Abstract A31: Monitoring the activity of various classes of methylltransferases in vitro using a universal, homogenous and bioluminescent assay

Abstract

Abstract Post transcriptional modifications of proteins and nucleic acids are well-recognized to play major role in mediation of extensive and broad range of cellular functions. Such modifications that include phosphorylation, acetylation, methylation, etc. are known to be critical for normal and abnormal cell growth, and thus, the enzymes involved in these reactions have been validated as drug targets resulting in the development of several drugs. Recent biochemical and biological data suggest that the enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in the development of novel therapeutics for multiple unmet medical needs. Of these, methyltransferases are known to alter the epigenome by altering the methylation status of nucleic acids or proteins resulting in changes in cellular phenotypes. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. The assay is universal since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, protein, RNA, and small molecules methyltransferases. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background and low C.V. The assay is robust (Z’ value > 0.7) and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates with no limitations on the use of high concentrations of substrates or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest. Citation Format: Said A. Goueli. Monitoring the activity of various classes of methylltransferases in vitro using a universal, homogenous and bioluminescent assay. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A31.