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

2017; Elsevier BV; Volume: 25; Issue: 2 Linguagem: Inglês

10.1016/j.chembiol.2017.10.010

2451-9456

James D. Vasta, Cesear Corona, Jennifer Wilkinson, Chad Zimprich, James R. Hartnett, Morgan R. Ingold, Kristopher Zimmerman, Thomas Machleidt, Thomas A. Kirkland, Kristin G. Huwiler, Rachel Friedman Ohana, Michael R. Slater, Paul Otto, Mei Cong, Carrow I. Wells, Benedict‐Tilman Berger, Thomas Hanke, Carina Glas, Ke Ding, David H. Drewry, K. Huber, Timothy M. Willson, Stefan Knapp, Susanne Müller, Poncho Meisenheimer, Frank Fan, Keith V. Wood, Matthew B. Robers,

HER2/EGFR in Cancer Research

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.