Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu (Estonia).
Chembiochem. 2014 Feb 10;15(3):443-50. doi: 10.1002/cbic.201300613. Epub 2014 Jan 8.
We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well-known inhibitors MLN8237 and VX-689 (MK-5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (kon 10(5)-10(7) M(-1) s(-1), koff 10(-3)-10(-4) s(-1); residence time 500-3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on-rate and increase in the off-rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX-689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data.
我们报告了三种荧光探针的开发，用于蛋白激酶 Aurora A，这些探针源自著名的抑制剂 MLN8237 和 VX-689（MK-5108）。其中两种探针针对 Aurora A 的 ATP 结合位点，一种探针同时针对激酶的 ATP 和底物结合位点。这些探针在荧光偏振/各向异性读取的测定中进行了测试，我们证明了探针的缓慢结合动力学和长驻留时间（kon 10(5)-10(7) M(-1) s(-1)，koff 10(-3)-10(-4) s(-1)；驻留时间 500-3000 s）。Aurora A 激活剂 TPX2 的存在导致针对 ATP 结合位点的荧光探针的结合速率显著降低，而解离速率增加。这些观察结果得到了用 MLN8237 和 VX-689 进行的 Aurora A 抑制测定的支持。总的来说，我们的结果强调了用这些化合物进行实验的合理设计的重要性，以及对获得的数据的正确解释。
