0037] (2) Place the system pump head in the buffer (20 mM pH 7.5 Tris-HCl, 200 mM NaCl, 1% DMSO) and perform two Prime processes.

[0038] (3) Select channels 1, 2 at the same time, and inject Ni 2+ solution for 120 s.

[0039] (4) Dilute the 1 mg / mL G3BP1 NTF2L protein stock solution to 10 pg / mL with the buffer (20 mM pH 7.5 Tris-HCl, 200 mM NaCl, 1% DMSO).

[0040] (5) Only select channel 2, inject protein solution for 100 s, and couple the protein to be tested to the chip.

[0041] (6) Flush to channel 2 baseline to be stable at a flow rate of 30 μL / s.

[0042] 2. Loading run

[0043] (1) The mother liquor concentration of UA-0007373 is 200 μM, which is diluted to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM by gradient dilution with buffer (20 mM pH 7.5 Tris-HCl, 200 mM NaCl, 1% DMSO), and each sample is 100 μL.

[0044] (2) After centrifuging the sample at 12000 rpm for 3 min, cut off the EP tube cap, and place it on the loading rack in the order of the program.

[0045] (3) Execute the program, and perform a round of gradient test from low concentration to high concentration.

[0046] 3. Data analysis

[0047] (1) Run Biacore T200 analysis software.

[0048] (2) Take the concentration as the X axis and the steady-state response value as the Y axis, and obtain the binding constant (KD) of 49.15 μM by fitting the “saturation curve”. Figure 3 ).

[0049] Example 2: UA-0007373 inhibits sorafenib-induced stress granules in MCF-7 cells

[0050] 1. Cell culture

[0051] (1) MCF-7 cells were cultured in a 10 cm culture dish with DMEM medium (added with 10% (v / v) fetal bovine serum) and placed in a constant temperature incubator with a culture condition of 37°C and 5% CO2.

[0052] (2) After the cell density reached about 80%, the culture medium was aspirated, and PBS buffer was added for washing.

[0053] (3) The PBS buffer was aspirated, 2 mL of trypsin was added, and the trypsin was allowed to digest for 3 min in the constant temperature incubator.

[0054] (4) 2 mL of DMEM medium (added with 10% (v / v) fetal bovine serum) was added to the culture dish to stop the digestion, and the culture dish was blown and mixed thoroughly.

[0055] (5) Take the cell suspension into 1.5 mL EP tube, centrifuge at 1200 r / min for 5 min, and discard the supernatant.

[0056] (6) Resuspend the cells with DMEM medium, inoculate into the 24-well culture plate with cell climbing sheet at a density of 60000 cells per well, 500 μL per well, and culture for 24 h to make the cells adhere completely.

[0057] 2. Drug treatment

[0058] Experimental group treatment: aspirate the cell culture medium of MCF-7 cells, wash with PBS buffer, and divide into three experimental groups, respectively, add DMEM medium containing 5 μM, 10 μM, and 50 μM of UA-0007373, and place in a 37℃, 5% CO2 incubator for 1 h.

[0059] Control group (Control) treatment: aspirate the cell culture medium of MCF-7 cells, wash with PBS buffer, and add DMEM medium containing 20 μM of Sorafenib to each well, and place in a 37℃, 5% CO2 incubator for 1 h.

[0060] 3. Fluorescence microscope shooting

[0061] The cells after culture of the experimental group and the control group are subjected to fluorescence microscope shooting, and the method is as follows:

[0062] (1) Aspirate the culture medium of the experimental group or the control group, wash with PBS buffer for three times. Add 300 μL of 4% paraformaldehyde to each well, and place in a horizontal shaking bed at room temperature for 15 min.

[0063] Aspirate the paraformaldehyde, and add 300 μL of -20℃ pre-cooled methanol to each well, and incubate on a horizontal shaking bed at room temperature for 10 min.

[0064] (2) Aspirate the methanol, and wash with PBS buffer for three times. Take the cell climbing sheet from the cell culture plate to a hydrophobic sealing film, and place in a light-proof wet box.

[0065] (3) Add 200 μL of blocking solution (1% BSA, 10% goat serum (Gibco)) to the cell climbing sheet, and incubate on a horizontal shaking bed at room temperature for 45 min.

[0066] (4) Aspirate the blocking solution, and prepare the primary antibody G3BP1 (Santa Cruz Biotechnology, Inc. Mouse sc-81940) (1:200 dilution) with the blocking solution. Add 150 μL of the primary antibody diluent to the cell climbing sheet, and incubate on a horizontal shaking bed at 4℃ overnight.

[0067] (5) Aspirate the primary antibody and wash the cell slides three times with PBS buffer. Prepare secondary antibody Goat anti mouse 555 (Thermo A32727) (1:200 dilution) with blocking solution, add 150 μL of the secondary antibody dilution to the cell slides and incubate for 60 min at room temperature on a horizontal shaker.

[0068] (6) Aspirate the secondary antibody and wash the cell slides three times with PBS buffer. Add 200 μL of 1 μg / mL 4',6-diamidino-2-phenylindole (DAPI) to the cell slides and incubate for 5 min at room temperature on a horizontal shaker.

[0069] (7) Aspirate the DAPI and wash the cell slides three times with PBS buffer. Add 4 μL of anti-quenching reagent to the cell slides, invert the slides onto a glass slide and mount the slides.

[0070] Take pictures using a laser confocal microscope (Zesis LSM880) under a 25x objective lens. The results are shown in Figure 4 . The results show that the Control group with only sorafenib has a large number of stress granules, while the experimental group with the addition of UA-0007373 compound has a significantly reduced number of stress granules.

[0071] 4. Data analysis

[0072] Statistical analysis of the number of stress granules generated in the experimental and control groups of cells. The results Figure 5 show that UA-0007373 inhibits the formation of stress granules induced by sorafenib in MCF-7 cells. After the addition of UA-0007373, the number of stress granules in the cells is greatly reduced, and as the concentration of UA-0007373 increases, the number of stress granules decreases. The addition of 50 μM UA-0007373 essentially completely inhibits the stress granules in the cells.

[0073] Example 3. UA-0007373 and sorafenib are used synergistically to inhibit tumor cell growth

[0074] (1) MCF-7 cells were cultured in a 9 cm culture dish with DMEM medium (with the addition of 10% (v / v) fetal bovine serum) in a constant temperature incubator at 37°C and 5% CO2.

[0075] (2) When the cell density reached about 80%, the culture medium was aspirated and washed with PBS buffer.

[0076] (3) Aspirate the PBS buffer and add 2 mL of trypsin and incubate in a constant temperature incubator for 3 min.

[0077] (4) The cell suspension was aspirated and added to an EP tube, centrifuged at 1200 r / min for 5 min, and the supernatant was discarded.

[0078] (5) Resuspend the cells in 1 mL DMEM medium, dilute to a density of 6000 / mL, inoculate in a 96-well culture plate, 90 μL per well, and culture for 24 h to make the cells adhere completely.

[0079] (6) Add UA-0007373 solution to the corresponding wells of the 96-well plate to make the final concentration 5 μM.

[0080] (7) Gradient dilute sorafenib to concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 μM, and take 10 μL of each to the corresponding positions of the 96-well plate. (6 replicates per group).

[0081] (8) Incubate in a constant-temperature incubator for 72 h.

[0082] (9) Mix CCK8 cell viability detection reagent (Bi Yun Tian C0038) and DMEM medium at a ratio of 1:10, aspirate the original culture medium of the 96-well plate, and immediately add 100 μL of the mixed solution to each well, and incubate at 37°C for 1 h.

[0083] (10) Measure and record the absorbance of the 96-well plate at 450 nm using a microplate reader.

[0084] (11) Use GraphPad Prism 8.0 software to calculate the half maximal inhibitory concentration (IC 50 ) of the synergistic effect of the chemotherapeutic drug and the polypeptide on the cells.

[0085] The inhibitory effect of UA-0007373 and sorafenib on MCF-7 cells is shown in Figure 6 . The IC 50 of sorafenib on MCF-7 is 18.4 μM, and after adding 5 μM of UA-0007373, the IC 50 of sorafenib is reduced to 5.734 μM.

[0086] In summary, the small molecule compound UA-0007373 in the present application can effectively inhibit the stress granule formation induced by sorafenib. The compound in the present application and sorafenib are used synergistically, which can significantly reduce the IC 50 of sorafenib on breast cancer cells MCF-7.

[0087] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. The application of a small molecule inhibitor in the preparation of tumor therapeutic drugs, characterized in that, The active ingredient of the small molecule inhibitor includes compound UA-0007373; the structural formula of the compound UA-0007373 is as follows: ； The tumor is breast cancer.

2. An antitumor pharmaceutical composition, characterized by, The active ingredient includes an anticancer drug and a small molecule inhibitor; The active ingredient of the small molecule inhibitor includes compound UA-0007373; the structural formula of the compound UA-0007373 is as follows: ； The tumor is breast cancer. The anticancer drug is sorafenib.

3. Use of the pharmaceutical composition according to claim 2 for the manufacture of a medicament for the treatment of tumors, characterized in that, The tumor is breast cancer.

4. A medicament for treating a tumor, characterized by comprising the compound of claim 1. The active ingredient includes the pharmaceutical composition of claim 2; The tumor is breast cancer.

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