An Automated Profiling Application For G9a Histone Methyltransferase And Bromodomain Proteins

Histone modifications play an essential role in the regulation of eukaryotic gene expression and are driven by histone writer, eraser and reader proteins. These changes to the N-terminus of histone tails are dynamic in nature and are a normal part of the embryonic differentiation of cells from their original totipotent state. However, aberrant modifications have been linked to numerous disease states, and many human cancers. The SET domaincontaining G9a methyltransferase writer protein is over-expressed in various cancers, and is also deficient in CD4+ T helper cells, leading to increased rates of intestinal infection (Lehnertz et al., 2010). In addition, the BET bromodomain reader protein Brd4 was implicated in NUT midline carcinoma (NMC) through a fusion of the protein and NUT (French et al., 2007). Due to these and other findings, G9a and BET bromodomain proteins have become the target for numerous drug discovery projects.

Current assay technologies exist in a biochemical format to examine enzyme activity and inhibition of G9a and BET bromodomain proteins. The existing cell-based assays for these epigenetic players are only limited to the detection of specific histone modifications using antibodies, with antibody specifi city being an issue. Hence there still exists a need for a robust and high throughput assay that can identify the binding of compounds to the catalytic domain of these proteins in a cell-based format. In this study, we describe a novel, cell-based assay platform that uses the robust, yet simple Enzyme Fragment Complementation (EFC) Technology. The assay measures compound binding to an intracellular target by detecting changes in protein stability. In this assay, the target is fused to the enhanced ProLabel™ (ePL) enzyme fragment and expressed in the selected cell background. The amount of fusion is quantifi ed through the addition of substrate and com plementary enzyme acceptor fragment (EA). The assay can be used to detect the specifi c binding and direct protein engagement of potential small molecule inhibitors to the G9a methyltransferase and Brd2, Brd4, and BrdT bromodomain proteins.

The assay procedures were carried out in 384-well format using high throughput liquid handling and detection instrumentation. Initial experiments included optimization of cell number and post-plating incubation time, as well as a Z’-factor validation. Screening of a small focused library was then carried out with each target assay. Finally, dose response testing was completed using “hits” from the compound screen, as well as known inhibitor positive control compounds.