Tavares Clint D J, Devkota Ashwini K, Dalby Kevin N, Cho Eun Jeong
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA.
Texas Screening Alliance for Cancer Therapeutics, The University of Texas at Austin, Austin, TX, 78712, USA.
Methods Mol Biol. 2016;1360:19-33. doi: 10.1007/978-1-4939-3073-9_2.
Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.
蛋白激酶已成为一类重要的治疗靶点，因为它们参与了与多种人类疾病相关的病理途径。学术界和制药公司发现激酶抑制剂的主要努力集中在开发强大的检测方法和经济高效的分离方法上。药物发现过程通常从通过高通量方式使用适当的检测方法筛选化学文库来鉴定先导化合物开始。考虑到每种检测技术和筛选能力的独特局限性，除了化学文库的选择外，智能整合各种检测方案和通量水平是这一初始步骤成功的关键。在这里，我们描述了蛋白激酶eEF-2K的纯化以及在鉴定阻断其酶促反应的小分子过程中三种生化检测方法的应用。
