Compound RMY-186, preparation method thereof and application of compound RMY-186 in medicine for treating drug-resistant tumors

Abstract

The invention discloses a compound RMY-186, a preparation method of the compound RMY-186 and application of the compound RMY-186 in drugs for treating drug-resistant tumors. The drugs further comprise chemotherapeutic drugs (including fluorouracil and oxaliplatin) which are used in a combined mode. The compound RMY-186 provided by the invention can directly promote the degradation of CAD regardless of whether mutation occurs or not. The chemotherapy effects of fluorouracil and the like are enhanced by promoting GC and CRC cell death and GSDME splitting decomposition. In conclusion, the RMY-186 can be used as a drug and a lead compound for overcoming GC and CRC chemotherapy drug-resistant tumors caused by CAD cleavage site mutation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies and relates to the use of a flavonol compound RMY-186 in the preparation of a medicament for treating chemotherapy-resistant tumors. Background Art

[0002] Drug therapy (chemotherapy, targeted and immunological drugs) is one of the very important treatment means for cancer patients. However, currently, for almost all drugs, as the patients use them for a longer time, the cancer will develop drug resistance, causing the drug to stop acting on cancer cells. The drug resistance of cancer cells is one of the main reasons for the failure of cancer treatment, which may lead to the rapid recurrence / progression of the disease and ultimately result in the death of the patient. The mechanisms of tumor chemotherapy resistance include drug inactivation, efflux, epigenetic changes, DNA damage repair, inhibition of cell death, and alteration of drug targets, etc. The drug-resistant tumors will lose their responsiveness to general chemotherapy drugs and grow wantonly. With the increasing incidence of tumor chemotherapy resistance, it has become more and more urgent to discover new resistance mechanisms and develop new anti-drug-resistant chemotherapy drugs. Summary of the Invention

[0003] The proliferation of mammalian cells requires de novo synthesis of pyrimidine nucleotides. This gene encodes a trifunctional protein that is associated with the enzymatic activities of the first three enzymes in six steps of the pyrimidine biosynthetic pathway: carbamoyl phosphate synthetase (CPSII), aspartate transcarbamylase, and dihydroorotase. That is, the metabolic flux of the de novo pyrimidine synthesis pathway is regulated by the first rate-limiting enzyme, Cytosolic carbamyl-phosphate synthetase II, asparate transcarbamylase, and Dihydroorotase (CAD). In the preliminary study of the applicant, it was found that the multifunctional enzyme CAD is a physiological substrate of Caspase-3 in the endogenous apoptosis pathway induced by chemotherapy drugs. Targeting these metabolic perturbations in cancer cells is the basis of all effective treatment strategies.

[0004] In the preliminary study, the applicant found that the multifunctional enzyme CAD is a physiological substrate of Caspase-3 in the endogenous apoptosis pathway induced by chemotherapy drugs. Subsequently, the research results of the applicant showed that overexpression of CAD or gain-of-function (GOF) mutations at codon 1371 of the aspartic acid cleavage site enhanced chemotherapy resistance, which was confirmed in clinical samples. In addition, the applicant demonstrated that pharmacological inhibition of anti-cleavage CAD sensitized tumor cells to clinically relevant chemotherapy drugs. Therefore, CAD has become a key inhibitor of chemotherapy-induced cell death and a promising molecular target for treatment resistance.

[0005] The compound RMY-186 involved in the present invention refers to the small molecule compound 2-(4-(3-bromobenzyl)piperazin-1-yl)-3-hydroxy-4H-chromen-4-one, and its structural formula is as follows:

[0006]

[0007] This compound and its preparation method have been disclosed in the previous patent of the present applicant, patent number: ZL201710970315.2, application date: October 16, 2017, invention title: "A flavonoid compound and its preparation method and application", specifically refer to Example 17, Compound 12. However, in the patent of this prior application, the disclosed application is the treatment of common tumors, including liver cancer, uterine cancer, lung cancer, etc., while the present invention further discovers that the compound RMY-186 has an effect on treating drug-resistant tumors.

[0008] Therefore, the first object of the present invention is to provide the use of compound RMY-186 or its pharmaceutically acceptable salt in the preparation of drugs for drug-resistant tumors.

[0009] Further, the drug-resistant tumor is a chemotherapy drug-resistant tumor.

[0010] Further, the chemotherapy drug-resistant tumor is a chemotherapy drug-resistant gastrointestinal tumor.

[0011] Another object of the present invention is to provide a drug for treating chemotherapy drug-resistant tumors, which comprises compound RMY-186 or its pharmaceutically acceptable salt.

[0012] Further, the drug of the present invention further comprises a chemotherapy drug.

[0013] Further, the chemotherapy drug is at least one of 5-fluorouracil (5-FU) and oxaliplatin.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. The present invention discovers that compound RMY-186 can combat cancer drug resistance. The possible reason is that RMY-186 can act on the CAD target. The present invention discovers that compound RMY-186 can directly promote the degradation of CAD, regardless of whether it mutates. By promoting the death of GC and CRC cells and GSDME cleavage, the chemotherapy effect of fluorouracil and the like is enhanced. Therefore, RMY-186 can become a therapeutic drug and lead compound for overcoming GC and CRC chemotherapy drug resistance caused by CAD cleavage site mutation.

[0016] 2. The present invention further discovers that the combined use of RMY-186 and the chemotherapy drug fluorouracil (5-FU) or oxaliplatin (Oxa) has a good therapeutic effect on anti-chemotherapy tumors. Brief Description of the Drawings

[0017] The present invention will be further described below in conjunction with the drawings and embodiments.

[0018] The present invention will be further described below in conjunction with the drawings and embodiments.

[0019] Figure 1 :

[0020] (A) Chemical structure of RMY-186.

[0021] (B) Flag-CAD-WT or Flag-CAD-D1371A was transfected into MKN45 cells, and then treated with RMY-186 (10 μM) for 24 h.

[0022] (c) Microscale thermophoresis (MST) was used to determine the differential selectivity of RMY-186 and its binding to GFP-CAD-D1371A and GFP.

[0023] (d) MKN45 cells overexpressing Flag-CAD-D1371A were pretreated with MG-132 (5 μM), and then treated with RMY-186 (10 μM) for 24 hours. The overexpressed protein with Flag tag and ubiquitination were detected by immunoblotting.

[0024] (e) Co-immunoprecipitation showed the ubiquitination of CAD-D1371A protein after RMY-186 treatment in MKN45 cells overexpressing HA-Ub and Flag-CAD-D1371A.

[0025] (f) Co-immunoprecipitation showed the ubiquitination of CAD protein in MKN45 cells transfected with the indicated plasmids.

[0026] (g) Computational molecular docking analysis was performed to study the binding interaction of RMY-186 with CAD-D1371A (AlphaFold2). CPSase represents the indicated local polypeptide.

[0027] (h-i) Volcano plot (h) and heat map (i) showing differential E3 ubiquitin ligases in proteomic MS data.

[0028] (j) HEK-293T cells were transfected with Flag-CAD-D1371A. After 24 hours of transfection, CHX (20 μg / ml) was added, and the cells were collected in the presence or absence of RMY-186 (10 μM) treatment. Cells were collected at the indicated times (0, 3, 6, 9, 12, or 15 hours) and immunoblotted with anti-Flag antibody to detect changes in protein levels.

[0029] (k) Each Western blot shown in (j) was scanned for densitometry using ImageJ. Values represent the mean ± standard deviation of three independent experiments. The best-fit exponential decay line was plotted using GraphPad Prism, and the half-life could be read from the figure (marked with a dotted line). p < 0.001.

[0030] Figure 2 :

[0031] (a-b) Flag-CAD-D1371A was transfected into MKN45 (a) and HCT116 (b) cells and then treated with different small molecule compounds (10 μM) for 24 hours. The expression level of Flag-CAD-D1371A was measured.

[0032] (c) Flag-CAD-WT or Flag-CAD-D1371A was transfected into HCT116 cells and then treated with RMY-186 (10 μM) for 24 hours.

[0033] (d) Flag-CAD-D1371E or Flag-CAD-D1371Y was transfected into MKN45 and HCT116 cells and then treated with RMY-186 (10 μM) for 24 hours.

[0034] (e-f) MKN45 cells overexpressing Flag-CAD-D1371E (e) or Flag-CAD-D1371Y (f) were pretreated with MG-132 (5 μM) and then treated with RMY-186 (10 μM) for 24 hours. Overexpressed proteins with Flag tags and ubiquitination were detected by immunoblotting.

[0035] (g-h) Immunoprecipitation showed ubiquitination of CAD-D1371E or CAD-D1371Y proteins after RMY-186 treatment in MKN45 cells overexpressing HA-Ub and Flag-CAD-D1371E (g) or Flag-CAD-D1371Y (h).

[0036] (i) Immunoprecipitation showed ubiquitination of CAD proteins in MKN45 cells transfected with the indicated plasmids.

[0037] (j) A schematic diagram showing the binding of CAD-D1371A to RMY-186 was presented by LigPlot.

[0038] (k) Molecular docking of CAD mutants (AlphaFold2) with RMY-186 was predicted using Autodock.

[0039] (l-o) HEK-293T cells were transfected with Flag-CAD-D1371E (l-m) or Flag-CAD-D1371Y (n-o). After 24 hours of transfection, CHX (20 μg / ml) was added, and cells were collected in the presence or absence of RMY-186 (10 μM). Cells were collected at the indicated times (0, 3, 6, 9, 12, or 15 hours), and immunoblotting with anti-Flag antibody was performed to detect changes in protein levels. Each Western blot was scanned for densitometry using ImageJ. Values represent the mean ± standard deviation of three independent experiments. The best-fit exponential decay line was plotted using GraphPad Prism, and the half-life could be read from the figure (marked with a dotted line). p < 0.001.

[0040] Figure 3 :

[0041] (a) In MKN45 CADD1371A / D1371A cells, after treatment with DMSO, RMY-186 (10 μM), 5-FU (50 μM), or a combination of RMY-186 and 5-FU for 24 hours, the protein expression levels of CAD, cleaved PARP, P53, and p-H2A.X were detected by Western blot.

[0042] (b) As described in (a), the expression of p-H2A.X was detected by immunofluorescence.

[0043] (c-d) Microscopic images of pyroptotic cells. Arrows indicate the balloon-like cell membranes characteristic of pyroptotic cells. Scale bar is 25 μm.

[0044] (e) In MKN45 CADD1371A / D1371A cells, after treatment as described in (a), the expression level of GSDME was detected by Western blot.

[0045] (f) In CAD-KD MKN45 cells, after treatment with 5-FU, the expression level of GSDME was detected by Western blot.

[0046] (g) In (a), pyroptotic cells were detected by staining with PI (50 μg / ml).

[0047] (h) The culture supernatant in (a) was collected, and the percentage of LDH release was measured (n = 3; mean ± standard deviation). p < 0.001.

[0048] (i) In (f), pyroptotic cells were detected by staining with PI (50 μg / ml).

[0049] (j)Collect the culture supernatant in (f) and measure the percentage of LDH release (n = 3; mean ± standard deviation). p < 0.001.

[0050] (k)Gross images of the stomachs of Cldn18-ATK CadD1371A / D1371A mice untreated, treated with RMY-186, treated with 5-FU, or treated with a combination of RMY-186 and 5-FU. Tumors are marked with black dotted lines (n = 8).

[0051] (l)Statistical analysis of the maximum tumor area in the stomach (n = 8; mean ± standard deviation). p < 0.05, p < 0.001.

[0052] (m)In tumor samples, detect the protein expression levels of CAD, cleaved PARP, GSDME, and P53 by Western blot.

[0053] (n)Representative images of H&E, CAD, cleaved PARP, P53, and Ki67 staining of stomach sections. The scale bar is 50 μm.

[0054] (o)Quantitative and statistical analysis of the Ki67 staining images in (n). ns: no significant difference, p < 0.001.

[0055] (p)Analyze the concentration of serum LDH in (k) by ELISA (n = 8; mean ± standard deviation). p < 0.001.

[0056] Figure 4 :

[0057] (a)In HCT116 CADD1371A / D1371A cells, after treatment with DMSO, RMY-186 (10 μM), 5-FU (50 μM), or a combination of RMY-186 and 5-FU for 24 hours, detect the protein expression levels of CAD, cleaved PARP, P53, and p-H2A.X by Western blot.

[0058] (b)Detect the expression of p-H2A.X by immunofluorescence as described in (a).

[0059] (c)In MKN45 CADD1371A / D1371A cells and HCT116 CADD1371A cells, after treatment with DMSO, RMY-186 (10 μM), Oxa (20 μM), or a combination of RMY-186 and Oxa for 24 hours, detect the protein expression levels of CAD, cleaved PARP, P53, and p-H2A.X by Western blot.

[0060] (d) HEK-293T cells were transfected with siCAD-1, siCAD-2 or a universal negative control siRNA, and the expression level of CAD was detected by Western blot.

[0061] (e-f) Microscopic images of pyroptotic cells. Arrows indicate the balloon-like cell membranes characteristic of pyroptotic cells. Scale bar is 25 μm.

[0062] (g) The protein expression levels of GSDME were detected by Western blot in multiple gastric cancer (GC) and colorectal cancer (CRC) cell lines.

[0063] (h) In HCT116 CADD1371A / D1371A cells, after treatment as described in (a), the expression level of GSDME was detected by Western blot.

[0064] (i) In CAD-KD HCT116 cells, after treatment with 5-FU, the expression level of GSDME was detected by Western blot.

[0065] (j) In (a), pyroptotic cells were detected by staining with PI (50 μg / ml).

[0066] (k) The culture supernatant in (a) was collected, and the percentage of LDH release was measured (n = 3; mean ± standard deviation). p < 0.001.

[0067] (l) In (i), pyroptotic cells were detected by staining with PI (50 μg / ml).

[0068] (m) The culture supernatant in (i) was collected, and the percentage of LDH release was measured (n = 3; mean ± standard deviation). p < 0.001.

[0069] (n) The LD50 of RMY-186 was tested in C57BL / 6J mice, with n = 6 mice in each group, and the LD50 was estimated using a non-linear fitting method. Source data are available in the Source Data file.

[0070] (o) The small molecule compound RMY-186 showed good safety. In the acute toxicity test, after treating mice with different concentrations of RMY-186, the tissues were stained with H&E. This experiment was independently repeated 3 times with similar results. Scale bar is 100 μm. Detailed implementation methods

[0071] Given the rapid proliferation and nucleotide synthesis activity of cancer cells, disrupting nucleotide metabolism has emerged as a promising anti-cancer strategy. Increasing evidence indicates that dysregulation of pyrimidine nucleotide metabolism is prevalent in cancer. Several new dihydroorotate dehydrogenase (DHODH) inhibitors play a key role in pyrimidine synthesis and show anti-tumor effects in preclinical models. However, durable tumor regression remains elusive, mainly due to the emergence of drug-resistant tumor subclones.

[0072] Considering the crucial role of abnormal nucleotide metabolism in tumorigenesis and the importance of CAD in pyrimidine nucleotide metabolism, targeting CAD provides a new therapeutic approach for cancer, especially for cancers resistant to DNA damage therapy. N-(phosphonoacetyl)-L-aspartate (PALA), an analogue of the activated complex of the carbamoylase-catalyzed reaction of aspartate, has shown effective inhibition of de novo pyrimidine nucleotide synthesis. The applicant's results suggest that CAD mutations may be negative regulators of chemotherapy-induced apoptosis, indicating that a new CAD mutation inhibitor could counteract cancer drug resistance.

[0073] To obtain small molecules that can inhibit CAD D1371A expression without inducing apoptosis, the applicant has synthesized the compound RMY-186 in previous studies. See the aforementioned prior application for details.

[0074] Example 1 verifies the therapeutic effect of compound RMY-186

[0075] This application aims to develop a method capable of downregulating the expression of CAD-D1371 mutants to overcome chemotherapy resistance caused by the inability of these mutants to be cleaved by caspase-3. The applicant constructed MKN45 and HCT116 cells expressing Flag-tagged CAD-D1371A. In 6-well plates, 2 μg of Flag-CAD-D1371A plasmid was transfected into MKN45 and HCT116 cells using Lipofectamine 3000 transfection reagent, and the fresh medium was changed 24 hours after transfection. After treatment with compounds (final concentration of 10 μM each) for 24 hours, the cells were lysed, and compounds that significantly inhibited the protein level of CAD-D1371A were screened by Western blot (anti-Flag antibody) (here, RMY-186 showed the strongest inhibitory effect). Method for constructing Flag-CAD-D1371A plasmid: The cDNA encoding human CAD (GenBank accession number: D78586, from the gene library of the Biomedical Instrument Sharing Platform of Xiamen University) was cloned into the pCMV-C-3xFlag vector (purchased from Public Protein / Plasmid Library, Nanjing, China) through restriction enzyme sites (EcoR I / Xho I). All CAD point mutants were generated using the QuikChange site-directed mutagenesis kit (purchased from Stratagene, California, USA) and cloned into the pCMV-C-3xFlag vector using the same restriction enzyme sites (EcoR I / Xho I) as wild-type CAD.

[0076] An in-house compound library was screened using these cells. Among all the compounds screened, the applicant found that 24 compounds could significantly inhibit the protein level of CAD-D1371A (determined by immunoblotting, see Figure 2 a, b). Among these compounds, the applicant identified RMY-186 (2-(4-(3-bromobenzyl)-piperazin-1-yl)-3-hydroxy-4H-chromen-4-one) as the most effective compound for inhibiting the expression of CAD-D1371A ( Figure 1 a). RMY-186 also showed a broad inhibitory effect on CAD because it not only significantly downregulated the protein level of CAD-D1371A but also the protein levels of wild-type CAD, CAD-D1371E, and CAD-D1371Y mutants ( Figure 1 b and Figure 2c-d). Construction methods of wild-type CAD, CAD-D1371E, and CAD-D1371Y mutants: In a 6-well plate, 2 μg of Flag-CAD-WT or Flag-CAD-D1371E or Flag-CAD-D1371Y plasmid was transfected into MKN45 and HCT116 cells using Lipofectamine 3000 transfection reagent. After 24 hours of transfection, the fresh medium was replaced. After adding RMY-186 (10 μM) and treating for 24 hours, the cells were lysed, and the protein levels of various types of CAD were detected by Western blot (anti-Flag antibody).

[0077] Microscale thermophoresis (MST) experiments confirmed the direct interaction between RMY-186 and CAD-D1371A protein in vitro, with a Kd value of 6.78×10 -6 mol / L( Figure 1 c), which is close to the concentration of RMY-186 that effectively downregulates CAD in cells. The applicant also studied how RMY-186 downregulates CAD. The applicant found that under RMY-186 treatment, MG-132 could restore the protein levels of exogenously expressed CAD, including wild-type and Asp1371 mutants, and this phenomenon was observed in HEK-293T, gastric cancer (GC), and colorectal cancer (CRC) cells. Microscale thermophoresis (MST) method: Protein purification: His-CAD-D1371A protein was purified from HEK-293T cells. Fluorescent labeling: His-CAD-D1371A protein was labeled with a fluorescent dye (NT-647). Binding analysis: The fluorescence intensity change of the fluorescent dye-labeled His-CAD-D1371A protein under different concentrations of RMY-186 treatment was detected in an MST instrument, a binding curve was plotted, and the Kd value was calculated (buffer: PBS + 0.05% Tween-20).

[0078] In addition, the applicant observed an increase in the ubiquitination level of CAD protein in these cells ( Figure 1 d-e and Figure 2 e-h), indicating that RMY-186 promoted the degradation of CAD. Method: Cell treatment: In HEK-293T, MKN45, or HCT116 cells, Flag-CAD-WT or Flag-CAD-D1371A was co-transfected with HA-Ub plasmid, and then treated with RMY-186 (10 μM) for 24 hours. Proteasome inhibition: In some experimental groups, MG-132 (5 μM) was added for 1 hour of pretreatment to inhibit proteasome activity. Immunoprecipitation (Co-IP): IP was performed with anti-Flag antibody, and after lysis, the ubiquitination modification of CAD or CAD-D1371A was detected by Western blot (anti-HA antibody).

[0079] Example 2 Identification of the interaction interface between CAD and RMY-186 by Alphafold2 and CavityPlus servers

[0080] The applicant used Alphafold2 and CavityPlus servers to identify the interaction interface between CAD and RMY-186. (The three-dimensional structure of the CAD protein was predicted using AlphaFold2, and the prediction model was optimized by energy minimization and molecular dynamics pre-equilibration. The potential binding pockets and binding sites of RMY-186 and CAD, that is, the amino acid residues around the active site, were analyzed by CavityPlus and the semi-flexible molecular docking software AutoDock Vina) By analyzing surface feature matching, binding energy, and potential hydrogen bonds and non-covalent interactions, the applicant identified four potential binding pockets on CAD: Pocket 1 (Glu(E)513 and Arg(R)515), Pocket 2 (Asp(D)622 and Glu(E)630), Pocket 3 (Pro(P)741 and Trp(W)743), and Pocket 4 (Glu(E)1214 and Arg(R)1221). Subsequently, the applicant mutated these pockets separately to determine the effect of RMY-186 on the promotion of CAD ubiquitination. The applicant found that the double mutation of CAD-P741 and CAD-W743 (CAD-P741 / W743A) disrupted Pocket 3 and almost completely blocked the promotion of CAD ubiquitination by RMY-186 ( Figure 1 f and Figure 2 i). These data further indicate that RMY-186 binds directly and specifically to CAD, and P741 and W743 are key residues in this interaction. As shown in the figure, the active pocket of the CAD protein contains the residues Pro741 (P741) and Trp743 (W743), which play a key role in promoting the binding of RMY-186. Pro741 provides the structural basis for the docking of RMY-186, while Trp743 forms two hydrogen bonds with the hydroxyl group of RMY-186 through its carboxyl and amino groups, with lengths of and In addition, the keto group of RMY-186 also forms a hydrogen bond with the carboxyl group ( Figure 1 g and Figure 2 j). Since Pocket 3 is far from the Asp1371 residue ( Figure 2 k), these data may explain the indiscriminate inhibitory effect of RMY-186 on CAD and its Asp1371 mutant.

[0081] Example 3 Identification of the E3 ligase mediating RMY-186-induced CAD ubiquitination

[0082] Through proteomic mass spectrometry (MS) data analysis of Flag-CAD immunoprecipitates, it was found that the bifunctional E2 ubiquitin-conjugating enzyme / E3 ubiquitin-protein ligase BIRC6 or ubiquitin-protein ligase E3C (UBE3C) was most likely to be involved in the ubiquitination process of CAD ( Figure 1 h-i). HEK-293T cells overexpressing Flag-tagged CAD and its mutants (CAD-D1371A / E / Y) were treated with the protein translation inhibitor cycloheximide (100 μg / mL) in the presence or absence of RMY-186 (20 μM). Cells were collected at different times (0, 3, 6, 9, 12, 15 h), and the expression of Flag-tagged CAD and its mutants was detected by Western Blot. The difference in protein stability was quantitatively analyzed by ImageJ. Consistently, RMY-186 significantly shortened the half-life of CAD. In HEK-293T cells, the half-life of CAD-D1371A was shortened from 13 hours to 8 hours ( Figure 1 j-k), the half-life of CAD-D1371E was shortened from 12 hours to 8 hours, and the half-life of CAD-D1371Y was shortened from 12 hours to 6 hours ( Figure 2 l-o). Therefore, RMY-186 directly promoted the degradation of CAD, regardless of whether it was mutated.

[0083] Example 4 RMY-186 promoting CAD degradation can enhance the chemotherapy effect by promoting GC and CRC cell death

[0084] The effect of RMY-186 on chemotherapy was consistent with its role in promoting the degradation of exogenous CAD. The applicant observed that the endogenous CAD level was significantly reduced in gastric cancer (GC) and colorectal cancer (CRC) cells treated with RMY-186. RMY-186 alone did not promote the death of GC and CRC cells, but when combined with 5-FU, it significantly increased apoptosis. This effect could be reproduced by knocking down CAD in these cells, indicating that accelerating CAD degradation promoted cell death during chemotherapy. These observations were also consistent with the applicant's finding that CAD-mediated de novo pyrimidine synthesis was crucial for the survival of GC and CRC cells, especially in the case of chemotherapy drugs causing pyrimidine nucleotide shortage. Importantly, the applicant found that when RMY-186 was combined with 5-FU or oxaliplatin (Oxa), it could significantly induce apoptosis in MKN45 and HCT116 cells carrying the CAD-D1371A knock-in mutation, while these cells showed resistance to cell death when treated with chemotherapy drugs alone ( Figure 3 a-b and Figure 4a-c; as demonstrated by the phosphorylation levels of c-PARP, p53, and H2A.X. Interestingly, the applicant observed that these chemotherapy-sensitized cells (treated with RMY-186 or knocked down for CAD) exhibited obvious swelling and characteristic large bubbles on the plasma membrane ( Figure 3 c-d and Figure 4 d-f), similar to the occurrence of pyroptosis, rather than just apoptosis. In fact, by immunoblotting, various GC and CRC cell lines analyzed in the present invention (including HGC27, MKN45, HCT116, and SW480) all showed positive for gasdermin-E (GSDME) ( Figure 4 g, where the non-small cell lung cancer (NSCLC) cell line NCI-H1299 was used as a positive control, known to express high levels of GSDME). In addition, the applicant observed that in these chemotherapy-sensitized GC and CRC cells, GSDME was significantly cleaved, resulting in an increase in GSDME-N (a marker of pyroptosis) after 5-FU treatment ( Figure 3 e-f and Figure 4 h-i). In addition, the release of lactate dehydrogenase (LDH) (collect the culture supernatant of MKN45 CADD1371A / D1371A cell line treated with RMY-186 (20 μM) combined with 5-FU (50 μM), and detect the content of lactate dehydrogenase (LDH) using CytoTox 96(R) Non-Radioactive Cytotoxicity Assay Kit. Add 50 μL of the culture supernatant to each well of a 96-well plate, add 50 μL of the LDH reaction working solution (containing substrates such as lactate, INT, NAD + etc.), incubate in the dark at room temperature for 30 minutes, then add 50 μL of the stop solution, measure the absorbance values at 490 nm and 680 nm with an enzyme-labeled instrument for data analysis, and normalize the data with the LDH release of the positive control group (cells lysed with Triton X-100) as 100%) and the propidium iodide (PI) staining signal (stain pyroptotic cells with 50 μg / ml PI, observe the membrane vesicle structure with a fluorescence microscope as another marker of pyroptosis) were also significantly upregulated in 5-FU-treated chemotherapy-sensitized GC and CRC cells ( Figure 3 g-j and Figure 4 j-m). Therefore, the applicant found that promoting CAD degradation can enhance the chemotherapy effect by promoting the death of GC and CRC cells.

[0085] Example 5 Chemosensitization Effect of RMY-186 in Vivo

[0086] RMY-186 exhibited low acute toxicity, with an LD50 of approximately 1391 mg / kg (measured by single intraperitoneal injection, formulated in saline containing 5% DMSO and 1% carboxymethyl cellulose). No signs of toxicity, such as lethargy, weight loss, or other disease symptoms, were observed in mice after daily injection of 500 mg / kg RMY-186 for 7 consecutive days. Additionally, no obvious tissue damage was found during the late stage of RMY-186 administration ( Figure 4 o, after a 14-day washout period). Therefore, the applicant treated CadD1371A / D1371A; Cldn18-ATK mice with RMY-186. The treatment method for mice: dissolve RMY-186 in saline containing 5% DMSO and 1% carboxymethyl cellulose (CMC), and set five dose groups: 2500, 1000, 500, 250, and 100 mg / kg. Randomly assign 8-week-old female C57 / BL6 mice to different concentrations of RMY-186 groups or DMSO control groups (6 mice in each group), record the mortality within 24 hours by intraperitoneal injection, and calculate the median lethal dose (LD50).

[0087] In a gastric cancer mouse model, intraperitoneally co-inject 5-FU (25 mg / kg) and RMY-186 (22 mg / kg) once a week for 4 weeks, calculate the tumor size to evaluate the growth, perform histopathological section staining to determine the malignancy, conduct immunohistochemistry experiments to detect and quantify Ki-67-positive tumor cells, and take gastric tumor tissues for cleaved-PARP, p53, and CAD protein detection.

[0088] The results showed that they became sensitive to 5-FU administration, similar to their wild-type littermates. In particular, 5-FU reduced the tumor size by 70%, while significantly promoting cell death (including apoptosis and pyroptosis) and inhibiting proliferation in the tumor tissue ( Figure 3 k-p). In summary, RMY-186 is expected to become a treatment method to overcome the chemoresistance of GC and CRC caused by CAD cleavage site mutations.

[0089] As described above, it is only a preferred embodiment of the present invention, so the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. Use of compound RMY-186 or a pharmaceutically acceptable salt thereof in the preparation of a drug-resistant tumor drug, wherein the compound RMY-186 is a small molecule compound 2-(4-(3-bromobenzyl)-piperazine-1-yl)-3-hydroxy-4H-chromene-4-one, and its structural formula is as follows:

2. The use according to claim 1, characterized in that: The drug-resistant tumor is a tumor resistant to chemotherapy drugs.

3. The use according to claim 2, characterized in that: The chemotherapy-resistant tumor is a chemotherapy-resistant gastrointestinal tumor.

4. A drug for treating drug-resistant tumors, characterized in that: The invention comprises compound RMY-186 or a pharmaceutically acceptable salt thereof. The compound RMY-186 is a small molecule compound 2-(4-(3-bromobenzyl)-piperazine-1-yl)-3-hydroxy-4H-chromene-4-one, and its structural formula is as follows:

5. The drug according to claim 4, characterized in that: It also includes chemotherapy drugs used in combination.

6. The drug according to claim 5, characterized in that The chemotherapy drug is at least one of fluorouracil and oxaliplatin.

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