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

Poster Board Number: A101

Michael Rowley (University of British Columbia), Shona Taylor (University of British Columbia), Adam Frankel (University of British Columbia)

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-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

Results: FBAPS 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

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.

We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2020-04227 Discovery Grant (A.F.) and NSERC PGS-D (M.J.R) for support. Additionally, we thank Dr. Mark Bedford at The University of Texas MD Anderson Cancer Center for the GST-PABP1(437-488) expression clone, Shireen Anbardan for GST-PABP1(437-488) production, Dr. Eric Therrien at Schramp;ouml;dinger for Diamine 12, and Jeff McCarthy at adMare BioInnovations for Sapphire Biomolecular Imager usage.



Schematic illustrating the readout from the filter binding and phosphor screening (FBAPS) assay for the inhibition of PRMT1 via various chemical moieties. The phosphor screen image above was analyzed by densitometry to yield IC50 curves shown below.