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Nat Commun. 2015 Dec 3;6:10091. doi: 10.1038/ncomms10091.

Target engagement and drug residence time can be observed in living cells with BRET.

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Promega Corporation, 2800 Woods Hollow Road, Fitchburg, Wisconsin 53711, USA.
Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, USA.
UNT System College of Pharmacy, Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, Texas, USA.


The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.

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