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

Thursday, September 17, 2015 — Poster Session II

12:00 p.m. – 1:30 p.m.
FAES Terrace

* FARE Award Winner


  • JH Shrimp
  • DC Montgomery
  • JL Meier


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.

Category: Chemical Biology