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.

中文翻译：

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靶向Cys-Gatekeeper激酶的II型激酶抑制剂显示与野生型和Ala / Gly-Gatekeeper激酶的正交性

类似物敏感（AS）激酶在看门者位置包含大到小的突变，使其易于被基于吡唑并嘧啶的Src激酶抑制剂（例如PP1）的庞大类似物抑制。这种“突如其来的”方法已被用于约520种激酶中的至少85种，但是许多激酶对此方法不耐受。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（类似物的“ DFG-out”激酶抑制剂2和6），其目标是Cys-gatekeeper激酶的“ DFG-out”构象与亚微摩尔效价。我们在体外验证了该系统使用表达M110C-GWL的小鼠胚胎成纤维细胞对抗长壁激酶（GWL），Aurora-A激酶和细胞周期蛋白依赖性激酶1的抗性。这些Cys-gatekeeper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。这些具有AS激酶抑制剂的化合物具有在系统水平和翻译激酶研究以及针对内源激酶的II型激酶抑制剂的合理设计中应用的潜力，可以独立抑制多种AS激酶。