Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany.
J Am Chem Soc. 2010 Mar 31;132(12):4152-60. doi: 10.1021/ja908083e.
Targeting protein kinases with small organic molecules is a promising strategy to regulate unwanted kinase activity in both chemical biology and medicinal chemistry research. Traditionally, kinase inhibitors are identified in activity-based screening assays using enzymatically active kinase preparations to measure the perturbation of substrate phosphorylation, often resulting in the enrichment of classical ATP competitive (Type I) inhibitors. However, addressing enzymatically incompetent kinase conformations offers new opportunities for targeted therapies and is moving to the forefront of kinase inhibitor research. Here we report the development of a new FLiK (Fluorescent Labels in Kinases) binding assay to detect small molecules that induce changes in the conformation of the glycine-rich loop. Due to cross-talk between the glycine-rich loop and the activation loop in kinases, this alternative labeling approach can also detect ligands that stabilize inactive kinase conformations, including slow-binding Type II and Type III kinase inhibitors. Protein X-ray crystallography validated the assay results and identified a novel DFG-out binding mode for a quinazoline-based inhibitor in p38alpha kinase. We also detected the high-affinity binding of a clinically relevant and specific VEGFR2 inhibitor, and we provide structural details of its binding mode in p38alpha, in which it stabilizes the DFG-out conformation. Last, we demonstrate the power of this new FLiK labeling strategy to detect the binding of Type I ligands that induce conformational changes in the glycine-rich loop as a means of gaining affinity for the target kinase. This approach may be a useful alternative to develop direct binding assays for kinases that do not adopt the DFG-out conformation while also avoiding the use of expensive kits, detection reagents, or radioactivity frequently employed with activity-based assays.
以小分子靶向蛋白激酶是一种很有前途的策略，可以调节化学生物学和药物化学研究中不需要的激酶活性。传统上，使用酶活性激酶制剂在基于活性的筛选测定中鉴定激酶抑制剂，以测量底物磷酸化的扰动，这通常导致经典的 ATP 竞争性（I 型）抑制剂的富集。然而，解决酶活性不足的激酶构象为靶向治疗提供了新的机会，并成为激酶抑制剂研究的前沿。在这里，我们报告了一种新的 FLiK（激酶中的荧光标签）结合测定法的开发，用于检测导致甘氨酸丰富环构象变化的小分子。由于甘氨酸丰富环和激酶中的激活环之间的串扰，这种替代标记方法还可以检测稳定无活性激酶构象的配体，包括慢结合的 II 型和 III 型激酶抑制剂。蛋白质 X 射线晶体学验证了该测定结果，并确定了一种新型基于喹唑啉的抑制剂在 p38α 激酶中的 DFG-out 结合模式。我们还检测到一种临床相关且特异性 VEGFR2 抑制剂的高亲和力结合，并提供了其在 p38α 中的结合模式的结构细节，其中它稳定了 DFG-out 构象。最后，我们证明了这种新的 FLiK 标记策略检测诱导甘氨酸丰富环构象变化的 I 型配体结合的能力，这是获得靶激酶亲和力的一种手段。这种方法可能是开发不采用 DFG-out 构象的激酶的直接结合测定法的有用替代方法，同时避免使用昂贵的试剂盒、检测试剂或经常用于基于活性的测定法的放射性物质。
