Application of small-molecule inhibitor of stress particles in preparation of tumor treatment drugs

Abstract

The invention discloses application of a small-molecule inhibitor of stress particles in preparation of tumor treatment drugs, and relates to the technical field of biological medicines. According to the present invention, the high throughput screening is performed on the 'ChemDiv Protein-Protein Interaction (PPI) Library', such that the small molecule inhibitor of the stress particle, i.e., the compound UA-0007373, is obtained; the research finds that the small-molecule inhibitor can effectively inhibit the formation of cell stress particles caused by G3BP1, so that the drug resistance of tumor cells is reduced. Cell experiments prove that the small-molecule inhibitor can effectively inhibit the formation of stress particles induced by anti-cancer drugs. Therefore, a brand new auxiliary compound is provided for chemical drug treatment of cancers, and a new technical thought can be provided for research and development of cancer drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application of a small molecule inhibitor of stress granules in the preparation of anti-tumor drugs. Background Art

[0002] Stress granules (SGs) are membraneless organelles formed by phase separation in cells in response to external stress, and promote cell survival by suspending the translation of non-essential mRNAs. It has been found that SGs are widely present in various tumor cells and are involved in the regulation of oncogene expression, tumor metabolism regulation and adaptation to the tumor microenvironment, directly affecting tumor development and the efficacy of anti-cancer drugs. Some tumor cells resist the translational stress induced by chemotherapy and radiotherapy by enhancing the formation of SGs, improving the drug resistance to anti-cancer drugs, resulting in poor clinical therapeutic effects of cancer in patients. Therefore, inhibiting the formation of SGs is a novel anti-tumor strategy.

[0003] G3BP1 is a core component of SGs, and its expression is up-regulated in various tumor cells. The formation of SGs can be effectively inhibited by knocking out G3BP1 in cells. It has been found that proteins such as USP10 and Caprin-1 can bind to the same site in the NTF2L domain of G3BP1 and can inhibit the formation of SGs. Therefore, screening for inhibitors of SGs by targeting the NTF2L domain of G3BP1 can provide new ideas for the development of cancer drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a small molecule inhibitor of stress granules in the preparation of anti-tumor drugs to solve the problems existing in the above-mentioned prior art. This small molecule inhibitor can effectively inhibit the formation of cellular stress granules caused by G3BP1 or anti-cancer drugs, thereby reducing the drug resistance of tumor cells.

[0005] To achieve the above purpose, the present invention provides the following solutions:

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

[0007]

[0008] Further, the cellular stress granules are mediated by G3BP1 or anti-cancer drugs.

[0009] The present invention also provides a small molecule inhibitor of cellular stress granules, the active ingredient of which includes compound UA-0007373; the structural formula of the compound UA-0007373 is as follows:

[0010]

[0011] The present invention also provides the use of the above-mentioned small molecule inhibitor in the preparation of a tumor treatment drug.

[0012] Furthermore, the tumor is breast cancer.

[0013] The present invention also provides an anti-tumor pharmaceutical composition, the active ingredients of which include an anti-cancer drug and the above-mentioned small molecule inhibitor.

[0014] Furthermore, the anti-cancer drug is sorafenib.

[0015] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a tumor treatment drug.

[0016] The present invention also provides a tumor treatment drug, the active ingredients of which include the above-mentioned pharmaceutical composition.

[0017] The present invention also provides the use of compound UA-0007373 in the preparation of an inhibitor of G3BP1, and the structural formula of the compound UA-0007373 is as follows:

[0018]

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

[0020] Through high-throughput screening of the "ChemDiv Protein-Protein Interaction (PPI) Library" ([[]] Figure 2 ), a compound that binds to G3BP1 NTF2L was obtained. This compound is (2-[bis(2-methylpropyl)amino]-5-(3-chlorobenzamido)benzoic acid) (named UA-0007373, CAS No.: 1223830-25-8, and the structural formula is shown in [[[]] Figure 1 ). The present invention has found through research that compound UA-0007373 can specifically bind to the G3BP1 NTF2L domain in cells, thereby inhibiting the formation of stress granules mediated by G3BP1. In this way, the present invention further discovers that this compound UA-0007373 can effectively inhibit the formation of stress granules caused by anti-cancer drugs, thereby reducing the drug resistance of tumor cells.

[0021] Confirmed by cell experiments, this compound UA-0007373 can effectively inhibit the formation of stress granules induced by sorafenib. When used in combination with sorafenib, it can significantly reduce the IC of sorafenib against breast cancer cell MCF-7 [[[]] 50Therefore, it can be seen that the present invention provides a brand-new auxiliary compound for the chemotherapy of cancer, which can provide new technical ideas for the research and development of cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

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

[0024] Figure 2 It is the experimental principle and flow chart for screening small molecule compounds of the present invention;

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

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

[0027] Figure 5 It is the statistical histogram of the number of stress granules inhibited by compound UA-0007373 induced by the chemotherapeutic drug sorafenib in human breast cancer cell line MCF-7;

[0028] Figure 6 It is the IC 50 detection result of the combined use of compound UA-0007373 and the chemotherapeutic drug sorafenib in human breast cancer cell line MCF-7. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] Example 1 Determination of the binding constant (KD) of UA - 0007373 to G3BP1 by surface plasmon resonance

[0035] 1. Protein conjugation

[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 and 2 simultaneously and introduce Ni 2+ solution for 120 s.

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

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

[0041] (6) Flush at a flow rate of 30 μL / s until the baseline of channel 2 is basically stable.

[0042] 2. Sample loading and running

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

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

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

[0046] 3. Data analysis

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

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

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

[0050] 1. Cell culture

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

[0052] (2) When the cell density reaches approximately 80%, aspirate the medium and add PBS buffer for washing.

[0053] (3) Aspirate the PBS buffer, add 2 mL of trypsin, and place it in the constant-temperature incubator for digestion for 3 min.

[0054] (4) Add 2 mL of DMEM medium (supplemented with 10% (v / v) fetal bovine serum) to the culture dish to stop digestion and pipette thoroughly.

[0055] (5) The cell suspension was aspirated and added to a 1.5 mL EP tube, centrifuged at 1200 r / min for 5 min, and the supernatant was discarded.

[0056] (6) The cells were resuspended thoroughly with DMEM medium and inoculated into a 24-well culture plate containing cell slides at a density of 60,000 cells / well, 500 μL per well, and cultured for 24 h to allow them to adhere completely.

[0057] 2. Drug treatment

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

[0059] Treatment of the control group: Aspirate the cell culture medium of MCF-7 cells, wash with PBS buffer, add DMEM medium containing 20 μM of sorafenib to each well, and place in an incubator at 37 °C and 5% CO2 for 1 h.

[0060] 3. Fluorescence microscopy imaging

[0061] The cells in the experimental group and the control group after culture were imaged under a fluorescence microscope, and the method was as follows:

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

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

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

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

[0066] (4) Aspirate the blocking solution, prepare a primary antibody G3BP1 (Santa Cruz Biotechnology, Inc. Mouse sc-81940) (diluted 1:200) with the blocking solution, add 150 μL of the primary antibody dilution to the cell slides, and incubate on a horizontal shaker at 4 °C overnight.

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

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

[0069] (7) Aspirate the DAPI and wash three times with PBS buffer. Add 4 μL of the anti-quencher to the cell smear, invert the cell smear onto the glass slide, and seal the slide.

[0070] Use a laser confocal microscope (Zesis LSM880) to take pictures under a 25× objective lens. The results are shown in Figure 4 . The results showed that the number of stress granules in the Control group with only sorafenib added was relatively large, while the number of stress granules in the experimental group with the UA-0007373 compound added was significantly reduced.

[0071] 4. Data analysis

[0072] Statistically analyze the number of stress granules generated in the cells of the experimental group and the control group. The results ( Figure 5 ) showed that UA-0007373 inhibited the formation of sorafenib-induced stress granules in MCF-7 cells. After adding UA-0007373, the number of intracellular stress granules was greatly reduced. As the concentration of UA-0007373 increased, the number of stress granules became fewer and fewer. 50 μM of UA-0007373 basically completely inhibited the intracellular stress granules.

[0073] Example 3 Inhibiting the growth of tumor cells by the synergistic use of UA-0007373 and sorafenib

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

[0075] (2) When the cell density reached about 80%, aspirate the medium and add PBS buffer to wash.

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

[0077] (4) Pipette the cell suspension into an EP tube, centrifuge at 1200 r / min for 5 min, and discard the supernatant.

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

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

[0080] (7) Perform gradient dilution of sorafenib with concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μM. Take 10 μL each and add them to the corresponding positions of the 96-well plate. (6 replicates for each group).

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

[0082] (9) Mix the CCK8 cell viability detection reagent (Beyotime C0038) and DMEM medium thoroughly at a ratio of 1:10. After aspirating the original medium in the 96-well plate, 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 an enzyme-linked immunosorbent assay (ELISA) reader.

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

[0085] The inhibitory effect of the combined use of UA-0007373 and sorafenib on MCF-7 cells is as Figure 6 shown. The IC 50 of sorafenib on MCF-7 is 18.4 μM. After adding 5 μM of UA-0007373, the IC 50 of sorafenib decreases to 5.734 μM.

[0086] In summary, the small molecule compound UA-0007373 in the present invention can effectively inhibit the formation of stress granules induced by sorafenib. The compound in the present invention, when used in combination with sorafenib, can significantly reduce the IC 50 of sorafenib on breast cancer cell MCF-7.

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

Claims

1. Use of compound UA-0007373 in the preparation of a small molecule inhibitor for inhibiting the formation of cellular stress granules, characterized in that, The structural formula of the compound UA-0007373 is as follows:

2. The application according to claim 1, wherein The cellular stress granules are formed by G3BP1 or anti-cancer drugs.

3. A small molecule inhibitor of cellular stress granules, characterized in that, The active ingredient includes the compound UA-0007373; the structural formula of the compound UA-0007373 is as follows:

4. Use of a small molecule inhibitor as described in claim 3 in the preparation of a tumor therapeutic agent.

5. The application according to claim 4, wherein The tumor is breast cancer.

6. An antitumor pharmaceutical composition, characterized in that, The active ingredient includes an anti-cancer drug and the small molecule inhibitor as described in claim 3.

7. The pharmaceutical composition according to claim 6, wherein, The anti-cancer drug is sorafenib.

8. Use of a pharmaceutical composition as described in claim 6 or 7 in the preparation of a tumor therapeutic agent.

9. A tumor treatment drug, characterized in that, The active ingredient includes the pharmaceutical composition as described in claim 6 or 7.

10. Use of compound UA-0007373 in the preparation of an inhibitor of G3BP1, characterized in that, The structural formula of the compound UA-0007373 is as follows:

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