Abstract:

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.

Key words: Bad, Pim, ELISA, Drug screening, Pim kinase inhibitors