Uses of deubiquitinating enzyme OTUB1 inhibitors

Abstract

The present invention relates to the field of biopharmaceuticals and discloses the use of inhibitors of the deubiquitinating enzyme OTUB1 (OTUB1). Specifically, it relates to the use of betafentanil or a pharmaceutically acceptable salt thereof in the preparation of inhibitors of the deubiquitinating enzyme OTUB1. This invention proposes for the first time the use of betafentanil as an OTUB1 inhibitor and the use of OTUB1 inhibitors to combat gemcitabine resistance, providing a novel approach for the treatment of gemcitabine-resistant cancers and possessing significant clinical value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of betafenalol or a pharmaceutically acceptable salt thereof in the preparation of a deubiquitinating enzyme (OTUB1) inhibitor, and the purpose of the deubiquitinating enzyme (OTUB1) inhibitor. Background Art

[0002] Gemcitabine (GEM) is a cytosine nucleoside derivative with the chemical formula C9H 11 F2N3O4. Like cytarabine, gemcitabine is activated by deoxycytidine kinase and metabolized by cytidine deaminase upon entry into the human body. Gemcitabine is a pyrimidine antitumor drug with the same mechanism of action as cytarabine. Its primary metabolite is incorporated into DNA intracellularly, acting at the G1 / S phase of the cell cycle. Clinically, gemcitabine and cytarabine have different antitumor spectrums and are both effective against a variety of solid tumors.

[0003] For example, gemcitabine is commonly used in patients with advanced pancreatic cancer, improving their quality of life. It is also used as a first-line treatment for locally advanced (stage III) and metastatic (stage IV) non-small cell lung cancer. Furthermore, studies have shown that gemcitabine has palliative effects in the treatment of 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 a major 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 the deubiquitinating enzyme OTUB1 inhibitor.

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

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

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

[0009] In a fourth aspect, the present invention provides the use of a deubiquitinating enzyme OTUB1 inhibitor in combination with gemcitabine or a gemcitabine salt in the preparation of a medicament for preventing and / or treating tumors, wherein the tumor is gemcitabine-resistant. Preferably, the tumor comprises pancreatic cancer and / or liver cancer.

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

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

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

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

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

[0015] In a seventh aspect, the present invention provides the use of a product for detecting the deubiquitinating enzyme OTUB1 in the preparation of a product for predicting the clinical prognosis of a tumor patient. Preferably, the tumor patient includes a pancreatic cancer patient and / or a liver cancer patient.

[0016] The product for detecting the 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:

[0018] Through research, this application discovered that the deubiquitinating enzyme OTUB1 is a promising therapeutic target for combating gemcitabine resistance in cancer. Abnormal expression of OTUB1 in cancer is significantly associated with low patient survival rate. Moreover, betafentanyl is an effective OTUB1 inhibitor and gemcitabine sensitizer. The combined use of betafentanyl and gemcitabine in cancer treatment may improve the therapeutic effect of cancer.

[0019] Therefore, this application proposes for the first time the use of betafentanyl as an OTUB1 inhibitor, and the use of OTUB1 inhibitors to combat gemcitabine resistance, providing a new approach for the treatment of gemcitabine-resistant cancers and having great clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Statistical graph of OTUB1 mRNA expression levels in normal pancreatic tissue and pancreatic tumor tissue;

[0022] Figure 2 Statistical graph of OTUB1 protein expression levels in normal pancreatic tissue and pancreatic tumor tissue;

[0023] Figure 3 This is a statistical chart of overall survival of pancreatic cancer patients in the OTUB1 high / low expression groups;

[0024] Figure 4 The figure shows the sensitivity of eight human pancreatic cancer cell lines to gemcitabine.

[0025] Figure 5 The figure shows the expression results of OTUB1 in eight human pancreatic cancer cell lines;

[0026] Figure 6 This figure shows the results of knocking out OTUB1 in pancreatic cancer cells using lentivirus;

[0027] Figure 7 This figure shows the changes in the survival rate of pancreatic cancer cells with OTUB1 knockout after gemcitabine treatment;

[0028] Figure 8 This figure shows the changes in apoptosis rate of pancreatic cancer cells with OTUB1 knockout after gemcitabine treatment;

[0029] Figure 9 This figure shows the effect of OTUB1 knockout on gemcitabine sensitivity (tumor volume) in tumor-bearing mice;

[0030] Figure 10 This figure shows the effect of OTUB1 knockout on gemcitabine sensitivity (tumor weight) in tumor-bearing mice;

[0031] Figure 11 This is a diagram showing the changes in gene transcriptome in pancreatic cancer cells after knocking out OTUB1;

[0032] Figure 12 This is the enrichment analysis result of genes whose expression changes in pancreatic cancer cells after knocking out OTUB1;

[0033] Figure 13 This figure shows the effect of OTUB1 knockout on the levels of dCTP (deoxycytidine triphosphate) and dTTP (deoxythymidine triphosphate) in pancreatic cancer cells;

[0034] Figure 14 This figure shows the effect of knocking out OTUB1 on the protein levels of several key enzymes in de novo pyrimidine synthesis in pancreatic cancer cells;

[0035] Figure 15 This figure shows the effect of OTUB1 knockout on DHODH mRNA levels in pancreatic cancer cells;

[0036] Figure 16 The results of mass spectrometry analysis to predict OTUB1 interacting proteins in pancreatic cancer cells;

[0037] Figure 17 This is the result of protein immunoprecipitation experiment;

[0038] Figure 18 This figure shows the effect of OTUB1 knockout on DDX3X protein expression in pancreatic cancer cells;

[0039] Figure 19 This figure shows the effect of OTUB1 knockout on the mRNA level of DDX3X in pancreatic cancer cells;

[0040] Figure 20 This figure shows the effect of DDX3X knockout on DHODH protein expression in pancreatic cancer cells;

[0041] Figure 21 This figure shows the effect of knocking out DDX3X on the mRNA level of DHODH in pancreatic cancer cells;

[0042] Figure 22 The diagram shows the interaction between DDX3X and DHODH mRNA;

[0043] Figure 23 This is the result diagram showing the effect of DDX3X knockout on the decay of DHODH gene mRNA;

[0044] Figure 24 This figure shows the effect of ectopic expression of DDX3X on DHODH expression in the absence of OTUB1;

[0045] Figure 25 This figure shows the effect of OTUB1 knockout on the mRNA level of DDX3X in pancreatic cancer cells;

[0046] Figure 26 This figure shows the effect of OTUB1 deubiquitination-deficient mutants on the half-life of DDX3X protein;

[0047] Figure 27 This is a statistical analysis of the effects of OTUB1 mutants on DDX3X protein degradation in pancreatic cancer cells;

[0048] Figure 28 This figure shows the effect of OTUB1 knockout on the polyubiquitination level of DHODH protein in pancreatic cancer cells;

[0049] Figure 29 This is the result of virtual screening of compounds docking with the OTUB1 protein binding site Cys91;

[0050] Figure 30 The diagram shows the binding pattern of OTUB1 protein and betafentanyl;

[0051] Figure 31 Surface plasmon resonance analysis of the compound betafentalol.

[0052] Figure 32 This is the result of the effect of betafenalol on gemcitabine resistance in pancreatic cancer cells with high OTUB1 expression;

[0053] Figure 33 This is a graph showing the inhibition of betafenalol on the DDX3X-DHODH axis;

[0054] Figure 34 This figure shows the effect of gemcitabine combined with betafentanyl on tumor growth in tumor-bearing mice;

[0055] Figure 35 This is a statistical analysis of the effects of gemcitabine and betafentanyl combined on tumor volume and weight in tumor-bearing mice. DETAILED DESCRIPTION

[0056] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0057] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0058] The present invention is further described in detail below with reference to specific examples. These examples should not be construed as limiting the scope of protection claimed in the present invention.

[0059] This application experimentally discovered 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), which 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 DDX3X-mediated stability of the DHODH gene mRNA. In addition, small molecule OTUB1 inhibitors combined with gemcitabine treatment can synergistically inhibit the growth of pancreatic tumors with high OTUB1 expression. In summary, OTUB1 can confer gemcitabine resistance by promoting pyrimidine de novo synthesis, and targeted inhibition of OTUB1 may be an effective strategy to overcome gemcitabine resistance in cancer. The specific experiments are as follows:

[0060] 1. Experimental Methods

[0061] 1. Patients and tumor specimens:

[0062] All formalin-fixed, rapidly frozen, and paraffin-embedded tumor and adjacent adjacent tissue samples were obtained from pancreatic cancer patients undergoing surgery at the Second Affiliated Hospital of Nanchang University. The Ethics and Research Committee of the Second Affiliated Hospital of Nanchang University approved this study, and all patients provided written informed consent for specimen collection. Table 1 summarizes the clinical characteristics and OTUB1 expression statistics for each pancreatic cancer patient.

[0063] Table 1 Relationship between OTUB1 expression and clinicopathological characteristics of pancreatic cancer patients

[0064]

[0065] 2. Cell culture:

[0066] Human pancreatic cancer (PC) cell lines, including CFPAC-1, PANC-1, AsPC-1, MIA PaCa-2, BXPC-3, HuPT-3, Panc0403, and SU86.86, were purchased from ATCC (Manassas, VA, USA). HEK-293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences. The culture medium used for HEK-293T, MIA PaCa-2, and PANC-1 included DMEM supplemented with 10% FBS. CFPAC-1, HuPT-3, Panc0403, SU86.86, AsPC-1, and BXPC-3 were cultured in RPMI 1640 medium (Gibco, Life Technologies) supplemented with 10% FBS. Furthermore, a gemcitabine (GEM)-resistant PANC-1 / GR cell line was generated by exposing the parental PANC-1 cells, which were sensitive to gemcitabine (GEM), to an initial concentration (0.002 μM) of GEM solution for 1 week. After a recovery period, the GEM concentration was gradually increased from an initial concentration of 0.1 μM over 10 months, allowing the cells to resume normal growth. All cell lines were maintained in a humidified incubator at 37°C and 5% CO2.

[0067] 3. Real-time quantitative PCR (qRT-PCR) analysis:

[0068] Total RNA was isolated from tumor cells and tissues using TRIzol reagent (Invitrogen, USA). The extracted mRNA was converted into cDNA according to the manufacturer's instructions and qRT-PCR was performed using a two-step SYBR Green qRT-PCR kit (Takara, Dalian, China). Gene expression levels were normalized to GAPDH expression levels in each sample. -∆Ct The relative levels of gene expression were calculated using the qRT-PCR method. The primer sequences used for qRT-PCR are shown in Table 2.

[0069] Table 2 Primer sequences used for qRT-PCR

[0070]

[0071] 4. Western blotting experiment:

[0072] Equal volumes of total protein were separated by SDS-PAGE and transferred to PVDF membranes for Western blot analysis. The membranes were treated with 5% skim milk to block nonspecific binding and then treated with primary antibodies and appropriate HRP-conjugated secondary antibodies for subsequent detection by chemiluminescence (Bio-Rad). The following antibodies were used: OTUB1 (1:1000, Abcam, ab270959), DDX3X (1:1000, Abcam, ab196032), DHODH (1:1000), Flag (1:100, Sigma, F1804), Caspase-3 (1:1000; Abcam, ab13847), and cleaved caspase-3.

[0073] 5. Overexpression plasmids and short hairpin RNA (shRNA):

[0074] Overexpression plasmids for producing recombinant lentivirus, including pLVX-Puro-OTUB1, pLVX-Pruro-OTUB1 C91A , pLVX-Puro-DHODH, and pLVX-Pro-DXX3X were constructed by GenePharma (Shanghai, China) and confirmed by direct DNA sequencing. Lentiviral-based shRNAs targeting DDX3X, DHODH, and OTUB1 or interference controls were synthesized by GenePharma (Shanghai, China) and cloned into the pLKO.1-1TRC vector. For stable knockdown, pancreatic cancer cell lines were lentivirally transduced with the corresponding gene shRNA for 24 hours and subsequently cultured in fresh medium containing puromycin for 1 week. After verification by Western blotting and qRT-PCR, selected cells were cultured and used for further experiments.

[0075] 6. Chemotherapy sensitivity test and apoptosis analysis test:

[0076] Chemosensitivity experiments were performed by gradually increasing concentrations of gemcitabine over 48 hours in designated cells. Cell viability was assessed using the CCK-8 assay according to the manufacturer's instructions. Data were analyzed using GraphPad Prism 8 to calculate IC50 values, and dose-response curves were plotted. For apoptosis analysis, gemcitabine was administered to designated cells at varying doses for 36 hours. Apoptosis was assessed using flow cytometry and the Annexin V-FITC / 7-AAD Apoptosis Detection Kit. Apoptotic cells were identified as those stained with both 7AAD and Annexin V, while viable cells were identified as those that did not stain with either substance.

[0077] 7. Deoxyribonucleoside triphosphate (dTTP) determination:

[0078] Deoxyribonucleoside ATP levels were measured as previously described. Briefly, pancreatic cancer cells were cultured in RPMI-1640 medium. To extract metabolites, the medium was aspirated after 24 hours and ice-cold 60% (v / v) methanol was added. The cells were then scraped and centrifuged at 15,000 rpm for 10 minutes at 4°C. The resulting metabolite-containing supernatant was evaporated and resuspended in water for further analysis.

[0079] 8. Cell line-derived tumor xenograft (CDX) and patient-derived tumor xenograft (PDX) models:

[0080] For the CDX model, the firefly luciferase gene was 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) instrument (PerkinElmer, MA, USA). Male BALB / c-nu / nu mice aged 6–8 weeks were injected subcutaneously in the flank with 1 × 10 6 When the tumor volume reaches about 100 mm 3 GEM (50 mg / kg / week) was intraperitoneally injected into mice. Tumor size was measured every five days. For the CDX model, tumor tissue was obtained from PC patients and cut into 3 mm 3 Small pieces of the tumor were implanted subcutaneously into the axilla of 6-week-old female M-NSG mice. 3 Afterwards, the mice were randomly divided into four groups: control group, GEM plus betafentanyl group, betafentanyl (intraperitoneal injection, 20 mg / kg / day) group, and GEM (peritoneal injection, 50 mg / kg / week) group. Before euthanasia, the tumor diameter was measured once a day, and the tumor volume was 1500 mm 3 The tumor tissues of PDX or CDX mouse models were embedded in paraffin and stored in 4% paraformaldehyde for further analysis. All animal experiments were performed in accordance with the protocols approved by the Animal Experimental Ethics Committee and the procedures specified in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0081] 9. Actinomycin D determination test:

[0082] Each well contains approximately 1 × 10 5 Pancreatic cancer cells were plated in 6-well plates and actinomycin D (APExBIO, #A4448) was added at the indicated time intervals. After harvesting the cells, total RNA was isolated and gene expression was assessed using qRT-PCR.

[0083] 10. In vivo ubiquitination assay and co-immunoprecipitation (co-IP) assay:

[0084] For in vivo ubiquitination assays, the indicated expression plasmids or recombinant lentivirus were transfected into HEK-293T or PC cells. After transfection, 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 assays, followed by inoculation with matching primary antibodies and α / G-Sepharose beads. Subsequently, the co-precipitated proteins were collected and analyzed by Western blotting.

[0085] 11. Virtual docking test:

[0086] Virtual docking 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, the compounds were screened using a high-throughput virtual screening program, and the top 3,000 candidates were selected based on their docking scores. Next, these were redocked using standard accuracy and Prime MM-GBSA studies. Finally, 20 compounds were selected and processed for subsequent activity evaluation.

[0087] 12. Surface plasmon resonance (SPR) test:

[0088] SPR detection of inhibitors targeting OTUB1 was performed using a CM5 sensor chip (GE Healthcare, USA) and a Biacore 8K (Cytiva). Recombinant human OTUB1 protein (Abcam, ab157086) was immobilized on the Fc2 channel of the CM5 chip via amino coupling, while the Fc1 channel served as a reference channel for blockade and activation. The protein coupling capacity of recombinant human OTUB1 was typically approximately 13,800 RU. Protein coupling conditions were as follows: sodium acetate solution at pH 4.0, a concentration of approximately 50 μg / mL, a chip activation time of 900 s, and a blocking duration of 420 s. Dissociation and association times of 60 s and a flow rate of 30 μL / min were used.

[0089] 13. Statistical analysis:

[0090] All data are expressed as mean ± standard error using GraphPad Prism 8 (GraphPad Software, USA). Significant differences were analyzed using a two-sided distribution analysis and Student's t-test. Survival curves were calculated using the Kaplan-Meier method, and significance was assessed using the log-rank test. Statistical significance was set at P < 0.05.

[0091] 2. Experimental Results

[0092] 1. OTUB1 gene identified as a predictor of poor prognosis in pancreatic cancer patients

[0093] The experimental results are as follows Figure 1-Figure 3 As shown. Among them, Figure 1 This is a statistical diagram of OTUB1 mRNA expression levels in normal pancreatic tissue and pancreatic tumor tissue. Figure 2 This is a statistical diagram of OTUB1 protein expression levels in normal pancreatic tissue and pancreatic tumor tissue. Figure 3 Statistical chart of overall survival of pancreatic cancer patients in the OTUB1 high / low expression groups.

[0094] To determine whether OTUB1 plays a role in pancreatic cancer tumorigenesis and progression, we first evaluated the expression levels of the OTUB1 gene in 86 pairs of pancreatic cancer samples. When pancreatic cancer tissue was compared with adjacent normal pancreatic tissue, 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 the patient's 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 findings suggest that elevated OTUB1 expression is closely associated with poor clinical prognosis in patients with pancreatic cancer.

[0095] 2. Overexpression of OTUB1 leads to gemcitabine resistance in pancreatic cancer cells

[0096] The experimental results are as follows Figure 4-Figure 8 As shown. Among them, Figure 4 This is a statistical chart showing the sensitivity of eight human pancreatic cancer cell lines to gemcitabine. Figure 5 The expression results of OTUB1 in eight human pancreatic cancer cell lines are shown in Figure 2. Figure 6 This is the result of knocking out OTUB1 in pancreatic cancer cells using lentivirus. Figure 7This is the result of the change in survival rate of pancreatic cancer cells with OTUB1 knockout after gemcitabine treatment. Figure 8 The graph shows the changes in apoptosis rate of pancreatic cancer cells with OTUB1 knockout after gemcitabine treatment.

[0097] To investigate whether OTUB1 is involved in the development of gemcitabine resistance in pancreatic cancer, we first tested 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. 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 In addition, pancreatic cancer cell lines with higher OTUB1 expression were more resistant to gemcitabine ( Figure 5 ), suggesting that OTUB1 may protect pancreatic cancer cells from the cytotoxic effects of gemcitabine.

[0098] To further investigate the functional role of OTUB1 in gemcitabine sensitivity, OTUB1-specific shRNA lentivirus was used to knock down OTUB1 in AsPC-1 cells ( Figure 6 After gemcitabine administration, the survival rate of OTUB1 knockout cells decreased significantly ( Figure 7 ), and enhanced resistance to gemcitabine-induced apoptosis ( Figure 8 These findings suggest that the effects of gemcitabine on pancreatic cancer cells are hindered by OTUB1, thus conferring a survival advantage to pancreatic cancer cells even under gemcitabine treatment.

[0099] 3. In vivo experiments show that OTUB1 inhibits the sensitivity of pancreatic cancer cells to gemcitabine

[0100] The experimental results are as follows Figure 9 and Figure 10 As shown. Among them, Figure 9 This is the result diagram of the effect of OTUB1 knockout on gemcitabine sensitivity (tumor volume) in tumor-bearing mice. Figure 10 The figure shows the effect of OTUB1 knockout on gemcitabine sensitivity (tumor weight) in tumor-bearing mice.

[0101] Mice were subcutaneously implanted with OTUB1-deficient pancreatic cancer cells to verify in vivo the effect of OTUB1 on gemcitabine resistance in pancreatic cancer. The results showed that gemcitabine treatment alone had no significant 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 of the OTUB1-deficient group were significantly reduced ( Figure 9and Figure 10 These results suggest that overexpression of OTUB1 contributes to poor response of pancreatic cancer to gemcitabine treatment and that inhibition of OTUB1 could improve the therapeutic efficacy of pancreatic tumors.

[0102] 4. OTUB1 confers gemcitabine resistance to pancreatic cancer cells by increasing pyrimidine metabolism

[0103] The experimental results are as follows Figure 11-Figure 15 As shown, Figure 11 This is a diagram showing changes in gene transcriptome in pancreatic cancer cells after knocking out OTUB1. Figure 12 This is the enrichment analysis result of genes whose expression changes in pancreatic cancer cells after knocking out OTUB1. Figure 13 This figure shows the effect of knocking out OTUB1 on the levels of dCTP (deoxycytidine triphosphate) and dTTP (deoxythymidine triphosphate) in pancreatic cancer cells. Figure 14 This figure shows the effect of knocking out OTUB1 on the levels of several key enzymes for de novo pyrimidine synthesis in pancreatic cancer cells. Figure 15 This figure shows the effect of OTUB1 knockout on the mRNA level of DHODH in pancreatic cancer cells.

[0104] To explore the mechanism by which OTUB1 regulates gemcitabine chemotherapy resistance in pancreatic cancer cells, RNA-Seq analysis was performed to identify the gene transcriptome changes caused by OTUB1 downregulation in pancreatic cancer cells. Figure 11 As shown in Figure 2, 608 upregulated genes and 576 downregulated genes were identified in gemcitabine-treated AsPC-1 cells after OTUB1 knockout. 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, they 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 changes in several key enzymes involved in de novo pyrimidine synthesis in pancreatic cancer cells, including UMPS, DHODH, and CAD, were detected. The results showed that OTUB1 silencing led to decreased DHODH protein and mRNA levels ( Figure 14 and Figure 15 However, after OTUB1 knockout, the expression levels of UMPS and CAD in pancreatic cancer cells remained unchanged ( Figure 14 Overall, our data suggest that OTUB1 inhibition can effectively suppress de novo pyrimidine synthesis from glucose in pancreatic cancer cells.

[0105] 5. OTUB1 upregulates DHODH expression by interacting with DDX3X

[0106] The experimental results are as follows Figure 16-Figure 25 As shown. Among them, Figure 16 This is the result of mass spectrometry analysis to predict OTUB1 interacting proteins in pancreatic cancer cells. Figure 17 The results of protein immunoprecipitation experiments are shown in Figure 2. Figure 18 This is the result of knocking out OTUB1 on the protein expression level of DDX3X in pancreatic cancer cells. Figure 19 This is the result of knocking out OTUB1 on the mRNA level of DDX3X in pancreatic cancer cells. Figure 20 This is the result of knocking out DDX3X on the protein expression level of DHODH in pancreatic cancer cells. Figure 21 This is the result of knocking out DDX3X on the mRNA level of DHODH in pancreatic cancer cells. Figure 22 The result diagram of the interaction between DDX3X and DHODH mRNA is shown. Figure 23 This is the result diagram of the effect of DDX3X knockout on the decay of DHODH gene mRNA. Figure 24 This is the result diagram showing the effect of ectopic expression of DDX3X on DHODH expression when OTUB1 is deficient. Figure 25 This figure shows the effect of OTUB1 knockout on the mRNA level of DDX3X in pancreatic cancer cells.

[0107] Mass spectrometry analysis was performed using OTUB1 immunoprecipitates from pancreatic cancer cells to investigate the mechanism by which OTUB1 causes DHODH elevation in pancreatic cancer cells. Based on the number of unique peptides identified, DDX3X was identified as a novel binding protein for OTUB1 in pancreatic cancer cells among the top eight OTUB1-interacting proteins ( 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, we found that DDX3X protein levels were significantly reduced in OTUB1-knockout pancreatic cancer cells, 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. Figure 20 and Figure 21 The results showed that the expression of DHODH in DDX3X knockout pancreatic cancer cells was reduced at both protein and mRNA levels. DDX3X is an RNA-binding protein involved in RNA splicing and mRNA degradation, and is crucial for tumorigenesis and chemotherapy resistance in many cancers, including pancreatic cancer. Figure 22As shown, RNA immunoprecipitation analysis demonstrated that DDX3X directly interacts with DHODH mRNA. Furthermore, after treatment with actinomycin D to block gene transcription, knockdown of DDX3X significantly accelerated the decay of DHODH gene mRNA in pancreatic cancer cells ( Figure 23 ).

[0108] To further validate this result, overexpression of DDX3X abolished the effect of OTUB1 deficiency on DHODH downregulation in pancreatic cancer cells ( Figure 24 and Figure 25 These findings indicate that OTUB1-mediated induction of DHODH expression and reprogramming of pyrimidine metabolism in pancreatic cancer cells is dependent on DDX3X.

[0109] 6. OTUB1 stabilizes DDX3X protein expression by inhibiting the K48-linked polyubiquitination level of DDX3X protein

[0110] The experimental results are as follows Figure 26-Figure 28 As shown. Among them, Figure 26 This is the result diagram showing the effect of OTUB1 deubiquitination defective mutant on the half-life of DDX3X protein. Figure 27 This is a statistical analysis of the effect of OTUB1 mutants on DDX3X protein degradation in pancreatic cancer cells. Figure 28 This figure shows the effect of OTUB1 knockout on the polyubiquitination level of DHODH protein in pancreatic cancer cells.

[0111] To explore the mechanism by which OTUB1 regulates DDX3X expression in pancreatic cancer cells, we first investigated the deubiquitination-deficient mutant OTUB1. WT or OTUB1 C91S Effects of cycloheximide (CHX) pulse chase assay on DDX3X expression in pancreatic cancer cells. WT Rather than OTUB1 C91S Ectopic expression of DDX3X inhibited the protein half-life of Figure 26 and Figure 27 ). In addition, OTUB1 WT Overexpression of OTUB1 rather than OTUB1 C91S The polyubiquitin chains of DDX3X were significantly removed both in vitro and in vivo, whereas silencing of OTUB1 increased the polyubiquitination level of DHODH protein ( Figure 28 Overall, our results suggest that OTUB1 promotes DDX3X protein stability by mediating DDX3X deubiquitination.

[0112] 7. Small molecule inhibitors of OTUB1 enhance the sensitivity of pancreatic cancer to gemcitabine treatment

[0113] The experimental results are as follows Figures 29-35 As shown. Among them, Figure 29 This is the result diagram of virtual screening of compounds docking with the OTUB1 protein binding site Cys91. Figure 30 This is the binding pattern diagram of OTUB1 protein and betafentanyl. Figure 31 The results of the surface plasmon resonance analysis of the compound betafentalol were selected. Figure 32 This is the result of the effect of betafenalol on gemcitabine resistance of pancreatic cancer cells in the OTUB1 high expression group. Figure 33 This is the result of betafenalol's inhibition on the DDX3X-DHODH axis. Figure 34 This is the result of the combined use of gemcitabine and betafentanyl on the growth of tumor in tumor-bearing mice. Figure 35 This is a statistical analysis of the effects of gemcitabine and betafentanyl combined on tumor volume and weight in tumor-bearing mice.

[0114] Recent studies have shown that pharmacological inhibition of cancer drivers improves the effectiveness of chemotherapy in a variety of cancer types. To determine this, Cys91, the active site 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, backbone diversity, and docking scores ( Figure 30 Subsequently, five compounds (aloxaline, betafenalol, endomorphin 2, BB-Cl-Amidine, and H-Arg-4MβNA) were further selected using surface plasmon resonance (SPR) analysis. SPR analysis results showed that betafenalol was able to assemble with OTUB1 with a dissociation constant (Kd) of 23.5 μM ( Figure 31 Furthermore, through efficacy assays, betafentanyl was identified as the best compound in pancreatic cancer cells with high OTUB1 levels. Figure 32 and Figure 33 As shown, betafenalol treatment significantly reduced gemcitabine resistance in pancreatic cancer cells ( Figure 32 ) and blocked the activation of the DDX3X-DHODH signaling axis ( Figure 33 ).

[0115] The pharmacological synergistic / antagonistic effects of gemcitabine and betafentanil were evaluated using pancreatic cancer patient-derived tumor xenograft (PDX) models. First, to clinically validate the effect of chemically targeting OTUB1 on gemcitabine response in pancreatic cancer, a subcutaneous PDX model was established based on the high expression of OTUB1 in pancreatic cancer tumor tissue. Then, the efficacy of combined treatment with betafentanil and gemcitabine was evaluated in PDX models with higher OTUB1 levels. Gemcitabine monotherapy had a slight or moderate effect on the development of high OTUB1-expressing PDX tumors; the combination of gemcitabine and betafentanil resulted in significant tumor regression ( Figure 34 and Figure 35 Collectively, these results suggest that benzylpenicillin is identified as a potent OTUB1 inhibitor and gemcitabine sensitizer, and combination therapy with benzylpenicillin and gemcitabine may enhance the efficacy of gemcitabine therapy in pancreatic cancer.

[0116] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a deubiquitinating enzyme OTUB1 inhibitor in the preparation of a synergist for gemcitabine or gemcitabine salts in the treatment of pancreatic cancer, wherein the deubiquitinating enzyme OTUB1 inhibitor is betafentalol or a pharmaceutically acceptable salt thereof.

2. Use of a deubiquitinating enzyme OTUB1 inhibitor in combination with gemcitabine or a gemcitabine salt in the preparation of a medicament for preventing and / or treating pancreatic cancer, wherein the deubiquitinating enzyme OTUB1 inhibitor is betafentalol or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition for preventing and / or treating pancreatic cancer, characterized in that: The pharmaceutical composition comprises a deubiquitinating enzyme OTUB1 inhibitor and gemcitabine or a gemcitabine salt. The deubiquitinating enzyme OTUB1 inhibitor is betafentalol or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that The molar ratio of the deubiquitinating enzyme OTUB1 inhibitor to the gemcitabine or gemcitabine salt is 1:3.

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