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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.

Original publication

DOI

10.1016/j.chembiol.2017.10.010

Type

Journal article

Journal

Cell Chem Biol

Publication Date

15/02/2018

Volume

25

Pages

206 - 214.e11

Keywords

ATP, BRET, NanoBRET, NanoLuc, crizotinib, dasatinib, kinase, profiling, selectivity, target engagement, Adenosine Triphosphate, Cell Survival, Dose-Response Relationship, Drug, Energy Transfer, Enzyme-Linked Immunosorbent Assay, HEK293 Cells, Humans, Mass Spectrometry, Molecular Structure, Phosphotransferases, Protein Kinase Inhibitors, Structure-Activity Relationship