ELISA高通量筛选PIM靶向抑制剂的研究

摘要

背景与目的：Pim家族是具有丝氨酸/苏氨酸激酶活性的原癌基因，包括Pim-1、Pim-2、Pim-3。研究发现，Pim能够抑制细胞凋亡，促进细胞增殖，导致肿瘤发生。本研究利用ELISA反应原理建立高通量筛选体系，对Pim抑制剂进行筛选，为药物的应用和解决恶性肿瘤临床治疗的难题提供研究基础。方法：通过计算机辅助药物设计的手段，以蔓生百部碱母核三环酰胺作为核心结构合成系列衍生物；利用Pim重组蛋白和Bad重组蛋白可以与Ni2+特异性结合的亲和层析原理进行重组蛋白的纯化精制；利用ELISA方法，通过Pim激酶磷酸化促凋亡蛋白分子Bad在112位丝氨酸的反应，建立筛选Pim靶向抑制剂的体系。结果：0.01 mmol/L的IPTG在37℃条件下，诱导Bad蛋白2 h，可以获得最大量的可溶性Bad重组蛋白；0.02%的阿拉伯糖在37℃条件下，诱导Pim-1、Pim-2、Pim-3蛋白3 h，可以获得最大量的Pim重组蛋白；ELISA结果显示，Pim激酶与Bad底物之间的作用存在剂量依赖性反应关系，进而成功建立药物筛选体系；初步筛选出低分子化合物T-18，在体外能有效抑制Pim激酶活性。结论：利用ELISA反应原理建立的筛选药物方法，是一种经济、简单、快速、高效、敏感的筛选手段，并初步筛选出小分子化合物T-18，能够抑制Pim激酶活性，阻断Pim对凋亡蛋白分子Bad的磷酸化过程，进而抑制肿瘤的发生。%　　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.