A signal-amplifiable, ligand-displacement assay for small molecule epigenetic modulator discovery

FARE Award Winner

Authors

• JH Shrimp
• DC Montgomery
• JL Meier

Abstract

Lysine acetyltransferase (KAT) enzymes are key regulators of gene expression programs in many diseases, including cancer. Few KAT inhibitors are currently known. To address current challenges, we have developed a highly sensitive, ligand-displacement assay for KAT enzymes. This strategy uses a proximity immunoassay approach known as AlphaScreen to detect the binding interaction of a biotin-labeled KAT cofactor analogue (biotin-H3K14-CoA) and a His-tagged KAT (Gcn5). This KAT-cofactor interaction brings a singlet-oxygen producing streptavidin “donor” bead into close proximity to an anti-His “acceptor” bead that upon reaction with singlet oxygen, produces an intense fluorescence signal. Since each KAT-cofactor interaction causes multiple singlet oxygen release events, the fluorescence signal is greatly amplified compared to traditional binding assays. This signal amplification permits highly sensitive detection of Gcn5 (signal: noise ratio >400:1), which is reduced in the presence of competitive inhibitors, thus providing a powerful platform for small-molecule inhibitor discovery. Using Gcn5 as a prototypical KAT we have demonstrated the assay is effective in a miniaturized (384-well) format and enables the rapid, quantitative determination of dissociation constants for known Gcn5-interacting ligands. Current efforts are focused on applying this approach in screening efforts to identify small molecules able to probe the targetable role of Gcn5 in c-Myc regulated gene expression. More broadly, we envision this approach may provide a general strategy for the development of highly sensitive activity assays for “orphan” chromatin modifiers that utilize a cofactor (i.e. acetyl-CoA, ATP, NAD+) but whose substrate is not known.

Scientific Focus Area: Chemical Biology

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