Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany.
J Med Chem. 2010 Jan 14;53(1):357-67. doi: 10.1021/jm901297e.
Targeting protein kinases with small molecules outside the highly conserved ATP pocket to stabilize inactive kinase conformations is becoming a more desirable approach in kinase inhibitor research, since these molecules have advanced pharmacological properties compared to compounds exclusively targeting the ATP pocket. Traditional screening approaches for kinase inhibitors are often based on enzyme activity, but they may miss inhibitors that stabilize inactive kinase conformations by enriching the active state of the kinase. Here we present the development of a kinase binding assay employing a pyrazolourea type III inhibitor and enzyme fragment complementation (EFC) technology that is suitable to screen stabilizers of enzymatically inactive kinases. To validate this assay system, we report the binding characteristics of a series of kinase inhibitors to inactive p38alpha and JNK2. Additionally, we present protein X-ray crystallography studies to examine the binding modes of potent quinoline-based DFG-out binders in p38alpha.
针对高度保守的 ATP 结合口袋以外的蛋白激酶小分子，以稳定非活性激酶构象，在激酶抑制剂研究中正成为一种更可取的方法，因为与专门针对 ATP 结合口袋的化合物相比，这些小分子具有先进的药理学特性。传统的激酶抑制剂筛选方法通常基于酶活性，但它们可能会错过通过富集激酶的活性状态来稳定非活性激酶构象的抑制剂。在这里，我们提出了一种激酶结合测定的开发，该测定采用吡唑并脲型 III 抑制剂和酶片段互补 (EFC) 技术，适用于筛选酶失活激酶的稳定剂。为了验证该测定系统，我们报告了一系列激酶抑制剂与失活的 p38alpha 和 JNK2 的结合特性。此外，我们还介绍了蛋白质 X 射线晶体学研究，以检查强效喹啉 DFG-out 结合物在 p38alpha 中的结合模式。
