Automating A Direct, Cell-Based, Target-Compound Interaction Assay For G9a Histone Methyltransferase And Bromodomain Proteins

By Brad Larson and Peter Banks, Applications Department, BioTek Instruments, Inc.

Current biochemical assay technologies examine enzyme activity and G9a and BET bromodomain protein inhibition. Cell-based assays for these epigenetic players were previously limited to the detection of specific histone modifications using antibodies, with antibody specificity being an issue. Hence there exists a need for a robust, high throughput cell-based assay that can identify compound binding to a protein’s catalytic domain. In this study, we describe a novel, cell-based assay platform that uses Enzyme Fragment Complementation (EFC) Technology to detect the specific binding and direct protein engagement of potential small molecule inhibitors to G9a methyltransferase and multiple bromodomain proteins.

Automated assay procedures were carried out in 384-well format using high-throughput liquid handling and detection instrumentation. Initial experiments included cell number optimization and post-plating optimization of compound incubation times. This was done to maximize the signal-to-background between wells containing compound-bound target cells, and wells with target cells only, as well as a Z’-factor4 validation.