App Note: Automating a Cell-Based Target-Compound Assay for Methyltransferase and Bromodomain Proteins using the InCELL Hunter Platform

App Note: Automating a Cell-Based Target-Compound Assay for Methyltransferase and Bromodomain Proteins using the InCELL Hunter Platform
Version:
21224

Year:
2021

Histone modifications are essential in eukaryotic gene expression and regulation, and are driven by histone writer, eraser, and reader proteins. These histone tail changes are dynamic, and a normal part of a cell’s embryonic differentiation from its original totipotent state. However, aberrant histone modifications are linked to numerous disease states1, including many human cancers. For example, the lysine-specific SET domain, containing G9a methyltransferase writer protein, is over-expressed in various cancers. It is also deficient in CD4+ T helper cells, leading to increased rates of intestinal infection2. In another example, the BET bromodomain reader protein Brd4 is implicated in NUT midline carcinoma (NMC) through a fusion of the protein and NUT3. Due to these and other findings, methlytransferase and bromodomain proteins are targets for numerous drug discovery projects.
 
Current biochemical assay technologies examine the enzyme activity of methlytransferase and bromodomain protein inhibition. These assays lack monitoring compound-target engagement in the cellular milieu, which is essential to validate the pharmacology of drug candidates. Additionally, cell-based assays for analyzing inhibitors to epigenetic proteins were previously limited to the detection of specific histone modifications using antibodies. When antibodies are not available, another method is needed. Hence there exists a need for a robust, high-throughput cell-based assay that can identify compound binding to a protein’s catalytic domain without the dependency on antibodies. In this study, we describe the antibody-free, cell-based assay platform called InCELL Hunter that enables the detection of specificity binding and direct protein engagement of potential small molecule inhibitors to G9a methyltransferase and multiple bromodomain proteins.