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  3. Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement

  • Cell Chem Biol. 2018 Feb 15;25(2):206-214.e11. doi: 10.1016/j.chembiol.2017.10.010.
James D Vasta 1,  Cesear R Corona 2,  Jennifer Wilkinson 1,  Chad A Zimprich 1,  James R Hartnett 1,  Morgan R Ingold 1,  Kristopher Zimmerman 1,  Thomas Machleidt 1,  Thomas A Kirkland 2,  Kristin G Huwiler 1,  Rachel Friedman Ohana 1,  Michael Slater 1,  Paul Otto 1,  Mei Cong 1,  Carrow I Wells 3,  Benedict-Tilman Berger 4,  Thomas Hanke 5,  Carina Glas 6,  Ke Ding 7,  David H Drewry 3,  Kilian V M Huber 6,  Timothy M Willson 3,  Stefan Knapp 4,  Susanne Müller 8,  Poncho L Meisenheimer 2,  Frank Fan 1,  Keith V Wood 1,  Matthew B Robers 9
Affiliations

Affiliations

  • 1 Promega Corporation, 2800 Woods Hollow Road, Fitchburg, WI 53711, USA.
  • 2 Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, CA 93401, USA.
  • 3 Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
  • 4 Structural Genomics Consortium, Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany; Structural Genomics Consortium, Buchmann Institute for Molecular Life Sciences, Johann Wolfgang Goethe-University, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany.
  • 5 Structural Genomics Consortium, Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
  • 6 Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
  • 7 State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou 510530, China; School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
  • 8 Structural Genomics Consortium, Buchmann Institute for Molecular Life Sciences, Johann Wolfgang Goethe-University, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany.
  • 9 Promega Corporation, 2800 Woods Hollow Road, Fitchburg, WI 53711, USA. Electronic address: matt.robers@promega.com.
PMID: 29174542 DOI: 10.1016/j.chembiol.2017.10.010
Abstract

For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.

Keywords

ATP; BRET; NanoBRET; NanoLuc; crizotinib; dasatinib; kinase; profiling; selectivity; target engagement.

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