An ELISA DYRK1A non-radioactive assay suitable for the

The DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer’s disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC s for EGCG and harmine determined by the ELISA method were found to be comparable to those previously measured by radioactive tracing methods. Furthermore, we determined the mode of inhibition for EGCG and harmine by a modification of the ELISA assay. This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors. Yu-Wen Hwang ( ) Corresponding author: [email protected] Liu Y, Adayev T and Hwang YW. How to cite this article: An ELISA DYRK1A non-radioactive assay suitable for the characterization of 2017, :42 (doi: ) inhibitors [version 1; referees: 2 approved] F1000Research 6 10.12688/f1000research.10582.1 © 2017 Liu Y . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver This work is supported by the New York State Office for People with Developmental Disabilities, the parent agency of New Grant information: York State Institute for Basic Research in Developmental Disabilities. No extramural fund was used to support this research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: No competing interests were disclosed. 13 Jan 2017, :42 (doi: ) First published: 6 10.12688/f1000research.10582.1 Referee Status: