Detection of PRMT1 inhibitors with stopped flow fluorescence

Kun Qian,Y George Zheng,Hao Hu,Hui Xu

doi:10.1038/s41392-018-0009-6

Abstract

Protein arginine methyltransferases (PRMTs) are crucial epigenetic regulators in eukaryotic organisms that serve as histone writers for chromatin remodeling. PRMTs also methylate a variety of non-histone protein substrates to modulate their function and activity. The development of potent PRMT inhibitors has become an emerging and imperative research area in the drug discovery field to provide novel therapeutic agents for treating diseases and as tools to investigate the biological functions of PRMTs. PRMT1 is the major type I enzyme that catalyzes the formation of asymmetric dimethyl arginine, and PRMT1 plays important regulatory roles in signal transduction, transcriptional activation, RNA splicing, and DNA repair. Aberrant expression of PRMT1 is found in many types of cancers, pulmonary diseases, cardiovascular disease, diabetes, and renal diseases. PRMT1 is a highly promising target for therapeutic development. We created a stopped flow fluorescence-based assay for PRMT1 inhibitor detection and characterization that has the advantages of being homogeneous, nonradioactive, and mix-and-measure in nature, allowing for continuous measurement of the methylation reaction and its inhibition. To our knowledge, this is the first continuous assay for PRMT1 reaction detection and inhibitor characterization. The approach is not only capable of quantitatively determining the potency (IC50) of PRMT1 inhibitors but can also distinguish cofactor-competitive inhibitors, substrate-competitive inhibitors, and mixed-type inhibitors.

Highlights

Protein arginine methylation is a type of universal posttranslational modification (PTM) that plays significant biological roles in eukaryotic organisms.[1]
Fluorescent changes of fluorescein-labeled histone H4 peptide during PRMT1 catalysis PRMT1 is the major type I enzyme responsible for asymmetric arginine dimethylation.[31]
PRMT1 transfers the methyl group from SAM to a guanidine nitrogen of arginine to form monomethyl arginine (MMA), which can be further methylated into asymmetric dimethyl arginine (ADMA) (Fig. 1a).[31]

Summary

Introduction

Protein arginine methylation is a type of universal posttranslational modification (PTM) that plays significant biological roles in eukaryotic organisms.[1] far, nine protein arginine methyltransferases (PRMTs) have been found in mammalian cells,[2] which are classified into three types: type I, type II and type III PRMTs. Type I enzymes (PRMT1, −2, −3, −4, −6, and −8) convert arginine residues to monomethyl arginine (MMA) and further modify them to asymmetric dimethyl arginine (ADMA); type II enzymes (PRMT5 and PRMT9) produce MMA and symmetric dimethyl arginine (SDMA); and PRMT7 is the only type III enzyme that generates MMA. The development of PRMT inhibitors has emerged as an imperative task to provide novel therapeutic agents to treat diseases and to find chemical probes to investigate the biological functions of PRMTs.[7,9,10]

Methods

Results

Conclusion