Versatile small molecule kinase assay through real-time, ratiometric fluorescent monitoring of interchange for ATP and ADP based on pyrene-Zn2+-DPA complex

Abstract

Since kinase, which induces the transfer of phosphate group, is associated with a biological process and various diseases, kinase has been recently identified as a major target in drug discovery and its activity measurement is of crucial importance. Herein, we introduced a versatile and real-time kinase assay platform using pyrene-based ratiometric fluorescence probe to monitor small molecule kinase activity. The fluorescent probe, pyrene-Zn2+-DPA complex, has a characteristic showing distinct excimer and monomer fluorescence according to the changed ratio of ATP and ADP for the kinase progression. This system was adapted to the various enzymes as shown by utilizing for the hexokinase and the creatine kinase which phosphorylates and dephosphorylates each substrate, respectively, and the quantitative analysis of each well-known inhibitor was estimated. Moreover, it could be monitored by the naked eye with a simple light source, indicating a powerful tool for inhibitor assay. As a result, the proposed assay system can be extended as a general platform applicable to various kinases and suitable for high throughput screening methods for inhibitor screening.