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

Lakshmi Prabhu; Lan Chen; Han Wei; Özlem Demir; Ahmad Safa; Lifan Zeng; Rommie E. Amaro; Bert H. O’Neil; Zhon-Yin Zhang; Tao Lu

doi:10.1039/C7MB00391A

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

Lakshmi Prabhu,

^a Lan Chen,‡^bc Han Wei,^a Özlem Demir,^d Ahmad Safa,^a Lifan Zeng,^bc Rommie E. Amaro,^d Bert H. O’Neil,^e Zhon-Yin Zhang

‡^c and Tao Lu*^acf

Author affiliations

* Corresponding authors

^a Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN, USA

^b Chemical Genomics Core Facility, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN, USA

^c Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA

^d Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

^e Department of Medicine, Indiana Cancer Pavilion, Indiana University School of Medicine, 535 Barnhill Drive, Indianapolis, IN 46202, USA

^f Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street, Medical Research and Library Building, IB 130, Indianapolis, IN 46202, USA
E-mail: lut@iupui.edu
Fax: +1317 274 7714
Tel: +1 317 278 0520

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. Importantly, these two compounds exhibited much more potent efficacy than the commercial PRMT5 inhibitor EPZ015666 in both pancreatic and colorectal cancer cells. Overall, our work highlights a novel, powerful, and sensitive approach to identify specific PRMT5 inhibitors. The general principle of this HTS screening method can not only be applied to PRMT5 and the PRMT superfamily, but may also be extended to other epigenetic targets. This approach allows us to identify compounds that inhibit the activity of their respective targets, and screening hits like P1608K04 and P1618J22 may serve as the basis for novel drug development to treat cancer and/or other diseases.

Molecular BioSystems

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

Abstract

Supplementary files

Article information

Download Citation

Permissions

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

Search articles by author

Spotlight

Advertisements