Lopez Michael S, Kliegman Joseph I, Shokat Kevan M
Howard Hughes Medical Institute and Department of Cellular & Molecular Pharmacology, University of California, San Francisco, California, USA.
Methods Enzymol. 2014;548:189-213. doi: 10.1016/B978-0-12-397918-6.00008-2.
Analog-sensitive AS Kinase technology allows for rapid, reversible, and highly specific inhibition of individual engineered kinases in cells and in mouse models of human diseases. The technique consists of two parts: a kinase containing a space-creating mutation in the ATP-binding pocket and a bulky ATP-competitive small molecule inhibitor that complements the shape of the mutant ATP pocket. This strategy enables dissection of phospho-signaling pathways, elucidation of the physiological function of individual kinases, and characterization of the pharmacology of clinical-kinase inhibitors. Here, we present an overview of AS technology and describe a stepwise approach for generating AS Kinase mutants and identifying appropriate small molecule inhibitors. We also describe commonly encountered technical obstacles and provide strategies to overcome them.
模拟敏感型（AS）激酶技术能够在细胞和人类疾病小鼠模型中对单个工程化激酶进行快速、可逆且高度特异性的抑制。该技术由两部分组成：一种在ATP结合口袋中含有产生空间突变的激酶，以及一种与突变型ATP口袋形状互补的大分子ATP竞争性小分子抑制剂。这种策略能够剖析磷酸信号通路，阐明单个激酶的生理功能，并表征临床激酶抑制剂的药理学特性。在此，我们概述了AS技术，并描述了生成AS激酶突变体和鉴定合适小分子抑制剂的逐步方法。我们还描述了常见的技术障碍并提供了克服这些障碍的策略。
