Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors

Methyltransferases
(MTases) play diverse roles in cellular processes. Aberrant methylation
levels have been implicated in many diseases, indicating the need
for the identification and development of small molecule inhibitors
for each MTase. Specific inhibitors can serve as probes to investigate
the function and validate therapeutic potential for the respective
MTase. High-throughput screening (HTS) is a powerful method to identify
initial hits for further optimization. Here, we report the development
of a fluorescence-based MTase assay and compare this format with the
recently developed MTase-Glo luminescence assay for application in
HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model
system, we miniaturized to 1536-well quantitative HTS format. Through
a pilot screen of 1428 pharmacologically active compounds and subsequent
validation, we discovered that MTase-Glo produced lower false positive
rates than the fluorescence-based MTase assay. Nevertheless, both
assays displayed robust performance along with low reagent requirements
and can potentially be employed as general HTS formats for the discovery
of inhibitors for any MTase.