title: A substrate-independent TR-FRET histone deacetylase inhibitor assay.
abstract: Developing molecularly targeted therapeutics with minimal off-target effects is facilitated by an understanding of compound selectivity. However, for HDAC inhibitors, a clear understanding of specificity has been challenging. In particular, it has been suggested that use of nonspecific substrates and the presence of multiple HDAC activities in enzyme preparations may complicate interpretation of inhibitor experiments. To overcome these and other potential limitations of activity-based HDAC assays, the authors have developed an assay format based on measurement of the binding affinity of inhibitors rather than measurement of enzyme activity. One advantage of this format is that it does not require use of a substrate and thus ameliorates concerns about lack of specificity of existing substrates. This assay is based on an Alexa Fluor® 647-labeled HDAC inhibitor or "tracer," which binds with a high affinity to Class I and Class IIb HDACs. Unlike activity assays, which can be affected by the presence of residual untagged endogenous HDACs from the host expression system, the signal in this format is dependent on the presence of an epitope tag on the specific HDAC of interest. The authors demonstrate the utility of this method by determining the potencies of commonly used inhibitors for six human HDACs.
authors.affiliation: Life Technologies, Madison, Wisconsin 53719, USA.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Marks
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Fakhoury
authors.forename: William J
authors.initials: WJ
authors.lastname: Frazee
authors.forename: Hildegard C
authors.initials: HC
authors.lastname: Eliason
authors.forename: Steven M
authors.initials: SM
authors.lastname: Riddle
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding / Q000187:drug effects
chemical_list: D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases