A small molecule inhibitor and its application

Abstract

This invention belongs to the field of biomedical technology and provides a small molecule inhibitor and its application. Specifically, this invention provides the application of an inhibitor that inhibits the expression and / or content of Integrin β1 protein in the preparation of drugs for treating breast cancer. The inhibitor used to inhibit the expression and / or content of Integrin β1 protein is a POH1 inhibitor. This invention is the first to elucidate the deubiquitination and stability regulation of the transmembrane receptor Integrin β1 by POH1, identifies the DUB of Integrin β1 as POH1, and utilizes a small molecule inhibitor of POH1 to block POH1 and target Integrin β1. This provides a new precise model and method for the development of cancer drugs targeting integrins, promotes the application and translation of basic research, and contributes to new breakthroughs in tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a small molecule inhibitor and its application. Background Technology

[0002] POH1 (PSMD14 / Rpn11) belongs to the JAB1 / MPN+ / MOV34 metalloproteinase (JAMM) deubiquitinating enzyme family. It participates in the regulation of many important biological processes, such as DNA double-strand break repair, intracellular protein stability, and embryonic stem cell differentiation. Recent studies have found that POH1 promotes the development and progression of various cancers, including liver cancer, gastric cancer, pancreatic cancer, and ovarian cancer, by regulating specific substrates. For example, POH1 can promote the growth and metastasis of liver cancer cells by stabilizing E2F1, TGF-β receptors, and caveolin-1, respectively; its role in promoting Snail stability has been found to be associated with poor prognosis in esophageal squamous cell carcinoma; and in breast cancer-related studies, POH1 expression in tumor samples is higher than in corresponding adjacent normal tissues, and it can promote the survival and motility of various breast cancer cells.

[0003] Existing technologies have confirmed that POH1 promotes lung cancer metastasis by mediating the deubiquitination and stabilization of Smad3. However, the mechanism by which POH1 promotes breast cancer is unclear. Previous studies in this invention discovered Integrin β1 in the mass spectrometry results of POH1. Integrin β1 has a significant pro-cancer effect due to its high expression in various tumors, including mastitis; however, its stability regulation mechanism remains unknown.

[0004] Thiolutin and Capzimin are two commonly used POH1 inhibitors. Therefore, this invention uses two small molecule inhibitors of POH1, Thiolutin and Capzimin, to explore their effects on delaying the progression of breast cancer. It is expected to discover new regulatory mechanisms of integrin signaling and provide new potential targets for breast cancer treatment. Summary of the Invention

[0005] To address the aforementioned technical issues, this invention discovered in breast cancer cells that the deubiquitinating enzyme POH1 interacts with Integrin β1, inhibiting its ubiquitination and enhancing its stability. POH1 promotes the motility and invasion of breast cancer cells, and CUL4A was preliminarily identified as the E3 ubiquitin ligase of Integrin β1. Therefore, the scientific hypothesis is that "POH1 promotes breast cancer progression by antagonizing CUL4A to stabilize Integrin β1." Different breast cancer cell lines were used to further elucidate the specific stability regulation mechanism of Integrin β1, and the role of this mechanism in breast cancer cell growth, adhesion, motility, invasion, and metastasis was studied at the cellular and animal levels. Microarray analysis of breast cancer patient tissues was used to analyze the correlation between POH1, CUL4A, and Integrin β1 expression, as well as their relationship with tumor progression and prognosis. Furthermore, by studying the inhibitors Thiolutin and Capzimin's inhibitory effects on breast cancer invasion and metastasis, it was confirmed that POH1 can increase the protein level of Integrin β1 in breast cancer cells, and the regulatory mechanism of POH1 on Integrin β1 and its role in breast cancer invasion and metastasis were investigated.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides the use of an inhibitor that suppresses the expression and / or content of Integrin β1 protein in the preparation of a drug for treating breast cancer.

[0008] Preferably, the inhibitor that suppresses the expression level and / or content of Integrin β1 protein is a POH1 inhibitor.

[0009] Preferably, the POH1 inhibitor affects the spread and movement, adhesion ability and plaque formation, migration and invasion of breast cancer cells by weakening the stability of Integrin β1 protein, thereby achieving the purpose of treating breast cancer.

[0010] Preferably, the POH1 inhibitor includes, but is not limited to, Thiolutin and Capzimin.

[0011] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0012] Preferably, the dosage form of the drug is an oral preparation or an injection.

[0013] Preferably, the oral dosage form includes a solid dosage form or a liquid dosage form.

[0014] Preferably, the solid dosage form includes tablets, powders, granules, capsules, or pills.

[0015] Preferably, the liquid dosage form includes a solution, an emulsion, or a suspension.

[0016] The beneficial effects of this invention are:

[0017] This invention is the first to elucidate the deubiquitination and stability regulation of the transmembrane receptor Integrin β1 by POH1, and identifies POH1 as the DUB of Integrin β1. By utilizing small molecule inhibitors of POH1, POH1 can be blocked to target Integrin β1, providing a new and precise model and means for the development of cancer drugs targeting integrins, promoting the application and translation of basic research, and contributing to new breakthroughs in tumor treatment. Attached Figure Description

[0018] Figure 1 The results show the effects of POH1 inhibitors on the downregulation of Integrin β1 protein levels (A and B are the Western blot and statistical results of the effect of THL on ITGB1 expression in MCF7 cells, respectively; D and E are the Western blot and statistical results of the effect of CZM on ITGB1 expression in MCF7 cells; C and F are the Western blots of the effects of THL and CZM on ITGB1 expression in MDA-MB-231 cells, respectively).

[0019] Figure 2 POH1 inhibitors reverse the deubiquitination effect of POH1 on Integrin β1;

[0020] Figure 3 POH1 inhibitors inhibit the movement, migration, and invasion of breast cancer cells by inhibiting POH1 activity and downregulating the protein expression of Integrin β1 (A is the cell scratch healing assay, B is the Transwell migration assay, and C is the Transwell invasion assay).

[0021] Figure 4 The effects of POH1 inhibitor administration on spontaneous breast cancer mice are shown in the following diagrams: (A: Subcutaneous breast cancer tumors dissected after mice were euthanized by administration; B: In vivo fluorescence imaging color thermograms of mice after administration; C: Sum of signals in the in vivo fluorescence imaging color thermograms of mice; D: Weight of dissected subcutaneous breast cancer tumors; E: Changes in body weight of mice during administration; F: Changes in tumor volume of mice during administration.) Detailed Implementation

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] The experimental materials and methods used in the following embodiments are as follows:

[0026] I. Experimental Materials

[0027] 1. Cell lines

[0028] Human breast cancer cells MCF7, SUM159, MDA-MB-231; human renal epithelial cells HEK-293T.

[0029] 2. Plasmids

[0030] HA-UB (ubiquitin), Flag-ITGB1 (Integrin β1), Myc-EV (no load), Myc-POH1 (PSMD14).

[0031] 3. Reagents

[0032] 1) Antibodies

[0033] β-Actin, Tubulin, Myc, Flag, HA, ITGB1 (CST, 9699S), POH1 (CST, 4197S).

[0034] 2) POH1 inhibitors

[0035] THL (Thiolutin, MCE, HY-N6712), CZM (Capzimin, MCE, HY-110404).

[0036] II. Experimental Methods

[0037] Western blot (WB)

[0038] 1) Vertical electrophoresis: Take protein samples, briefly centrifuge them in a 100℃ metal bath for 7 minutes, and set aside; load 15-40 μl of sample, and make up the difference with 2× Loading solution; in the stacking gel stage, electrophoresis at a constant voltage of 80V until the standard separates; until the Loading solution reaches the bottom of the protein gel.

[0039] 2) Horizontal electrophoresis: Take a PVDF membrane, cut it to the size of a gel, activate it with methanol for 1 min, transfer it to 1× electrophoresis buffer, and let it stand for equilibration for 5 min; stack the membranes in the order of sponge-filter paper-protein gel-PVDF membrane-filter paper-sponge, and then insert them into the horizontal electrophoresis tank; transfer the membrane with a constant current of 200 mA for 90 min.

[0040] 3) Blocking & Antibody Incubation: Block with 5% skim milk at room temperature for 2 hours; cut the membrane to the desired position according to the required molecular weight, and incubate the resulting membrane with the primary antibody at 4°C overnight.

[0041] 4) Development: Incubate the PVDF membrane with the corresponding species secondary antibody at 4°C for 1 hour; add sufficient ECL luminescent solution, incubate for 30 seconds, and then use an exposure machine to detect fluorescence.

[0042] Example 1: Efficacy testing of POH1 inhibitors

[0043] In this embodiment, POH1 inhibitors THL and CZM were applied at concentration gradients (250 nM, 500 nM, and 750 nM) to MCF7 and MDA-MB-231 breast cancer cells, with DMSO as a control. Proteins were collected after 48 hours, and the expression of the exogenous POH1 activity marker protein Integrin β1 (ITGB1) was detected by Western blotting. The results showed that both THL and CZM effectively inhibited POH1 activity and also affected the stability of Integrin β1 protein by inhibiting POH1 activity. Figure 1 ).

[0044] This embodiment designed a transfection experiment in HEK-293T cells, divided into four groups: Flag-Integrin β1+HA-Ub+Myc-EV, Flag-Integrin β1+HA-Ub+Myc-POH1, Flag-Integrin β1+HA-Ub+Myc-POH1, and Flag-Integrin β1+HA-Ub+Myc-POH1. The medium was changed 6-8 hours after transfection. 24 hours after transfection, DMSO, DMSO, THL, and CZM were added to the four groups respectively. MG132 (25mM) was added 6 hours before sample collection. Cells were harvested, proteins were extracted, and immunoprecipitation was performed using anti-Flag M2 antibody. Ubiquitin binding to Flag-Integrin β1 was detected using HA antibody, and HA-Ub expression was detected by Western blotting. The results showed that both the POH1 inhibitors THL and CZM could reverse the deubiquitination of Integrin β1 by POH1. Figure 2 ).

[0045] In summary, the POH1 inhibitors THL and CZM can inhibit the activity of POH1, and can affect the stability of Integrin β1 protein by inhibiting the activity of POH1, and can also reverse the deubiquitination effect of POH1 on Integrin β1.

[0046] Example 2: Effects of POH1 inhibitors on the biological function of breast cancer cells

[0047] To investigate the effects of the POH1 inhibitors THL and CZM on the motility of breast cancer cells, a cell scratch healing experiment was conducted in this embodiment.

[0048] The cell scratch assay is a method for detecting cell migration and repair capabilities, similar to an in vitro wound healing model. In this method, a line is drawn in the central region of a monolayer of adherent cells cultured in a dish or plate using a micropipette tip or other hard object. The central cells are then removed, and the cells are cultured again. Normal control and experimental groups are set up. After the experiment, the cell culture plate is removed, and it is observed whether peripheral cells have grown to the central scratched area, thus assessing the cell's growth and migration capabilities.

[0049] Digest and resuspend breast cancer cells MCF7 at a ratio of 3 × 10⁻⁶. 5 Standard samples were seeded in equal volumes into six-well plates and cultured in complete culture medium. On the second day, group experiments were conducted: the experimental groups were the THL group and the CZM group, with THL and CZM added to the six-well plates respectively; the control group was the DMSO group, with DMSO added to the six-well plates. After 48 hours of culture, once the cells had largely merged, uniformly wide lines were drawn on the bottom of the plate using a clean pipette tip. Exfoliated cells were washed away with PBS, and complete culture medium was added for further culture. At 0, 12, 24, and 36 hours, the drawn areas were photographed using a 10× eyepiece microscope, and the images were analyzed using ImageJ to compare the confluence of the drawn areas in different treatment groups. The results showed that POH1 inhibitors (THL and CZM) could affect the motility of breast cancer cells by altering the stability of Integrin β1 protein. Figure 3 A).

[0050] To investigate the effects of the POH1 inhibitors THL and CZM on the migration and invasion abilities of breast cancer cells, the following experiments were conducted in this embodiment:

[0051] Cell migration assay: MCF7 breast cancer cells were digested and resuspended, counted, and the suspension concentration was calibrated to 5 × 10⁻⁶. 5 The drug was administered to MCF7 breast cancer cells in two groups: the experimental groups were THL and CZM, and the control group was DMSO. 700 μl / well of medium containing 20% ​​FBS was added to each well of a 24-well plate as the lower chamber chemokine. 5 × 10⁻⁶ cells / ml were added to each Transwell chamber. 4Cells were evenly distributed; the Transwell chambers were moved and placed in wells containing chemokines, and stained after 6-8 hours; the chambers were removed, rinsed twice with PBS, and the upper chamber was gently wiped off with a cotton swab; 700 μl / well of 4% paraformaldehyde was added to a 24-well plate, and the chambers were placed in it for fixation for 20 min; the chambers were removed, rinsed twice with PBS, and the upper chamber was gently wiped off with a cotton swab; 700 μl / well of 5% crystal violet solution was added to a 24-well plate, and the chambers were stained for 20 min; the chambers were removed, rinsed twice with ultrapure water, and the upper chamber was gently wiped off with a cotton swab; after the chambers dried, 5 fields of view were randomly selected from each group and photographed under a 20X microscope, and the cells were counted and compared using ImageJ software.

[0052] Cell invasion assay: Except for the need for pre-coating the Transwell chambers with Matrigel and the difference in cell transmembrane time, the other experimental procedures are the same as those for the cell migration assay, so they will not be repeated here. The transmembrane time for the invasion assay varies from 24 to 48 hours depending on the cell state. The Matrigel pre-coating procedure is also noted below:

[0053] 1) Take the matrix gel that was frozen at -20℃ the day before the experiment and place it at 4℃ to allow it to thaw completely;

[0054] 2) Mix the matrix gel and serum-free culture medium thoroughly at a ratio of 1:4;

[0055] 3) Add 50 μl of matrix gel dilution solution evenly to the upper chamber of the Transwell chamber and gel at 37°C for 5 h.

[0056] (The base adhesive must be in an ice bath environment during the above steps, and the nozzle in contact with the base adhesive must be pre-cooled.)

[0057] The results showed that POH1 inhibitors could affect the migration and invasion abilities of breast cancer cells by altering the stability of Integrin β1 protein. Figure 3 B).

[0058] To investigate the effects of the POH1 inhibitors THL and CZM on the extension and adhesion abilities of breast cancer cells, the following experiments were conducted in this study:

[0059] After coating cells with a layer of Fibronectin in a cell culture dish, cells were seeded. Cell spreading and cell attachment assays were used to detect the cell spread and adhesion abilities after the addition of the POH1 inhibitor. The results showed that the POH1 inhibitor could affect the spread and adhesion of breast cancer cells by altering the stability of Integrin β1 protein (see...). Figure 3 C).

[0060] In summary, POH1 inhibitors can affect the motility, extension, adhesion, migration, and invasion capabilities of breast cancer cells by altering the stability of Integrin β1 protein.

[0061] Example 3: Effects of POH1 inhibitors on breast cancer growth and metastasis in vivo

[0062] I. Experimental Methods

[0063] 1) Take 6-8 week old MMTV-PyMT spontaneous breast cancer mice with similar body weight and divide them into DMSO injection mouse group and POH1 inhibitor CZM injection mouse group. The breast pad injection method is selected. The injection is carried out once every 3 days, with 6 mice in each group. The appropriate concentration of the inhibitor is injected according to the IC50. At the same time, spontaneous breast cancer is induced in the mice. The mice are cultured for another 3-4 weeks.

[0064] 2) Record the progression of breast cancer in mice in different treatment groups every 3 days, including tumor formation time, volume, mouse weight, and tumor-bearing survival time.

[0065] 3) Mice were euthanized by cervical dislocation, and organs and tissues were dissected to observe the metastasis of breast cancer to other organs and tissues.

[0066] 4) Prepare breast tissue sections, perform HE staining, and detect the metastasis of breast cancer cells to other organs and tissues.

[0067] II. Experimental Results

[0068] Experimental results are as follows Figure 4 As shown, the results indicate that there was no significant difference in body weight between the control and experimental groups during the drug administration period. The POH1 inhibitor CZM significantly inhibited the growth of breast cancer tumors. The breast cancer tumors in the experimental group were significantly smaller than those in the control group after drug administration.

[0069] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Application of inhibitors that suppress the expression and / or content of Integrin β1 protein in the preparation of drugs for treating breast cancer.

2. The application according to claim 1, characterized in that, The inhibitor that suppresses the expression and / or content of Integrin β1 protein is a POH1 inhibitor.

3. The application according to claim 2, characterized in that, The POH1 inhibitors affect the spread and movement, adhesion and plaque formation, migration and invasion of breast cancer cells by weakening the stability of Integrin β1 protein, thereby achieving the goal of treating breast cancer.

4. The application according to claim 2, characterized in that, The POH1 inhibitors include, but are not limited to, Thiolutin and Capzimin.

5. The application according to any one of claims 1 to 4, characterized in that, The drug also contains pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The drug is available in oral or injectable form.

7. The application according to claim 6, characterized in that, The oral dosage form includes solid dosage form or liquid dosage form.

8. The application according to claim 7, characterized in that, The solid dosage forms include tablets, powders, granules, capsules, or pills.

9. The application according to claim 7, characterized in that, The liquid dosage form includes solutions, emulsions, or suspensions.

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