Methyltransferase inhibition assay development

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DESCRIPTION (provided by applicant): Methyltransferases mediate biotransformations in a wide range of important processes - these include biosynthesis of neurotransmitters, and steroid hormones, as well as post-synthetic modifications of DNA, RNA, and protein. Methyltransferases also play a major role in epigenetic alterations in the genome seen in a growing list of cancer cell types. The diverse critical roles played by methyltransferases makes them attractive targets for the discovery of compounds that differentially modulate their activity. The goal of this proposal will be to design a simple reproducible high throughput enzyme-based assay to facilitate the discovery of agents that inhibit methylation of the 5'- mRNA "cap", a sequence, 5'-GpppA, present in eucaryotic mRNA's. In the proposed studies, a Dengue virus-encoded cap-methyltransferase will be used as a model enzyme target that converts the partially methylated to fully methylated cap with S-adenosyl-methionine serving as methyl donor. In the proposed assay, the association of a fluorescent "surrogate" peptide aptamer with the target methyltransferase will be measured using fluorescence polarization. The proposed assay is a "mix- and-measure" type of determination that is based on the principle that perturbation of a protein by a drug-like compound at a discrete site can perturb the binding of another ligand, the surrogate ligand, bound elsewhere. Unfortunately, commonly used assays that measure cap-methyltransferase activity are best suited for low-throughput screening applications because they: (a) use radioactivity, (b) require a substrate that is laborious to prepare, (c) measure a product that requires chromatographic characterization, or, must be precipitated, filtered, and counted. The surrogate ligand assay that we propose to develop: (a) does not require radioactive reagents, (b) can be implemented robotically with simple liquid addition transfers ("mix and measure"), and (c) gives a reproducible readout that is stable over several hours. The long-range goal of these studies is to extend the type of primary assay described herein to the discovery of a broader range of active compounds that includes methyltransferases capable of modifying other substrates - proteins, RNA, or DNA. PUBLIC HEALTH RELEVANCE: High throughput screening is an essential part of the discovery of new drugs. The process starts with the discovery/design of compounds that affect physiological functions at the cell or enzyme level, followed by determination of their suitability for use as drugs. We propose to develop high throughput screens for the first phase of this process to enable the discovery of compounds that inhibit methyltransferases, a group of enzymes that play a direct role in various aspects of stroke, heart attack, cancer, and infectious diseases.