摘要：

Background and purpose: Pim family is the proto-oncogene that exhibits serine/threonine kinase activity,containing Pim-1,Pim-2,Pim-3.Pim family has potent anti-apoptotic capacity,ultimately promoting tumor cells survival.This study aimed to establish a high-throughput system to screen the anti-cancer drugs targeting Pim kinase by ELISA.Methods: The stemonamide synthetic intermediates were synthesized using a radical cascade.The expression of Pim kinase proteins and Bad proteins were purified by bacterial system.A high-throughput ELISA screening was performed for in vitro Pim kinase assay.Results: Treatment with 0.01 mmol/L of IPTG for 2 hours at 37?℃,the induction of Bad recombinant proteins was the maximum;Treatment with 0.02% of arabinose for 3 hours at 37?℃,the induction of Pim-1,Pim-2,Pim-3 recombinant proteins was the maximum.ELISA results showed that the Pim kinase could phosphorylate Bad in a dose-dependent manner;we had found a low molecular weight compounds T-18,which could effective inhibit Pim kinase activity in vitro.Conclusion: We successfully established a screening system with Bad and Pim by ELISA.ELISA is a method for screening drug with high throughput,effect and sensitivity.Moreover,small molecule the compound T-18 that is screened by ELISA,can inhibit Pim kinase activities,ultimately reduce the amount of phosphorylated Bad and could induce apoptosis.

展开