Application of deubiquitinating enzyme OTUB1 inhibitor

Abstract

The invention relates to the technical field of biological medicine, discloses application of a deubiquitinating enzyme OTUB1 inhibitor, and particularly relates to application of betfenoterol or pharmaceutically acceptable salt thereof in preparation of the deubiquitinating enzyme OTUB1 inhibitor. According to the application disclosed by the invention, the application of the betfenoterol as the OTUB1 inhibitor and the application of the OTUB1 inhibitor in resisting gemcitabine drug resistance are proposed for the first time, a new thought is provided for treating cancers with gemcitabine drug resistance, and the clinical value is relatively high.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and specifically relates to the use of batefenterol or a pharmaceutically acceptable salt thereof in the preparation of a deubiquitinating enzyme OTUB1 inhibitor, and the use of a deubiquitinating enzyme OTUB1 inhibitor. Background Art

[0002] Gemcitabine is a cytosine nucleoside derivative with the chemical formula C9H 11 F2N3O4. Similar to cytarabine, gemcitabine is activated by deoxycytidine kinase after entering the human body and metabolized by cytosine nucleoside deaminase. Gemcitabine is a pyrimidine antitumor drug with the same mechanism of action as cytarabine. Its main metabolite is incorporated into DNA in cells and acts on the G1 / S phase of cells. Clinically, gemcitabine and cytarabine have different antitumor spectra and are effective against a variety of solid tumors.

[0003] For example, gemcitabine is commonly used in patients with advanced pancreatic cancer and can improve the quality of life of patients; secondly, it is used as a first-line drug for locally advanced (stage III) and metastatic (stage IV) non-small cell lung cancer. In addition, studies have shown that gemcitabine also has palliative effects on tumors such as ovarian cancer, breast cancer, bladder cancer, cervical cancer, liver cancer, biliary tract cancer, nasopharyngeal cancer, testicular tumors, lymphoma, mesothelioma, and head and neck cancer.

[0004] Currently, gemcitabine resistance is the main clinical challenge in the treatment of pancreatic cancer (PC). Therefore, there is an urgent need to explore and find strategies to reverse gemcitabine resistance in pancreatic cancer. Summary of the Invention

[0005] In view of this, the present invention provides the use of batefenterol or a pharmaceutically acceptable salt thereof in the preparation of a deubiquitinating enzyme OTUB1 inhibitor, and the use of a deubiquitinating enzyme OTUB1 inhibitor.

[0006] In the first aspect, the present invention provides the use of batefenterol or a pharmaceutically acceptable salt thereof in the preparation of a deubiquitinating enzyme OTUB1 inhibitor.

[0007] In the second aspect, the present invention provides the use of a deubiquitinating enzyme OTUB1 inhibitor in the preparation of a drug for inhibiting pyrimidine metabolism.

[0008] In the third aspect, the present invention provides the use of a deubiquitinating enzyme OTUB1 inhibitor in the preparation of a synergist for gemcitabine or a gemcitabine salt.

[0009] Fourth aspect, the present invention provides an application of a deubiquitinating enzyme OTUB1 inhibitor in combination with gemcitabine or a gemcitabine salt in the preparation of a drug for preventing and / or treating tumors, wherein the tumor is a tumor with gemcitabine resistance. Preferably, the tumor includes pancreatic cancer and / or liver cancer.

[0010] Fifth aspect, the present invention provides an application of a deubiquitinating enzyme OTUB1 inhibitor in the preparation of a drug for improving the clinical prognosis of tumor patients. Preferably, the tumor includes pancreatic cancer and / or liver cancer.

[0011] Sixth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating tumors, which includes a deubiquitinating enzyme OTUB1 inhibitor and gemcitabine or a gemcitabine salt.

[0012] In an alternative embodiment, the molar ratio of the deubiquitinating enzyme OTUB1 inhibitor to the gemcitabine or the gemcitabine salt is 1:3.

[0013] In an alternative embodiment, the deubiquitinating enzyme OTUB1 inhibitor is Batefenterol or a pharmaceutically acceptable salt thereof.

[0014] In an alternative embodiment, the tumor includes pancreatic cancer and / or liver cancer.

[0015] Seventh aspect, the present invention provides an application of a product for detecting deubiquitinating enzyme OTUB1 in the preparation of a product for predicting the clinical prognosis of tumor patients. Preferably, the tumor patients include pancreatic cancer patients and / or liver cancer patients.

[0016] Wherein, the product for detecting deubiquitinating enzyme OTUB1 may include any reagent or product for detecting the activity and / or expression level of OTUB1.

[0017] The technical solution provided by the present invention has at least the following beneficial effects: Through research, it is found in this application that the deubiquitinating enzyme OTUB1 is a promising therapeutic target against gemcitabine resistance in cancer. OTUB1 is abnormally expressed in cancer and is significantly correlated with a low patient survival rate. Moreover, Batefenterol is an effective OTUB1 inhibitor and gemcitabine sensitizer. Combining Batefenterol and gemcitabine for cancer treatment may improve the treatment effect of cancer.

[0018] Therefore, this application first proposes using Batefenterol as an OTUB1 inhibitor and using an OTUB1 inhibitor to combat gemcitabine resistance, providing a new idea for the treatment of cancers with gemcitabine resistance and having great clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is a statistical chart of the mRNA expression levels of OTUB1 in normal pancreatic tissues and pancreatic tumor tissues; Figure 2 It is a statistical chart of the protein expression levels of OTUB1 in normal pancreatic tissues and pancreatic tumor tissues; Figure 3 It is a statistical chart of the overall survival of pancreatic cancer patients in the OTUB1 high / low expression group; Figure 4 It is a statistical chart of the sensitivity of eight human pancreatic cancer cell lines to gemcitabine; Figure 5 It is a result chart of the expression of OTUB1 in eight human pancreatic cancer cell lines; Figure 6 It is a result chart of knocking out OTUB1 in pancreatic cancer cells using lentivirus; Figure 7 It is a result chart of the change in the survival rate of pancreatic cancer cells with OTUB1 knocked out after treatment with gemcitabine; Figure 8 It is a result chart of the change in the apoptosis rate of pancreatic cancer cells with OTUB1 knocked out after treatment with gemcitabine; Figure 9 It is a result chart of the effect of knocking out OTUB1 on the sensitivity of gemcitabine in tumor-bearing mice (tumor volume); Figure 10 It is a result chart of the effect of knocking out OTUB1 on the sensitivity of gemcitabine in tumor-bearing mice (tumor weight); Figure 11 It is a chart of the changes in the gene transcriptome in pancreatic cancer cells after knocking out OTUB1; Figure 12 It is a result chart of the enrichment analysis of genes with expression changes in pancreatic cancer cells after knocking out OTUB1; Figure 13 It is a result chart of the effect of knocking out OTUB1 on the levels of dCTP (deoxycytidine triphosphate) and dTTP (deoxythymidine triphosphate) in pancreatic cancer cells; Figure 14 It is a result chart of the effect of knocking out OTUB1 on the protein levels of several key enzymes in the de novo synthesis of pyrimidines in pancreatic cancer cells; Figure 15Results of the effect of OTUB1 knockout on the mRNA level of DHODH in pancreatic cancer cells; Figure 16 Results of mass spectrometry analysis predicting the interacting proteins of OTUB1 in pancreatic cancer cells; Figure 17 Results of the protein immunoprecipitation experiment; Figure 18 Results of the effect of OTUB1 knockout on the protein expression level of DDX3X in pancreatic cancer cells; Figure 19 Results of the effect of OTUB1 knockout on the mRNA level of DDX3X in pancreatic cancer cells; Figure 20 Results of the effect of DDX3X knockout on the protein expression level of DHODH in pancreatic cancer cells; Figure 21 Results of the effect of DDX3X knockout on the mRNA level of DHODH in pancreatic cancer cells; Figure 22 Results of the interaction between DDX3X and DHODH mRNA; Figure 23 Results of the effect of DDX3X knockout on the mRNA decay of the DHODH gene; Figure 24 Results of the effect of ectopic expression of DDX3X on the expression of DHODH in the absence of OTUB1; Figure 25 Results of the effect of OTUB1 knockout on the mRNA level of DDX3X in pancreatic cancer cells; Figure 26 Results of the effect of the OTUB1 deubiquitination-deficient mutant on the protein half-life of DDX3X; Figure 27 Results of the statistical analysis of the effect of the OTUB1 mutant on the protein degradation of DDX3X in pancreatic cancer cells; Figure 28 Results of the effect of OTUB1 knockout on the polyubiquitination level of DHODH protein in pancreatic cancer cells; Figure 29 Results of virtual screening for compounds docking with the Cys91 binding site of OTUB1 protein; Figure 30 Results of the binding mode of OTUB1 protein with beclofenac; Figure 31 Results of surface plasmon resonance analysis for selecting the compound beclofenac; Figure 32 Results of the effect of beclofenac on the gemcitabine resistance of pancreatic cancer cells in the OTUB1 high-expression group; Figure 33 Figure showing the inhibitory effect of beclofenac on the DDX3X-DHODH axis; Figure 34 Figure showing the effect of the combined use of gemcitabine and beclofenac on tumor growth in tumor-bearing mice; Figure 35 Figure showing the statistical analysis results of the effect of the combined use of gemcitabine and beclofenac on tumor volume and weight in tumor-bearing mice. Detailed implementation manners

[0021] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0022] For those not specifying specific experimental steps or conditions in the embodiments, operations or conditions of conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0023] The following further describes the present invention in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed by the present invention.

[0024] This application found through experiments that knocking out OTUB1 can inhibit pyrimidine metabolism in vivo and in vitro, thereby improving the efficacy of gemcitabine in cancer cells. In addition, OTUB1 enhances de novo nucleotide pyrimidine synthesis in cancer cells by upregulating dihydroorotate dehydrogenase (DHODH), and DHODH is a key rate-limiting enzyme in de novo pyrimidine biosynthesis. Mechanistically, OTUB1 inhibits the degradation and polyubiquitination of the RNA-binding protein DEAD-box helicase 3X-linked (DDX3X), thereby enhancing the stability of DDX3X-mediated DHODH gene mRNA. In addition, treatment with a small molecule OTUB1 inhibitor combined with gemcitabine can synergistically inhibit the growth of pancreatic tumors with high OTUB1 expression. Generally speaking, OTUB1 can confer gemcitabine resistance by promoting de novo pyrimidine synthesis, and targeted inhibition of OTUB1 may be an effective strategy to overcome gemcitabine resistance in cancer. The specific experiments are as follows: I. Experimental methods 1. Patients and tumor specimens: All formalin-fixed, snap-frozen, and paraffin-embedded tumor and adjacent cancer tissue samples were obtained from pancreatic cancer patients who underwent surgery at the Second Affiliated Hospital of Nanchang University. The research process was authorized by the Ethics and Research Committee of the Second Affiliated Hospital of Nanchang University, and all patients provided written informed consent for specimen collection. Table 1 summarizes the clinical characteristics of each pancreatic cancer patient and the statistical data of OTUB1 expression.

[0025] Table 1 Relationship between OTUB1 expression and clinicopathological characteristics of pancreatic cancer patients

[0026]

[0027] 2. Cell culture: Human pancreatic cancer (PC) cell lines, including CFPAC-1, PANC-1, AsPC-1, MIAPaCa-2, BXPC-3, HuPT-3, Panc0403, and SU86.86 were purchased from ATCC (Manassas, VA, USA), and HEK-293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences. The culture media for HEK-293T, MIA PaCa-2, and PANC-1 included 10% FBS in addition to DMEM. CFPAC-1, HuPT-3, Panc0403, SU86.86, AsPC-1, and BXPC-3 were cultured in RPMI 1640 medium (Gibco, Life Technologies) containing 10% FBS. In addition, a gemcitabine (GEM)-resistant PANC-1 / GR cell line was generated by exposing the GEM-sensitive PANC-1 parental cells to an initial concentration (0.002 μM) of GEM solution for 1 week. When the cells resumed normal growth after the recovery period, the GEM concentration was gradually increased from the initial concentration of 0.1 μM over 10 months. All cell lines were maintained in a humidified incubator at 37 °C and 5% CO2.

[0028] 3. Real-time quantitative PCR (qRT-PCR) analysis: Total RNA was isolated from tumor cells and tissues using TRIzol reagent (Invitrogen, USA). According to the manufacturer's instructions, the extracted mRNA was converted into cDNA, and qRT-PCR was performed using a two-step SYBR Green qRT-PCR kit (Takara, Dalian, China). The gene expression levels were normalized to the GAPDH expression level in each sample. The relative levels of gene expression were calculated using the 2 -∆Ct -ΔΔCt method. The primer sequences used for qRT-PCR are shown in Table 2.

[0029] Table 2 Primer sequences for qRT-PCR

[0030] 4. Western blotting assay: Equal volumes of total proteins were separated by SDS-PAGE and transferred onto PVDF membranes for Western blotting analysis. The membranes were treated with 5% non-fat milk to block non-specific binding and then treated with the primary antibody and the appropriate HRP-conjugated secondary antibody for subsequent detection by chemiluminescence (Bio-Rad). The following antibodies were applied: against OTUB1 (1:1000, Abcam, ab270959), DDX3X (1:1000, Abcam, ab196032), DHODH (1:1000), Flag (1:100, Sigma, F1804), Caspase-3 (1:1000; Abcam, ab13847), cleaved caspase-3.

[0031] 5. Overexpression plasmids and short hairpin RNAs (shRNAs): Overexpression plasmids for generating recombinant lentiviruses included pLVX-Puro-OTUB1, pLVX-Pruro-OTUB1 C91A , pLVX-Puro-DHODH, and pLVX-Pro-DXX3X, which were constructed by GenePharma (Shanghai, China) and confirmed by direct DNA sequencing. GenePharma (Shanghai, China) synthesized lentivirus-based shRNAs against DDX3X, DHODH, and OTUB1 or an interference control and cloned them into the pLKO.1-1TRC vector. For stable knockdown, pancreatic cancer cell lines were transfected with the corresponding gene shRNA lentiviruses for 24 hours and then cultured in fresh medium containing puromycin for 1 week. After verification by Western blotting and qRT-PCR, the selected cells were cultured and used for further experiments.

[0032] 6. Chemosensitivity test and apoptosis analysis test: The chemosensitivity experiment was performed by gradually increasing the concentration of gemcitabine in specific cells for 48 hours. The cell viability was examined using the CCK-8 assay according to the manufacturer's instructions. To calculate the IC50 value, the data were analyzed using GraphPad Prism 8 and a dose-response curve was plotted. For apoptosis analysis, gemcitabine was administered to the designated cells at different doses for 36 hours. Cell apoptosis was detected using a flow cytometer and an Annexin V-FITC / 7-AAD apoptosis detection kit. Apoptotic cells were those stained with both 7AAD and Annexin V, while live cells were those not stained with either of these two substances.

[0033] 7. Determination of deoxythymidine triphosphate (dTTP): Measure the level of deoxythymidine triphosphate as described above. Briefly, pancreatic cancer cells are cultured in RPMI-1640 medium. To extract metabolites, the medium is aspirated after 24 hours, and ice-cold 60% (v / v) methanol is added. Subsequently, the cells are scraped off and centrifuged at 15,000 rpm for 10 minutes at 4 °C. The resulting metabolite-containing supernatant is evaporated and resuspended in water for further analysis.

[0034] 8. Cell line-derived tumor xenograft model (CDX) and patient-derived tumor xenograft (PDX) models: For the CDX model, the firefly luciferase gene is stably transduced into pancreatic cancer cells for injection into the CDX animal model, allowing for frequent monitoring of tumor growth by bioluminescence imaging using a Lumina Series III IVIS (in vivo imaging system) device (PerkinElmer, Massachusetts, USA). Six- to eight-week-old male BALB / c-nu / nu mice are subcutaneously injected in the flank with 1×10 6 cells. When the tumor volume reaches approximately 100 mm 3 , GEM (50 mg / kg / week) is intraperitoneally injected into the mice. The tumor size is measured every five days. For the CDX model, tumor tissues are obtained from PC patients, cut into small pieces of 3 mm 3 , and subcutaneously implanted into the axilla of 6-week-old female M-NSG mice. After the tumor volume reaches 150 mm 3 , the mice are randomly divided into four groups, namely the control group, the GEM plus betafarnesol group, the betafarnesol (intraperitoneal injection, 20 mg / kg / day) group, and the GEM (intraperitoneal injection, 50 mg / kg / week) group. Before euthanasia, the tumor diameter is measured daily, and a tumor volume of 1500 mm 3 is considered to indicate death. In addition, the tumor tissues of the PDX or CDX mouse models are embedded in paraffin and stored in 4% paraformaldehyde for further analysis. All animal experiments are conducted in accordance with the protocols approved by the Animal Experiment Ethics Committee and the procedures specified in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States.

[0035] 9. Actinomycin D assay: Prepare a 6-well plate containing approximately 1×10 5 pancreatic cancer cells per well, and add actinomycin D (APExBIO, #A4448) at specified time intervals. After harvesting the cells, total RNA is isolated using qRT-PCR to evaluate gene expression.

[0036] 10. In vivo ubiquitination assay and co-immunoprecipitation (co-IP) assay: For the in vivo ubiquitination assay, the designated expression plasmid or recombinant lentivirus was transfected into HEK-293T or PC cells. After transfection, the cells were exposed to the proteasome inhibitor MG132 for 6 hours, and the following steps were performed as previously described. Cell lysates were extracted from the indicated cells for co-IP assay, and then the matching primary antibody and a / G-Sepharose protein beads were inoculated. Subsequently, the co-precipitated proteins were collected and assayed by Western blotting.

[0037] 11. Docking simulation: Docking simulation was performed using Schrödinger Suite 2021-2 (Schrödinger, LLC). Briefly, (1) the OTUB structural model (PDB: 4DDI) was processed using the default settings in the Protein Preparation Wizard; (2) the active site Cys91 was identified as the binding region for small molecule compound screening using the Receptor Grid Generation Wizard; (3) the compounds used for this screening were from the MCE Bioactive Compound Library (HY-L001V, a total of 23,562 compounds), and the small molecule structures were generated and optimized using the OPLS4 force field of the LigPrep program; (4) molecular docking was performed using the Glide program through a three-step screening process. First, a high-throughput virtual screening program was used to screen the compounds, and the top 3000 candidates were selected based on their docking scores. Next, these were redocked using the standard accuracy and Prime MM-GBSA studies. Finally, 20 compounds were selected and processed for subsequent activity evaluation.

[0038] 12. Surface plasmon resonance (SPR) assay: SPR detection of inhibitors targeting OTUB1 was performed using a sensor chip CM5 (GE Healthcare, USA) and a Biacore 8 K (Cytiva). Recombinant human OTUB1 protein (Abcam, ab157086) was immobilized on the Fc2 channel by the CM5 chip amine coupling method, while the Fc1 channel served as a reference channel for blocking and activation. The protein coupling capacity of recombinant human OTUB1 was typically about 13,800 RU. The following were the conditions for protein coupling: the system was a sodium acetate solution at pH 4.0, with a concentration of about 50 μg / mL, the chip activation time was 900 s, and the blocking duration was 420 s. A dissociation and binding time of 60 s and a flow rate of 30 μL / min were used.

[0039] 13. Statistical analysis: All data were expressed as mean ± standard error using GraphPad Prism 8 (GraphPad Software, USA). Two-tailed distribution and Student's t-test were used to analyze significant differences. Survival curves were calculated using the Kaplan-Meier method, and significance was evaluated using the log-rank test. Statistical significance was set at P < 0.05.

[0040] II. Experimental Results 1. The OTUB1 gene was identified as a predictor of poor prognosis in pancreatic cancer patients The experimental results are as Figures 1 - 3 shown. Among them, Figure 1 is a statistical chart of the mRNA expression levels of OTUB1 in normal pancreatic tissues and pancreatic tumor tissues, Figure 2 is a statistical chart of the protein expression levels of OTUB1 in normal pancreatic tissues and pancreatic tumor tissues, Figure 3 is a statistical chart of the overall survival of pancreatic cancer patients in the OTUB1 high / low expression group.

[0041] To clarify whether OTUB1 plays a role in the tumorigenesis and development of pancreatic cancer, the expression levels of the OTUB1 gene were first evaluated in 86 pairs of pancreatic cancer samples. When pancreatic cancer tissues were compared with adjacent normal pancreatic tissues, the mRNA and protein levels of the OTUB1 gene were significantly increased ( Figure 1 and Figure 2 ). Next, the relationship between the clinical characteristics of pancreatic cancer patients and the OTUB1 expression pattern was evaluated. The data showed that high OTUB1 levels were positively correlated with patient tumor stage (P = 0.0027) and tumor size (P = 0.0213) (Table 1). Kaplan-Meier analysis showed that, consistent with the results of the TCGA database, the overall survival (OS) of pancreatic cancer patients with high OTUB1 expression was significantly shorter than that of patients with low OTUB1 expression ( Figure 3 ). Overall, the research results indicate that elevated OTUB1 expression is closely associated with poor clinical prognosis in pancreatic cancer patients.

[0042] 2. High expression of OTUB1 leads to gemcitabine resistance in pancreatic cancer cells The experimental results are as Figures 4 - 8 shown. Among them, Figure 4 is a statistical chart of the sensitivity of eight human pancreatic cancer cell lines to gemcitabine, Figure 5 is a result chart of the expression of OTUB1 in eight human pancreatic cancer cell lines, Figure 6 is a result chart of knocking out OTUB1 in pancreatic cancer cells using lentivirus, Figure 7 is a result chart of the change in the survival rate of pancreatic cancer cells with knocked-out OTUB1 after gemcitabine treatment, Figure 8Results of the change in the apoptosis rate of pancreatic cancer cells with OTUB1 knocked out after gemcitabine treatment.

[0043] To investigate whether OTUB1 is involved in the development of gemcitabine resistance in pancreatic cancer, the sensitivity of eight human pancreatic cancer cell lines (i.e., AsPC-1, PANC-1, CFPAC-1, HuPT-3, BxPC-3, MIA PaCa-2, SU86.86, and Panc0403) to gemcitabine was first detected. CCK-8 assay data showed that AsPC-1 and PANC-1 cells were the most resistant to gemcitabine, while SU86.86 and MIA PaCa-2 cells were the most sensitive to gemcitabine ( Figure 4 ). Additionally, pancreatic cancer cell lines with higher OTUB1 expression were more resistant to gemcitabine ( Figure 5 ), indicating that OTUB1 may protect pancreatic cancer cells from the cytotoxic effects of gemcitabine.

[0044] To further investigate the functional role of OTUB1 in gemcitabine sensitivity, OTUB1 was knocked out in AsPC-1 cells using OTUB1-specific shRNA lentivirus ( Figure 6 ). After gemcitabine administration, there was a significant decrease in the survival rate of OTUB1-knockout cells ( Figure 7 ), and an enhanced resistance to gemcitabine-induced apoptosis ( Figure 8 ). These findings suggest that the effect of gemcitabine on pancreatic cancer cells is hindered by OTUB1, and thus pancreatic cancer cells have a survival advantage even under gemcitabine treatment.

[0045] 3. In vivo experiments show that OTUB1 hinders the sensitivity of pancreatic cancer cells to gemcitabine The experimental results are shown in Figure 9 and Figure 10 . Among them, Figure 9 is the result graph of the effect of knocking out OTUB1 on the sensitivity of gemcitabine in tumor-bearing mice (tumor volume), Figure 10 is the result graph of the effect of knocking out OTUB1 on the sensitivity of gemcitabine in tumor-bearing mice (tumor weight).

[0046] OTUB1-deficient pancreatic cancer cells were subcutaneously implanted into mice to verify the effect of OTUB1 on gemcitabine resistance in pancreatic cancer in vivo. The results showed that gemcitabine treatment alone had no obvious effect on pancreatic tumor growth, while in the OTUB1-deficient group without gemcitabine treatment, tumor growth was slightly slower; under gemcitabine treatment, the tumor weight and volume in the OTUB1-deficient group were significantly reduced ( Figure 9 and Figure 10). These results indicate that high expression of OTUB1 leads to poor response of pancreatic cancer to gemcitabine treatment, and inhibition of OTUB1 can improve the therapeutic effect of pancreatic tumors.

[0047] 4. OTUB1 confers gemcitabine resistance to pancreatic cancer cells by increasing pyrimidine metabolism The experimental results are as Figures 11 - 15 shown, where Figure 11 is the gene transcriptome change map in pancreatic cancer cells after knocking out OTUB1, Figure 12 is the enrichment analysis result map of genes with changed expression in pancreatic cancer cells after knocking out OTUB1, Figure 13 is the result map of the effect of knocking out OTUB1 on the levels of dCTP (deoxycytidine triphosphate) and dTTP (deoxythymidine triphosphate) in pancreatic cancer cells, Figure 14 is the result map of the effect of knocking out OTUB1 on the protein levels of several key enzymes in de novo pyrimidine synthesis in pancreatic cancer cells, Figure 15 is the result map of the effect of knocking out OTUB1 on the mRNA level of DHODH in pancreatic cancer cells.

[0048] To explore the mechanism by which OTUB1 regulates gemcitabine chemoresistance in pancreatic cancer cells, RNA-Seq analysis was performed to determine the gene transcriptome changes caused by downregulation of OTUB1 in pancreatic cancer cells. As Figure 11 shown, after knocking out OTUB1, 608 upregulated genes and 576 downregulated genes were identified in gemcitabine-treated AsPC-1 cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed a strong correlation between OTUB1 knockout and the pyrimidine metabolism pathway ( Figure 12 ). Next, using a sensitive fluorescence-based detection method, it was found that OTUB1 silencing significantly reduced the levels of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) in pancreatic cancer cells ( Figure 13 ). Finally, the expression of OTUB1 in pancreatic cancer cells was inhibited, and the changes in several key enzymes involved in de novo pyrimidine synthesis in pancreatic cancer cells were detected, including UMPS, DHODH, and CAD. It was found that OTUB1 silencing led to a decrease in the protein and mRNA levels of DHODH ( Figure 14 and Figure 15 ). However, after knocking out OTUB1, the expression levels of UMPS and CAD in pancreatic cancer cells remained unchanged ( Figure 14 ). Overall, the data indicate that OTUB1 inhibition can effectively inhibit de novo pyrimidine synthesis from glucose in pancreatic cancer cells.

[0049] 5. OTUB1 upregulates DHODH expression by interacting with DDX3X The experimental results are as Figures 16 - 25As shown in the figure. Among them, Figure 16 is the result graph of mass spectrometry analysis predicting the interacting proteins of OTUB1 in pancreatic cancer cells, Figure 17 is the result graph of the protein immunoprecipitation experiment, Figure 18 is the result graph of the effect of knocking out OTUB1 on the protein expression level of DDX3X in pancreatic cancer cells, Figure 19 is the result graph of the effect of knocking out OTUB1 on the mRNA level of DDX3X in pancreatic cancer cells, Figure 20 is the result graph of the effect of knocking out DDX3X on the protein expression level of DHODH in pancreatic cancer cells, Figure 21 is the result graph of the effect of knocking out DDX3X on the mRNA level of DHODH in pancreatic cancer cells, Figure 22 is the result graph of the interaction between DDX3X and DHODH mRNA, Figure 23 is the result graph of the effect of knocking out DDX3X on the mRNA decay of the DHODH gene, Figure 24 is the result graph of the effect of ectopic expression of DDX3X on the expression of DHODH in the absence of OTUB1, Figure 25 is the result graph of the effect of knocking out OTUB1 on the mRNA level of DDX3X in pancreatic cancer cells.

[0050] Mass spectrometry analysis was performed using the OTUB1 immunoprecipitates of pancreatic cancer cells to study the mechanism by which OTUB1 causes the increase of DHODH in pancreatic cancer cells. According to the number of identified unique peptides, among the top eight OTUB1 interacting proteins, DDX3X is a new binding protein of OTUB1 in pancreatic cancer cells ( Figure 16 ). Co-IP analysis showed that there was an interaction between endogenous or exogenous DDX3X and OTUB1 ( Figure 17 ). Next, to determine whether OTUB1 regulates DDX3X expression, in OTUB1-knockout pancreatic cancer cells, the protein level of DDX3X was significantly reduced, but not at the mRNA level ( Figure 18 and Figure 19 ). At the same time, the correlation between DDX3X and DHODH in pancreatic cancer cells was detected. As shown in Figure 20 and Figure 21 results, the expression of DHODH in DDX3X-knockout pancreatic cancer cells was reduced at both the protein and mRNA levels. DDX3X is an RNA-binding protein that participates in RNA splicing and mRNA degradation and is crucial for tumorigenesis and chemoresistance in many cancers including pancreatic cancer. As shown in Figure 22 shown, RNA immunoprecipitation analysis showed that DDX3X directly interacted with DHODH mRNA. In addition, after treatment with actinomycin D to block gene transcription, the knockout of DDX3X significantly accelerated the mRNA decay of the DHODH gene in pancreatic cancer cells (Figure 23 ).

[0051] To further verify this result, overexpression of DDX3X eliminated the effect of OTUB1 deficiency on the downregulation of DHODH in pancreatic cancer cells ( Figure 24 and Figure 25 ). These findings indicate that in pancreatic cancer cells, the induction of DHODH expression and the reprogramming of pyrimidine metabolism mediated by OTUB1 depend on DDX3X.

[0052] 6. OTUB1 stabilizes DDX3X protein expression by inhibiting the K48-linked polyubiquitination level of DDX3X protein The experimental results are as Figures 26 - 28 shown. Among them, Figure 26 is the result graph of the effect of the OTUB1 deubiquitination-deficient mutant on the half-life of the DDX3X protein, Figure 27 is the statistical analysis result graph of the effect of the OTUB1 mutant on the degradation of the DDX3X protein in pancreatic cancer cells, Figure 28 is the result graph of the effect of knocking out OTUB1 on the polyubiquitination level of the DHODH protein in pancreatic cancer cells.

[0053] To explore the mechanism by which OTUB1 regulates DDX3X expression in pancreatic cancer cells. First, the deubiquitination-deficient mutant OTUB1 WT or OTUB1 C91S was studied for its effect on DDX3X expression in pancreatic cancer cells. The results showed that in the cycloheximide (CHX) pulse-chase assay, the ectopic expression of OTUB1 WT but not OTUB1 C91S inhibited the protein half-life of DDX3X ( Figure 26 and Figure 27 ). In addition, overexpression of OTUB1 WT but not OTUB1 C91S significantly removed the polyubiquitin chains of DDX3X both in vivo and in vitro, while silencing of OTUB1 increased the polyubiquitination level of the DHODH protein ( Figure 28 ). Overall, the research results indicate that OTUB1 promotes the stability of DDX3X protein by mediating DDX3X deubiquitination.

[0054] 7. Small molecule inhibitors of OTUB1 increase the sensitivity of pancreatic cancer to gemcitabine treatment The experimental results are as Figures 29 - 35 shown. Among them, Figure 29 is the result graph of virtual screening for compounds docked to the Cys91 binding site of the OTUB1 protein, Figure 30 is the binding mode graph of the OTUB1 protein and beclofenac, Figure 31Result graph of selecting compound beclofenac for surface plasmon resonance analysis Figure 32 Result graph of the effect of beclofenac on the gemcitabine resistance of pancreatic cancer cells in the OTUB1 high-expression group Figure 33 Result graph of the inhibition of beclofenac on the DDX3X-DHODH axis Figure 34 Result graph of the effect of the combination of gemcitabine and beclofenac on tumor growth in tumor-bearing mice Figure 35 Statistical analysis result graph of the effect of the combination of gemcitabine and beclofenac on tumor volume and weight in tumor-bearing mice

[0055] Recent studies have shown that pharmacological inhibition of cancer drivers improves the efficacy of chemotherapy in multiple cancer types. To determine this, the active site Cys91 of the OTUB1 protein was identified as an inhibitor-binding pocket for small molecule compound screening ( Figure 29 ). From the MCE bioactive chemical library, including 23,562 compounds using virtual screening, 25 possible compounds were selected based on their molecular weight, scaffold diversity, and docking scores ( Figure 30 ). Subsequently, 5 compounds (afloqualone, beclofenac, endomorphin-2, BB-Cl-Amidine, and H-Arg-4MβNA) were further selected using surface plasmon resonance (SPR) analysis. The SPR analysis results showed that beclofenac could assemble with OTUB1, and the dissociation constant (Kd) was 23.5 μM ( Figure 31 ). In addition, through efficacy determination, beclofenac was identified as the best compound in pancreatic cancer cells with a higher OTUB1 level. As shown in Figure 32 and Figure 33 , beclofenac treatment significantly reduced gemcitabine resistance in pancreatic cancer cells ( Figure 32 ) and blocked the activation of the DDX3X-DHODH signaling axis ( Figure 33 ).

[0056] The pharmacological synergy / antagonism of gemcitabine and beclofenac was evaluated using a patient-derived tumor xenograft (PDX) model of pancreatic cancer. First, to clinically verify the effect of chemical targeting of OTUB1 on the response to gemcitabine in pancreatic cancer, a subcutaneous PDX model was established based on the high expression of OTUB1 in pancreatic cancer tumor tissues. Then, the efficacy of the combination treatment of beclofenac and gemcitabine was evaluated in the PDX model with a higher OTUB1 level. Gemcitabine monotherapy had a mild or moderate effect on the development of PDX tumors with high OTUB1 expression; the combination of gemcitabine and beclofenac led to significant tumor regression ( Figure 34 and Figure 35). Generally speaking, these results indicate that betefenalol has been identified as an effective OTUB1 inhibitor and gemcitabine sensitizer, and the combination treatment of betefenalol and gemcitabine may improve the therapeutic effect of gemcitabine in pancreatic cancer.

[0057] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of betefenalol or a pharmaceutically acceptable salt thereof in the preparation of a deubiquitinating enzyme OTUB1 inhibitor.

2. Use of a deubiquitinating enzyme OTUB1 inhibitor in the preparation of a drug for inhibiting pyrimidine metabolism.

3. Use of a deubiquitinating enzyme OTUB1 inhibitor in the preparation of a synergist for gemcitabine or a gemcitabine salt.

4. Use of a deubiquitinating enzyme OTUB1 inhibitor in combination with gemcitabine or a gemcitabine salt in the preparation of a drug for preventing and / or treating tumors, wherein, The tumor is a tumor with gemcitabine resistance.

5. Use of a deubiquitinating enzyme OTUB1 inhibitor in the preparation of a drug for improving the clinical prognosis of tumor patients.

6. A pharmaceutical composition for preventing and / or treating tumors, characterized in that, The pharmaceutical composition comprises a deubiquitinating enzyme OTUB1 inhibitor and gemcitabine or a gemcitabine salt.

7. The pharmaceutical composition according to claim 6, characterized in that, The molar ratio of the deubiquitinating enzyme OTUB1 inhibitor to the gemcitabine or gemcitabine salt is 1:

3.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that, The deubiquitinating enzyme OTUB1 inhibitor is betefenalol or a pharmaceutically acceptable salt thereof.

9. The pharmaceutical composition according to claim 6 or 7, characterized in that, The tumor includes pancreatic cancer and / or liver cancer.

10. Use of a product for detecting deubiquitinating enzyme OTUB1 in the preparation of a product for predicting the clinical prognosis of tumor patients.

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