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

Matthew B. Robers (), Melanie L. Dart, Carolyn C. Woodroofe, Chad A. Zimprich, Thomas A. Kirkland, Thomas Machleidt, Kevin R. Kupcho, Sergiy Levin, James R. Hartnett, Kristopher Zimmerman, Andrew L. Niles, Rachel Friedman Ohana, Danette L. Daniels, Michael Slater, Monika G. Wood, Mei Cong, Yi-Qiang Cheng and Keith V. Wood
Additional contact information
Matthew B. Robers: Promega Corporation
Melanie L. Dart: Promega Corporation
Carolyn C. Woodroofe: Promega Biosciences Incorporated
Chad A. Zimprich: Promega Corporation
Thomas A. Kirkland: Promega Biosciences Incorporated
Thomas Machleidt: Promega Corporation
Kevin R. Kupcho: Promega Corporation
Sergiy Levin: Promega Biosciences Incorporated
James R. Hartnett: Promega Corporation
Kristopher Zimmerman: Promega Corporation
Andrew L. Niles: Promega Corporation
Rachel Friedman Ohana: Promega Corporation
Danette L. Daniels: Promega Corporation
Michael Slater: Promega Corporation
Monika G. Wood: Promega Corporation
Mei Cong: Promega Corporation
Yi-Qiang Cheng: UNT System College of Pharmacy, University of North Texas Health Science Center
Keith V. Wood: Promega Corporation

Nature Communications, 2015, vol. 6, issue 1, 1-10

Abstract: Abstract 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.

Date: 2015
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DOI: 10.1038/ncomms10091

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