ELISA高通量筛选PIM靶向抑制剂的研究

doi:10.3969/j.issn.1007-3969.2013.04.004

中国癌症杂志 ›› 2013, Vol. 23 ›› Issue (4): 260-266.doi: 10.3969/j.issn.1007-3969.2013.04.004

ELISA高通量筛选PIM靶向抑制剂的研究

尚琨¹,张鹏²,周璐²,王瑧¹,曹越¹,李影奕¹

1.复旦大学附属肿瘤医院实验研究中心，复旦大学上海医学院肿瘤学系，上海 200032；
2.复旦大学药学院，上海 201203

出版日期:2013-04-25 发布日期:2014-11-19
通信作者: 李影奕　E-mail:liyingyi@fudan.edu.cn
基金资助:
国家自然科学基金面上项目(No：30973476)；上海市浦江人才计划(No：10PJ1402100)；复旦大学“985工程”三期肿瘤研究项目Ⅱ (No：985Ⅲ-YFX0102)

Screening of Pim kinase inhibitors by the establishment of high-throughput ELISA system

SHANG Kun¹, ZHANG Peng², ZHOU Lu², WANG Zhen¹, CAO Yue¹, LI Ying-yi¹

1.Cancer Research Laboratory, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China; 2.School of Pharmacy, Fudan University, Shanghai 201203, China

Published:2013-04-25 Online:2014-11-19
Contact: LI Ying-yi E-mail: liyingyi@fudan.edu.cn

摘要/Abstract

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。

关键词: Bad, Pim, ELISA, 药物筛选, Pim靶向抑制剂

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

尚琨,张鹏,周璐,王瑧,曹越,李影奕. ELISA高通量筛选PIM靶向抑制剂的研究[J]. 中国癌症杂志, 2013, 23(4): 260-266.

SHANG Kun, ZHANG Peng, ZHOU Lu, WANG Zhen, CAO Yue, LI Ying-yi. Screening of Pim kinase inhibitors by the establishment of high-throughput ELISA system[J]. China Oncology, 2013, 23(4): 260-266.

ELISA高通量筛选PIM靶向抑制剂的研究

Screening of Pim kinase inhibitors by the establishment of high-throughput ELISA system

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