Wang Weixue, Krosky Daniel, Ahn Kay
Molecular & Cellular Pharmacology, Lead Discovery, Janssen Research and Development, LLC , Spring House, Pennsylvania 19477, United States.
Biochemistry. 2017 Aug 29;56(34):4449-4456. doi: 10.1021/acs.biochem.7b00521. Epub 2017 Jun 14.
Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Targeting inactive (vs active) kinase conformations offers advantages in achieving selectivity because of their more diversified structures. Discovery of inactive conformation-selective inhibitors, however, has been hampered partly by the lack of general assay methods. Herein, we show that such inhibitors can be discovered by utilizing kinase cascade assays. This type of assay is initiated with the target kinase in its unphosphorylated, inactive conformation, which is activated during the assay. Inactive conformation-selective inhibitors stabilize the inactive kinase, block activation, and yield reduced kinase activity. We investigate the properties of the assay by mathematical modeling, as well as by proof-of-concept experiments using the BRAF-MEK1 cascade. This study demonstrates effective identification of inactive conformation-selective inhibitors by cascade assays, reveals key factors that impact results, and provides guidelines for successful cascade assay development.
在激酶抑制剂药物研发中，实现对人类激酶组的选择性是一个主要障碍。由于非活性（相对于活性）激酶构象具有更多样化的结构，靶向这些构象在实现选择性方面具有优势。然而，非活性构象选择性抑制剂的发现部分受到缺乏通用检测方法的阻碍。在此，我们表明可以通过利用激酶级联检测来发现此类抑制剂。这种检测类型以处于未磷酸化、非活性构象的靶激酶开始，该构象在检测过程中被激活。非活性构象选择性抑制剂可稳定非活性激酶，阻断激活，并降低激酶活性。我们通过数学建模以及使用BRAF-MEK1级联的概念验证实验来研究该检测的特性。这项研究证明了通过级联检测可有效鉴定非活性构象选择性抑制剂，揭示了影响结果的关键因素，并为成功开发级联检测提供了指导。
