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High Throughput Screening of Protein Arginine N ‐Methyltransferases – Filter Binding and Phosphor Screening (FBAPS) assay
Author(s) -
Rowley Michael J.,
Taylor Shona A.,
Frankel Adam
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r1963
Subject(s) - arginine , high throughput screening , methyltransferase , chemistry , ligand binding assay , western blot , enzyme , biochemistry , methylation , amino acid , receptor , gene
Study Objective We 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. Hypothesis A 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 methods Recombinantly‐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 ‐ 14 C]‐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 (K M , k cat ) for enzymes and substrates were determined, and IC 50 values were obtained for well‐characterized inhibitors S ‐adenosyl‐L‐homocysteine (SAH), MS023, EPZ020411, and Diamine 12 using FBAPS. Summary of Results FBAPS yielded kinetic parameters with no statistically significant differences to what was obtained using liquid scintillation counting. The IC 50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature. Statement of Conclusions The 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.