Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure–activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.

蛋白激酶经历大规模的结构变化，这些结构紧密调节功能并控制小分子抑制剂的识别。量化抑制剂构象效应并将其与选择性模式理解联系起来的方法长期以来难以捉摸。我们已经开发了一种超快的时间分辨荧光方法，可以以埃级精度跟踪溶液中激酶激活环的结构运动，并且可以解析多个结构状态并量化状态之间的构象变化。针对有丝分裂激酶Aurora A分析一组临床相关的Aurora激酶抑制剂揭示了广泛的构象偏好，所有抑制剂均促进DFG处于活跃状态或DFG处于非活跃状态，但程度不同。值得注意的是 这些构象偏好解释了在Aurora A的不同激活状态下抑制剂选择性的广泛模式，其中DFG-out抑制剂优先结合通过激活环上的磷酸化激活的Aurora A，该环可动态采样DFG-out状态，而DFG-in抑制剂则优先结合到Aurora A的变构活化剂Tpx2限制在DFG-in状态。结果表明，目前正在临床开发中的许多抑制剂可能能够区分与正常有丝分裂控制以及黑色素瘤，成神经细胞瘤和前列腺癌有关的Aurora A信号传导途径。