High Throughput Screening of Protein Arginine N‐Methyltransferases – Filter Binding and Phosphor Screening (FBAPS) assay

Abstract

Study ObjectiveWe seek to develop a cost‐effective assay to screen protein arginine N‐methyltransferase (PRMT) enzyme activity in a high throughput manner by combining P81 filter binding with phosphor imaging.HypothesisA combination of the P81 filter binding assay and phosphor screening (FBAPS) will provide improved throughput for testing inhibitors against PRMTs while eliminating the economic and environmental costs incurred with current PRMT assays.Statement of methodsRecombinantly‐expressed PRMT1 and coactivator‐associated arginine methyltransferase 1 (CARM1) were used to develop the FBAPS assay using GST fusions of polyA binding protein 1 (PABP1(437‐488)) and glycine‐ and arginine‐rich (GAR) protein as substrates, respectively, and radiolabelled S‐adenosyl‐L‐[methyl‐14C]‐methionine as cofactor. Methylation reactions were spotted onto P81 filter paper in a dot blot apparatus and radioactive signal was measured both by phosphor imaging and liquid scintillation counting. Kinetic parameters (KM, kcat) for enzymes and substrates were determined, and IC50 values were obtained for well‐characterized inhibitors S‐adenosyl‐L‐homocysteine (SAH), MS023, EPZ020411, and Diamine 12 using FBAPS.Summary of ResultsFBAPS yielded kinetic parameters with no statistically significant differences to what was obtained using liquid scintillation counting. The IC50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature.Statement of ConclusionsThe FBAPS assay is a modification to the P81 filter binding assay with a dot blot apparatus that allows for parallel processing of samples in a multi‐well format, dramatically increasing throughput. Signal detection by phosphor imaging offers a cost effective and quantitative method that can be used to screen several inhibitors simultaneously against PRMT enzymes with high accuracy.