Versatile small molecule kinase assay through real-time, ratiometric fluorescence changes based on a pyrene-DPA-Zn2+ complex.

Jihoon Kim,Min Su Han,Jinyoung Oh

doi:10.1039/d1ra01547h

Abstract

A real-time kinase assay method based on a ratiometric fluorescence probe that can be applied to various small-molecule kinases is described herein. The probe can trace the reversible interchange of ATP and ADP, which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric fluorescence change. This property facilitates the monitoring of phosphorylation and dephosphorylation in small-molecule kinases, whereas most of the existing methods focus on one of these reactions. To prove the applicability of this method for small-molecule kinase assays, hexokinase and creatine kinase, which phosphorylate and dephosphorylate substrates, respectively, were analyzed. The ratiometric fluorescence change was correlated with the enzyme activity, and the inhibition efficiencies of the well-known inhibitors, N-benzoyl-d-glucosamine and iodoacetamide, were also monitored. Notably, the change in fluorescence can be observed with a simple light source by the naked eye.

Highlights

Kinases mediate the transfer of a phosphate group to an acceptor for phosphorylation or dephosphorylation of various substrates
The probe can trace the reversible interchange of ATP and ADP, which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric fluorescence change
Many kinase assay methods have been reported for the rapid detection of enzyme malfunctions and inhibitor screening, most of them have focused on protein kinases, despite the importance of small molecule kinases, which utilize small molecules as their substrates

Summary

Introduction

Kinases mediate the transfer of a phosphate group to an acceptor for phosphorylation or dephosphorylation of various substrates. The probe can trace the reversible interchange of ATP and ADP, which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric fluorescence change. The reversible uorescence change of the pyrene-DPA-Zn2+ complex would facilitate monitoring of the dephosphorylation as well as phosphorylation of the substrate by various small molecule kinases.

Results

Conclusion