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Automation of a Generic Fluorescent Methyltransferase Activity Assay
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BioTek Instruments

Abstract

Epigenetic processes are attracting considerable attention in drug discovery as their fundamental roles in controlling normal cell development and contributions to disease states become more clearly defined. Methylation is a known ubiquitous covalent modification involved in regulation of a diverse range of biomolecules. As histone methylation is linked to certain disease states, including many cancer types, Histone methyltransferases (HMTs) are of particular interest as drug targets. A high-throughput screening (HTS)-ready, universal methyltransferase activity assay was recently developed based on competitive fluorescent polarization immunodetection of adenosine monophosphate (AMP), formed from the methyltransferase (MT) reaction product S-adenosylhomocysteine (SAH) in a dual enzyme coupling step.

Here we demonstrate automation of the assay in a 384-well microplate format suitable for HTS. A combination 8-channel and single channel liquid handling instrument was used to automate histone methyltransferase (HMT) G9a titration and transfer to the assay plate in quadruplicate. Automated serial dilution and transfer of the HMT inhibitor sinefungin was also performed using the liquid handler’s 8-channel head. During HMT EC80 determination, cofactor, substrate, stop buffer and detection reagent additions were automated using a non-contact dispenser. Dispensing of the EC80 concentration of HMT enzyme and all assay components were automated during HMT inhibitor IC50 determination. The G9a EC80 was determined to be approximately 80 ng/mL and used for subsequent inhibition studies, while the sinefungin IC50 value was determined to be 14.5 μM, showing excellent correlation with published values.

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