Virtual Screening Of PI3Kα Inhibitors And Studying The Mechanism Of Their Anti-tumor Activity

Background:Mutations and aberrant activation of the PI3K/AKT/mTOR signaling pathway are often found in ovarian and breast cancers,which are strongly linked with tumor growth,prolifration,migration,metabolism,and drugs resistance.Therefore,PI3K/AKT/mTOR pathway were considered as a potential target for anti-tumor drug development.In this paper,the PI3Ka was used as the research target,finding highly effective PI3K inhibitor through virtual screening.What’s more,we study the molecular mechanism of Cucurbitacin B(CuB)inhibits the proliferation of cisplatin-resistant ovarian cancer cells and CP068 inhibits the growth of breast cancer.Methods:(1)According to the PI3Ka protein structure,virtual screening was employed with Targetmol and Chemdiv databases on Schording software,compounds were filtered by HTVS,SP and XP firstly,and the effective PI3K inhibitors were choosed by hierarchical clustering and docking.By using the CCK-8 reagent,we determined the viability of cisplatin-resistant ovarian cancer cells and breast cancer cells after treatment with CuB and compound CP068,respectively.(2)Hoechst staining and flow cytometry experiments was conducted to determine the effect of CuB on cisplatin-resistant ovarian cancer cells’ proliferation and apoptosis.A cell colony-forming experiment and flow cytometry were used to examine the effect of CP068 on the proliferation and cell cycle of breast cancer cells.(3)Constructing nude mouse xenograft tumor model of ovarian cancer with cisplatin resistance and breast cancer tumor model in mouse to observe the effects of CuB and CP068 in mice vivo.(4)The molecular mechanisms of CP068 inhibiting the proliferation of breast cancer were analyzed by proteomics analysis,and the influence of CuB and CP068 or combined with the PI3K inhibitor LY294002 on the PI3K/AKT/mTOR pathway by western blotting,and further investigated downstream regulated molecular signals.Results:(1)CP068 and CuB were screened from Chemdiv and Targetmol databases,which have strong affinity with PI3Kα,and their binding energy were-9.0 kcal/mol and-9.14 kcal/mol,respectively.CuB and CP068 inhibited proliferation of cisplatin-resistant ovarian cancer cells and breast cancer cells in dose-dependent manner.(2)Accroding to Hoechst staining and Flow cytometry analysis of Annexin-V FITC/PI staining data,CuB could inhibit the proliferation and promote the apoptosis of cisplatin-resistant ovarian cancer cells in a concentration-dependent manner.As shown by cell colony-forming and flow cytometry PI staining results,CP068 was able to inhibit cell proliferation depending on concentration and induce G2/M phase arrest in breast cancer cell.(3)CuB can’t overcome drug resistance and inhibit ovarian tumor growth in nude mice.And CP068 effect on breast cancer tumor-bearing mice was not statistically different from the model group.(4)CuB could down-regulate PI3K/AKT/mTOR signaling pathway,induce DNA damage,activate cGAS and recruit IKBa to inhibit proliferation of cisplatin-resistant ovarian cancer cells;CP068 can inhibit PI3K/AKT/mTOR pathway,activate P53/P21 and regulate ATR and Cyclin D signaling to induce cell arrest and inhibit proliferation of breast cancer.Conclusion:This topic combining virtual screening,docking and ant-itumor activity evaluation discovered the PI3K inhibitors CuB and CP068.CuB inhibited PI3K/AKT/mTOR pathway,induced DNA damage,activated cGAS to recruit iKBα,playing a crucial role in promoting apoptosis,and providing a new approach for overcoming chemotherapy drug resistance.CP068 inhibited PI3K/AKT/mTOR pathway,activated P53/P21 pathway and regulated the expression of ATR and Cyclin D,thereby the breast cancer cells are induced to cell cycle arrest and stop growth,providing a novel choose for the development of PI3K inhibitors.