Optimization and validation of a DYRK1A TR-FRET assay for high-throughput screening.

Due to its role in brain development, the DYRK1A kinase (dual-specificity tyrosine phosphorylation-regulated kinase 1a) has been proposed as a drug target for Down syndrome, and diseases associated with neurodegeneration including Alzheimer's and Parkinson's. Other diseases in which DYRK1A is implicated include cancer and diabetes. Hence, there is need for potent and selective DYRK1A inhibitors. To screen large diversity compound libraries versus DYRK1A requires the development of a cost-effective high-throughput screen. In this study, we have taken a commercial time-resolved fluorescence energy transfer (TR-FRET)-based assay for DYRK1A and optimized for smaller volumes and homogenous format at room temperature. Tracer and enzyme concentrations were determined. DYRK1A-GST, anti-GST Ab and tracer were pre-combined and total assay volume reduced 2-fold. The assay was validated using whole plate minimum and maximum signal wells with a Z' of 0.7-0.8 determined. Overall, this method:•Results in an optimized low volume, homogenous and validated assay for DYRK1A.•Delivers a cost effective high-throughput assay format for DYRK1A inhibitor screening.