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

Author

Listed:
  • Matthew B. Robers

    (Promega Corporation)

  • Melanie L. Dart

    (Promega Corporation)

  • Carolyn C. Woodroofe

    (Promega Biosciences Incorporated
    Present address: National Institutes of Health, 9800 Medical Center Drive, Rockville MD 20850, USA.)

  • 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)

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.

Suggested Citation

  • 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 & Andr, 2015. "Target engagement and drug residence time can be observed in living cells with BRET," Nature Communications, Nature, vol. 6(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms10091
    DOI: 10.1038/ncomms10091
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