1. Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement.
- Author
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Vasta JD, Corona CR, Wilkinson J, Zimprich CA, Hartnett JR, Ingold MR, Zimmerman K, Machleidt T, Kirkland TA, Huwiler KG, Ohana RF, Slater M, Otto P, Cong M, Wells CI, Berger BT, Hanke T, Glas C, Ding K, Drewry DH, Huber KVM, Willson TM, Knapp S, Müller S, Meisenheimer PL, Fan F, Wood KV, and Robers MB
- Subjects
- Cell Survival, Dose-Response Relationship, Drug, Energy Transfer, Enzyme-Linked Immunosorbent Assay, HEK293 Cells, Humans, Mass Spectrometry, Molecular Structure, Phosphotransferases metabolism, Protein Kinase Inhibitors chemistry, Structure-Activity Relationship, Adenosine Triphosphate metabolism, Phosphotransferases antagonists & inhibitors, Protein Kinase Inhibitors pharmacology
- 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., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
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