Screening And Validation Of Small Molecule Inhibitors Against Lung Cancer Targeting CYTH2

Along with the development of industrialization,urbanization and pollution of the environment,Lung cancer is the most common and deadliest cancer in hospitals.Lung cancer countermeasures are attracting attention around the world.Non-small cell lung cancer accounts for about 80%of all lung cancers.The most common types of non-small cell lung cancer are squamous cell carcinoma,large cell carcinoma,and adenocarcinoma.The expenditure on cancer drugs in the United States reached 71 billion US dollars in2020,and the number and speed of research and development of anti tumor drugs in China is far lower than that in Europe and the United States.Molecularly targeted drug therapy has gradually become the main method of cancer treatment,and the application of targeted drugs has broad prospects.Cytophysin-2(CYTH2)is widely expressed in organisms and is very conservative.It is mainly involved in the transformation between GTP and GDP in ADP ribosylation factor(ARF),and plays a role in the signaling of cancer cell migration,apoptosis,proliferation,differentiation,and immune response.CYTH2 mediates EGFR and IGF-1R signaling pathways to promote the migration,proliferation and inhibit apoptosis of lung cancer cells.CYTH2 is a prospective target of cancerβIntegrin is often expressed in malignant tumors and participates in cell migration,proliferation,tumor angiogenesis and other functions.Integrin 1 recycling is regulated by CYTH2.CYTH2 knockout can reduce the adhesion,diffusion and migration of lung cancer cells.CYTH2 is a potential target of cancer.In this paper,we searched for inhibitors of CYTH2 protein based on the crystal structure of CYTH2protein through computer virtual screening technology,and achieved the following results:1.CYTH2 target is a key target in the development and progression of A549 lung cancer cellsDesign and purchase small RNA,and relevant tests verify that small RNA interference CYTH2 can inhibit the proliferation,migration,and promote apoptosis of A549 lung cancer cells.Construction of pc DNA3.0(+)-CYTH2 eukaryotic expression vector.Overexpression of CYTH2 can promote the proliferation and migration of A549lung cancer cells.The effects of si RNA interference with CYTH2 and overexpression of CYTH2 on its upstream and downstream pathways were verified by fluorescence quantitative PCR:si RNA interference with CYTH2 can significantly inhibit the transcription level of CYTH2 upstream and downstream related signal pathways MAP2K3,IGF-1R,IRS and AKT genes.Overexpression of CYTH2 can significantly up-regulate the transcriptional level of CYTH2 gene,and promote the transcriptional level of IGF-1R,IRS,AKT genes in its upstream and downstream pathways.2.In vitro and in vivo pharmacodynamic study of oleuropein and icariin I on A549cells based on CYTH2Molecular docking technology found two small molecular compounds,oleuropein and icariin I,and the CCK8 test looked for subsequent experimental concentrations of two compounds on A549 lung cancer cells.Using scratch test,cell colony test,Ed U test,and flow cytometry to detect apoptosis,its effect on A549 non small cell lung cancer cells was verified.It was found that oleuropei,icariin I 5,10μM can significantly inhibit the proliferation,migration,and promote apoptosis of A549 lung cancer cells,,and its anti lung cancer effect is better than that of the known CYTH2 target inhibitor Secin H3,which is equivalent to cisplatin,an anticancer drug often used in clinic.The subcutaneous tumorigenesis test in nude mice in vivo verified that oleuropein(20 mg/kg)could significantly inhibit the growth rate of tumor,the tumor volume,weight of oleuropein group were significantly reduced compared with those of 20-30 days after33-40 days(administration period of oleuropein).Oleuropein 20 mg/kg has no obvious toxicity to the heart,spleen liver,kidney and lung of nude mice.The immunohistochemical results showed that the 20 mg/kg group of oleuroside significantly inhibited the expression of tumor growth index Ki67 in tumor cells,and its inhibitory ability was better than that of the 20 mg/kg group of cisplatin.The 20 mg/kg group of oleurosiden can significantly inhibit the expression of CYTH2 and promote the expression of RASSF4.3.Study on the mechanism of action of oleuropein and icariin I on A549 cells based on CYTH2To explore the mechanism of oleuropein and icariin I acting on A549 lung cancer cells,RT-q PCR results showed that Secin H3 40,80,160μM.Oleuropein 1.25,2.5,5,10μM can significantly inhibit the transcription level of CYTH2 gene,icariin I 5,10μM can extremely significantly inhibit the transcription level of CYTH2 gene.Oleuropein 10μM,icariin I 10μM can extremely significantly promote the transcription level of EGFR,RASSF4 and MAP2K3;It can extremely significantly promote the transcription level of IGF-1R gene;icariin I 10μM can extremely significantly promote the transcription level of IRS gene;Oleuropein 10μM can extremely significantly promote the transcription level of AKT gene.Western blot results show that Secin H3 80μM,oleuroside 10μM.icariin I 10μM can extremely inhibit the expression of CYTH2 protein,oleuropein and icariin I 10μM can extremely promote the expression of its downstream RASSF4-MAP2K3-p38 protein,the expression of Bax and Bim pro-apoptotic proteins downstream of RASSF4,and the expression of caspase-9 pro-apoptotic related protein,thus promoting the apoptosis of A549 lung cancer cells.4.Oleuropein and icariin I can bind to CYTH2 and RASSF4Construct prokaryotic expression vectors of pET28a-CYTH2 and p Cold I-CYTH2and analyze their solubility:CYTH2 protein has the best expression effect in p Cold I prokaryotic expression vector,the optimal induction expression condition is 16℃,shaking table 140 r/min,IPTG concentration is 0.2 m M.Using the induced expression conditions,a large number of expanded bacteria were prepared and CYTH2 protein was purified to obtain a concentration of 3.930 mg/m L.Isothermal titration calorimetry(ITC)proved that Secin H3,oleuropein,and icariin I can bind to CYTH2 protein with binding constants of 2.246×10~5,1.161×10~5,4.648×10~4and all of them are exothermic reactions with medium binding strength.Cellular thermal shift assay(CETSA)and Drug affinity responsive target stability(DARTS)tests showed that oleuroside,icariin I and RASSF4protein had binding potential.