Simard Jeffrey R, Rauh Daniel
Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany.
Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Fakultät Chemie, Chemische Biologie, Technische Universität Dortmund, Dortmund, Germany.
Methods Enzymol. 2014;548:147-71. doi: 10.1016/B978-0-12-397918-6.00006-9.
Despite the hundreds of kinase inhibitors currently in discovery and preclinical phases, the number of FDA-approved kinase inhibitors remains very low by comparison, a discrepancy which reflects the challenges which accompanies kinase inhibitor development. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibit kinase activity, but the highly conserved nature of the ATP-binding site often contributes to the poor inhibitor selectivity. To address this problem, we developed a high-throughput screening technology that can discriminate for inhibitors, which stabilize inactive kinase conformations by binding within allosteric pockets in the kinase domain. Here, we describe how to use the Fluorescence Labels in Kinases approach to measure the K(d) of ligands as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries in high throughput.
尽管目前有数百种激酶抑制剂正处于发现和临床前阶段，但相比之下，美国食品药品监督管理局（FDA）批准的激酶抑制剂数量仍然非常少，这种差异反映了激酶抑制剂开发所面临的挑战。用ATP竞争性抑制剂靶向蛋白激酶一直是抑制激酶活性的经典方法，但ATP结合位点的高度保守性质常常导致抑制剂选择性较差。为了解决这个问题，我们开发了一种高通量筛选技术，该技术可以区分通过结合激酶结构域的变构口袋来稳定无活性激酶构象的抑制剂。在这里，我们描述了如何使用激酶中的荧光标记方法来测量配体的解离常数（K(d)），以及如何从动力学角度表征配体与激酶的结合和解离。我们还描述了如何利用该技术在高通量条件下快速筛选小分子文库。
