Genome Damage and Stability Centre, School of Life Sciences , University of Sussex , Falmer, Brighton BN1 9RQ , U.K.
Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology , University of California, San Francisco , 600 16th Street , San Francisco , California 94158-2280 , United States.
ACS Chem Biol. 2018 Oct 19;13(10):2956-2965. doi: 10.1021/acschembio.8b00592. Epub 2018 Oct 9.
Analogue-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogues of pyrazolopyrimidine-based Src kinase inhibitors (e.g., PP1). This "bump-hole" method has been utilized for at least 85 of ∼520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS kinase technology, we designed type II kinase inhibitors, ASDO2/6 (analogue-sensitive "DFG-out" kinase inhibitors 2 and 6), that target the "DFG-out" conformation of Cys-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase, and cyclin-dependent kinase-1 and in cells using M110C-GWL-expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6 inhibition but not AS kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS kinase inhibitors, have the potential to inhibit multiple AS kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.
模拟敏感 (AS) 激酶在门控位置含有大到小的突变，使它们容易受到吡唑并嘧啶基Src 激酶抑制剂（例如 PP1）的大体积类似物的抑制。这种“凹凸”方法已被用于至少 85 种约 520 种激酶，但许多激酶对这种方法不耐受。为了扩大 AS 激酶技术的范围，我们设计了 II 型激酶抑制剂 ASDO2/6（模拟敏感的“DFG-out”激酶抑制剂 2 和 6），它们以亚微摩尔的效力针对 Cys-门控激酶的“DFG-out”构象。我们在体外使用 Greatwall 激酶 (GWL)、Aurora-A 激酶和细胞周期蛋白依赖性激酶-1 以及使用表达 M110C-GWL 的小鼠胚胎成纤维细胞验证了该系统。这些 Cys-门控激酶对 ASDO2/6 抑制敏感，但对 AS 激酶抑制剂 3MB-PP1 不敏感，反之亦然。这些化合物与 AS 激酶抑制剂一起，有可能独立抑制多种 AS 激酶，可应用于系统水平和转化激酶研究以及针对内源性激酶的 II 型激酶抑制剂的合理设计。
