Development of an AlphaLISA high throughput technique to screen for small molecule inhibitors targeting protein arginine methyltransferases

Molecular BioSystems
Lakshmi PrabhuTao Lu

Abstract

The protein arginine methyltransferase (PRMT) family of enzymes comprises nine family members in mammals. They catalyze arginine methylation, either monomethylation or symmetric/asymmetric dimethylation of histone and non-histone proteins. PRMT methylation of its substrate proteins modulates cellular processes such as signal transduction, transcription, and mRNA splicing. Recent studies have linked overexpression of PRMT5, a member of the PRMT superfamily, to oncogenesis, making it a potential target for cancer therapy. In this study, we developed a highly sensitive (Z' score = 0.7) robotic high throughput screening (HTS) platform to discover small molecule inhibitors of PRMT5 by adapting the AlphaLISA™ technology. Using biotinylated histone H4 as a substrate, and S-adenosyl-l-methionine as a methyl donor, PRMT5 symmetrically dimethylated H4 at arginine (R) 3. Highly specific acceptor beads for symmetrically dimethylated H4R3 and streptavidin-coated donor beads bound the substrate, emitting a signal that is proportional to the methyltransferase activity. Using this powerful approach, we identified specific PRMT5 inhibitors P1608K04 and P1618J22, and further validated their efficacy and specificity for inhibiting PRMT5. Importan...Continue Reading

References

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Citations

Apr 2, 2019·Chemical & Pharmaceutical Bulletin·Hongrui TaoHua Zhang
Sep 15, 2020·SLAS Discovery·Michael PlatchekWensheng Xie
Jul 28, 2020·The Biochemical Journal·Sarah A MannBryan Knuckley
Jan 23, 2021·RSC Medicinal Chemistry·Jufeng SunAdam McCluskey
Mar 7, 2021·Journal of Cellular and Molecular Medicine·Xiangwei LiJingjing Wu
Jun 3, 2021·International Journal of Molecular Sciences·Maxime BlijlevensVictor W van Beusechem
May 30, 2021·Applied Microbiology and Biotechnology·Dongjie ChenShaoqiang Wu
Jul 25, 2021·International Journal of Molecular Sciences·Matthew MartinTao Lu
Aug 3, 2020·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Sidong CaiPeiqing Liu
Aug 14, 2020·Journal of Medicinal Chemistry·Yudao ShenJian Jin

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Methods Mentioned

BETA
Assay-Linked ImmunoSorbent Assay
AlphaLISA
Assay
transfection
PCR
co-immunoprecipitation
ELISA
xenograft

Software Mentioned

AlphaLISA
Primer Express
GraphPad Prism
Glide

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