Application of MX1 or its expression product as a target in the preparation of ovarian cancer drugs

Abstract

The present invention uses MX1 or its expression product as a target in the preparation of ovarian cancer drugs. Through virtual screening and molecular dynamics simulation technology and in vitro cytological verification, the present invention reveals the MX1 gene associated with ovarian cancer and its use as a potential drug target, providing a new theoretical basis for disease drug discovery.

Description

Technical Field

[0001] The present invention relates to the field of bioinformation technology, and in particular to the use of gene MX1 or its expression product as a target in the preparation of ovarian cancer drugs and the ovarian cancer drugs. Background Art

[0002] Ovarian cancer is a common malignant tumor of the female reproductive system that can occur at any age. Due to its atypical early symptoms, rapid progression, and early pelvic implantation and metastasis, its mortality rate has always ranked first among gynecological tumors, seriously threatening women's lives. Tumor cell reduction surgery combined with platinum-based chemotherapy is the main clinical treatment option. With the improvement of surgical techniques and chemotherapy regimens, the short-term survival rate of ovarian cancer patients has increased significantly. However, its long-term recurrence rate is high and the prognosis remains poor. The extremely high metastatic ability and extremely high drug resistance of ovarian cancer are one of the main causes of death in patients. Therefore, research on ovarian cancer is of great significance to help find drug targets to improve cure rates. Summary of the Invention

[0003] This study aims to use bioinformatics methods to analyze ovarian cancer-related genes and to find closely related compounds through virtual screening technology, thereby exploring potential therapeutic drugs.

[0004] The first invention objective of the present invention is to use the Gly-188 and Glu-227 sites of the gene MX1 or its expression product as targets in the preparation of drugs for the prevention and / or treatment of ovarian cancer.

[0005] Furthermore, the ovarian cancer prevention and / or treatment drug also contains pharmaceutically acceptable excipients. The excipients are adjusted according to the dosage form and application of the drug, and this application does not limit the specific types and amounts of the pharmaceutical excipients contained therein.

[0006] Furthermore, the ovarian cancer drug is used to inhibit the proliferation, migration and invasion of ovarian cancer cells.

[0007] Furthermore, the invention can be used in the preparation of drugs for inhibiting breast proliferation, invasion and / or migration.

[0008] The second invention objective of the present invention is to provide a drug for preventing and / or treating ovarian cancer, wherein the drug is an expression inhibitor of the gene MX1 or its expression product, and the expression inhibitor is selected from the group consisting of:

[0009] .

[0010] The compound binds to the target of MX1 or its expression product; the target is located at the Gly-188 and Glu-227 sites of MX1 or its expression product.

[0011] Furthermore, the drug may contain pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients may be added to the drug for its final application to prepare dosage forms including, but not limited to, tablets, capsules, injections, oral solutions, and pills. Suitable excipients are selected based on the specific formulation. Examples of acceptable excipients include disintegrants and fillers.

[0012] Furthermore, the drug is mainly used to inhibit the proliferation, invasion and / or migration of ovarian cancer cells.

[0013] This study reveals key genes such as MX1 related to ovarian cancer and their potential drug targets by comprehensively applying bioinformatics and virtual screening technology, providing a new theoretical basis for disease drug discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For virtual screening and kinetic simulation cases.

[0015] Figure 2 The inhibition of ovarian cancer cells.

[0016] Figure 3 The expression of MX1 gene protein.

[0017] Figure 4 The results of in vivo anticancer activity experiments are shown.

[0018] Figure 5 For biosafety verification. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0020] 1. Analysis Methods:

[0021] 1. Virtual screening and molecular dynamics simulation

[0022] Druggable pocket discovery: The complete MX1 protein structure was obtained for molecular dynamics simulations. A topology file was generated in gromacs. A simulated water box was constructed, and appropriate sodium and chloride ions were added to balance the charge. The system then underwent energy minimization, NVT temperature equilibration, and NPT pressure equilibration. A 500ns long-range molecular dynamics simulation was then performed. The protein simulation was confirmed to have reached equilibrium by observing the RMSD of the simulated system. Dimensionality reduction of the protein's stable trajectory revealed multiple stable low-energy conformational regions. Thousands of low-energy conformational frames with energy differences of less than 0.2 kcal / mol relative to the lowest energy conformation were extracted for subsequent small molecule binding pocket analysis. Small molecule binding pockets were tracked and discovered using the MDpocket algorithm, and each frame was assigned an algorithmic druggability score. The five highest-scoring conformations were reviewed, and the highest-priority pocket with the best drug-like properties was selected for subsequent docking and small molecule drug screening.

[0023] Large-scale docking screening of small molecule compound libraries: The compounds are first processed by SP high-precision docking to obtain the docking score, and then the 1% compounds with the highest docking scores are extracted. Their docking scores should be less than or equal to -7. The conformational depth search is performed again in Schrodinger through the MacroModel module, and the global molecular stress is calculated and optimized to not exceed 6kcal / mol, so that the compounds interact with the target protein and dock with the target protein in the lowest energy state possible. The ranking results are closer to the physiological experimental environment.

[0024] High-precision molecular dynamics simulation evaluation of compounds: After the high-precision docking evaluation in the previous step, the top three small molecules are evaluated for their MM / GBSA free energy (molecular mechanics / Poisson-Boltzmann generalized Born surface area). Compared to static docking scores, the free energy values ​​(kcal / mol) of the dynamic simulation of the protein-ligand complex can more accurately describe the strength of ligand binding to the target protein, the stability of the simulated system (RMSD), and the key interaction force occupancy (Hbondoccupancy), thereby screening for potent small molecule inhibitors.

[0025] Central nervous system permeability assessment: Thousands of top-screened compounds were evaluated for their central nervous system permeability (CNSMPOscore). CNSMPO is a comprehensive score based on ClogP (oil-water partition coefficient), ClogD (oil-water partition coefficient at neutral pH), MW (molecular weight), HBD (number of hydrogen bond donors), pKa (dissociation constant), and TPSA (polar surface area). It is a continuous score ranging from 0 to 6. Compounds with scores above 5 are considered highly likely to cross the blood-brain barrier, while compounds with scores below 3 are considered unlikely to cross the barrier.

[0026] 2. Mutant protein design and expression

[0027] The mutant proteins were designed and expressed using the gene from Heyuan Biotechnology (Shanghai, China). Mutations were introduced into the pSLenti-CMV-MX1(p.G188A,E227A)-6xHis-PGK-Puro-WPRE vector using overlap extension PCR. Two pairs of overlapping primers were designed, each containing the target mutation site and surrounding sequences. PCR reactions were performed using a high-fidelity DNA polymerase. After restriction digestion and ligation, the reaction products were transformed into Escherichia coli DH5α strains for amplification, ultimately yielding the mutant plasmid DNA. A control plasmid, pSLenti-CMV-MCS-6xHis-PGK-Puro-WPRE, remained unmutated and served as a control. The mutant plasmid pSLenti-CMV-MX1(p.G188A,E227A)-6xHis-PGK-Puro-WPRE and the control plasmid pSLenti-CMV-MCS-6xHis-PGK-Puro-WPRE were transfected into HEK293T cells using polyethylenimine (PEI)-mediated transfection. After transfection, the cells were incubated at 37°C in a 5% CO2 incubator for 48 hours. Western blot analysis was performed to detect target protein expression. Anti-His tag antibodies were used to specifically detect MX1 protein expression, and β-actin was used as a loading control.

[0028] 3. Animal experiments

[0029] Nude mice (Jicui Yaokang, Nanjing) were implanted with human ovarian cancer cell line Caov-3 by subcutaneous injection. 6 After the injection of 100 cells per mouse, the tumor growth of the mice was observed regularly and the experimental data were recorded. At the end of the experiment, the tumor tissue of the mice was taken out and weighed. At the same time, in order to evaluate the therapeutic effect of the compound in vivo, the tumor was grown to 60 mm. 3 Afterwards, the drug was administered via intraperitoneal injection at a concentration of 5 mg / kg and its effect on the tumor was observed. Finally, the corresponding concentrations of the compound were administered via intraperitoneal injection to nude mice and C57 mice, and the changes in the mice's body weight and HE staining of important organs were observed.

[0030] 4. Data Analysis

[0031] Data were normalized to control values ​​and presented as mean ± SD. The chi-square test, Fisher's exact test, and two-tailed Student's t-test were used to compare the day groups. GraphPad Prism 9.0 software facilitated statistical analysis and graphical presentation. A multivariate Cox regression model was used to identify independent prognostic factors. P < 0.05 was considered statistically significant, and specific values ​​are reported in the figure legends or indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0032] 2. Results

[0033] 1. Virtual screening for small molecule inhibitors of MX1

[0034] Dynamics simulation and pocket discovery: After 100ns dynamics simulation, the movement of MX1 protein tends to be stable, and the RMSD fluctuation is less than 0.2nm, which can be considered to have reached a relatively stable conformational state ( Figure 1 A). After dimensionality reduction and potential energy surface analysis of the stabilized protein trajectory, multiple low-energy stable regions were observed. After extracting all frames with a difference of 0.2 kcalmol from the lowest energy point (a total of 2867 frames), the MDpocket algorithm was used to dynamically track the stable potential druggable pocket regions ( Figure 1 B). MDpocket was used to analyze the frequency of pockets in low-energy stable conformations. Pockets with a frequency of 10%, 30%, and 50% were selected to identify stable druggable pockets. Their spatial positions are shown in Figure 2. Figure 1 C) 10% occupied pocket space; D) 30% occupied pocket space; E) 50% occupied pocket space; F) The final focused pocket region space. After reviewing the final top 10 conformation frames, frame-14668 ( Figure 1 H) GTP regions close to magenta.

[0035] Lead Compound MM / GBSA: After high-precision docking and optimization of the docking results, three compounds were screened and the binding free energies of the small molecule-target complexes were analyzed. The analysis results for the three molecules are shown below (Table 1). For early-stage drug lead discovery, the MM / GBSA rule of thumb is that a binding free energy less than -35 kcal / mol is considered excellent, between -25 and -35 kcal / mol is considered good, and greater than -25 kcal / mol is considered poor. Therefore, all three molecules were considered excellent. While the docking scores of -8.95, -8.27, and -8.1 were quite different, the MM / GBSA results indicate that the actual differences between the three molecules are not significant. Furthermore, hydrogen bond stability was continuously monitored during the MD simulation to verify the reliability of the docking results. The docking pose of Ligand1 is as follows. The molecule forms two hydrogen bonds with the protein. The occupancy rates of these two hydrogen bonds in MD are Gly-188 (95%) and Glu-227 (87%). In addition, the hydrogen bond of Ala-228, which was not formed during docking due to the long distance, was found in MD and has an occupancy rate of up to 87%. This indicates that Gly188, Ala-228 and Glu-227 are relatively important hydrogen bonds that determine the interaction force between the ligand and the protein. Figure 1 I).

[0036] Table 1

[0037]

[0038] 2. In vitro experiments confirmed that compound 1 is the best inhibitor of MX1

[0039] In order to verify the inhibitory effect of the three compounds screened out from virtual screening on ovarian cancer cells, in vitro validation was performed in SK-OV-3 and Caov-3 cells. Compound 1 was purchased from ChemDiv (Cat. No. 4296-0995), Compound 4 was purchased from Vitas-M (Cat. No. STK779354), and Compound 6 was purchased from ChemDiv (Cat. No. 8005-1096). The best inhibitor was determined by measuring the IC50 of each compound. The results showed that compound 1 had the most significant inhibitory effect on tumor cells, followed by compound 6, and compound 4 had the worst inhibitory efficiency ( Figure 2 AB). Therefore, in subsequent experiments, we will use compound 1 as the most suitable candidate drug for experiments. WB results showed that in SK-OV-3 and Caov-3 cell lines, the addition of compound 1 significantly inhibited the expression of MX1 compared with the DMSO group (P < 0.05) ( Figure 2 CD). At the same time, compared with the DMSO group, the addition of compound 1 also significantly reduced the clone formation ability, migration and invasion ability of SK-OV-3 and Caov-3 cells (P < 0.05) ( Figure 2EF). The above experiments all confirmed that compound 1 has a good tumor inhibitory effect in vitro.

[0040] 3. In vitro experiments verify the optimal binding site of MX1 and compound 1

[0041] In order to determine the optimal binding site of MX1 and compound 1, the constructed MX1 specific point mutation plasmid and wild-type MX1 overexpression plasmid were transfected into SK-OV-3 and Caov-3 cells. The results showed that in both cells, the expression of MX1 protein in the mutant MX1-MT group was not significantly different from that in the wild-type MX1-WT group, but showed a significant increase in expression relative to the NC group (P < 0.05) ( Figure 3 AB), indicating that the point mutation has no significant effect on the MX1 protein itself. At the same time, after the addition of compound 1, the expression level of MX1 in the wild-type MX1-WT group was significantly reduced compared with the mutant MX1-MT group in both SK-OV-3 and Caov-3 cells (P < 0.05) ( Figure 3 CD). The cell viability of the wild-type MX1-WT group was also significantly lower than that of the mutant MX1-MT group (P<0.05) ( Figure 3 E). At the same time, the addition of compound 1 also significantly reduced the clone formation ability, migration and invasion ability of the MX1-WT group in SK-OV-3 and Caov-3 cells compared with the MX1-MT group (P < 0.05) ( Figure 3 The above experiments all proved that Gly-188 and Glu-227 are the optimal binding sites for MX1 ​​and compound 1.

[0042] 4. In vivo experiments verify the cancer-promoting effect of MX1 and the in vivo anticancer activity of compound 1

[0043] To confirm that MX1 has the same in vitro and in vivo tumor-promoting effects, Caov-3 cells overexpressing MX1 and control cells were subcutaneously injected into nude mice. The subcutaneous tumor volume was measured every two days after tumor formation. The nude mice were sacrificed three weeks later and the tumors were removed and weighed. The results showed that the growth trend and tumor weight of the subcutaneous tumors in the nude mice injected with the overexpressed MX1 group were significantly greater than those in the NC group (P < 0.05). Figure 4 AC), indicating that MX1 also has a carcinogenic effect in vivo. At the same time, in order to determine whether compound 1 also has an anti-cancer effect in vivo, the same wild-type Caov-3 cells were subcutaneously injected into nude mice. After tumor formation, compound 1 was administered intraperitoneally at a dose of 5 mg / kg every two days, and the same dose of DMSO was administered to the WT group. The tumor volume was measured every two days, and the mice were killed after 3 weeks and the tumors were removed and weighed. The results showed that the growth trend and tumor weight of the subcutaneous tumors in nude mice given compound 1 were significantly smaller than those in the WT group (P < 0.05) ( Figure 4DF), indicating that compound 1 also has anticancer activity in vivo.

[0044] 5. In vivo experiments confirmed that compound 1 has good biosafety

[0045] In order to determine the safety of compound 1 as a candidate anticancer drug for normal organisms, nude mice were intraperitoneally injected every two days at a concentration gradient of 5, 10, and 20 mg / kg, and the weight of the four groups of mice was measured every week. The results showed that there was no significant trend in the weight change of mice in the drug-treated group compared with the control group. Subsequently, wild-type C57 mice were treated with the same concentration of 10 mg / kg, and the results were consistent with the experimental conclusions of nude mice. Finally, after the mice were sacrificed, the important organs (heart, liver, spleen, lung, and kidney) in their bodies were removed and fixed, embedded, and sliced ​​for HE staining, which showed that there was no significant difference in the organ structure between the drug-treated group and the normal group ( Figure 5 AC). In summary, it can be shown that compound 1 has good biosafety in vivo as a candidate anticancer drug.

[0046] Pharmaceutically acceptable excipients are added to the end-use drug to be formulated into dosage forms including, but not limited to, tablets, capsules, injections, oral solutions, and pills. Appropriate excipients are selected based on the specific formulation. The drug is primarily used to inhibit the proliferation, invasion, and / or migration of ovarian cancer cells.

Claims

1. Use of an expression inhibitor of gene MX1 or its expression product in the preparation of a drug for the prevention and / or treatment of ovarian cancer, characterized in that: The expression inhibitor is selected from the group consisting of: 。 2. The use according to claim 1, characterized in that: The ovarian cancer therapeutic drug is used to inhibit the proliferation, migration and invasion of ovarian cancer cells.

3. The use according to claim 1, characterized in that: The drug for preventing and / or treating ovarian cancer further contains pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that The compound binds to the target of MX1 or its expression product; the target is located at the Gly-188 and Glu-227 sites of MX1 or its expression product.

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