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

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2509-2520
Number of pages12
JournalMolecular BioSystems
Volume13
Issue number12
DOIs
StatePublished - Jan 1 2017

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Protein-Arginine N-Methyltransferases
Histones
Methylation
Arginine
Streptavidin
Methyltransferases
Robotics
Pancreatic Neoplasms
Epigenomics
Methionine
Mammals
Colorectal Neoplasms
Signal Transduction
Neoplasms
Carcinogenesis
Proteins
Technology
Messenger RNA
Enzymes
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

Cite this

Development of an AlphaLISA high throughput technique to screen for small molecule inhibitors targeting protein arginine methyltransferases. / Prabhu, Lakshmi; Chen, Lan; Wei, Han; Demir, Özlem; Safa, Ahmad; Zeng, Lifan; Amaro, Rommie E.; O'Neil, Bert H.; Zhang, Zhon Yin; Lu, Tao.

In: Molecular BioSystems, Vol. 13, No. 12, 01.01.2017, p. 2509-2520.

Research output: Contribution to journalArticle

Prabhu, Lakshmi ; Chen, Lan ; Wei, Han ; Demir, Özlem ; Safa, Ahmad ; Zeng, Lifan ; Amaro, Rommie E. ; O'Neil, Bert H. ; Zhang, Zhon Yin ; Lu, Tao. / Development of an AlphaLISA high throughput technique to screen for small molecule inhibitors targeting protein arginine methyltransferases. In: Molecular BioSystems. 2017 ; Vol. 13, No. 12. pp. 2509-2520.
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AU - Chen, Lan

AU - Wei, Han

AU - Demir, Özlem

AU - Safa, Ahmad

AU - Zeng, Lifan

AU - Amaro, Rommie E.

AU - O'Neil, Bert H.

AU - Zhang, Zhon Yin

AU - Lu, Tao

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