Abstract
SummaryFor 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.
Highlights
Kinases represent the largest group of enzymes in the human proteome and are functionally integral to signal transduction
Intracellular target selectivity is fundamental to pharmacological mechanism
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
Summary
Kinases represent the largest group of enzymes in the human proteome and are functionally integral to signal transduction. The majority of clinically relevant kinase inhibitors engage their target proteins at the kinase active site, and compete with high intracellular concentrations (1–10 mM) of the cosubstrate ATP (Wu et al, 2016; Becher et al, 2013). Selective modulation of individual kinases represents a challenge due to the highly conserved structure of the ATP binding pocket, most notably within the protein kinase family. Kinase inhibitors may demonstrate polypharmacology over a number of intracellular targets, a feature that often underlies the therapeutic potential of lead drug candidates. As our understanding of the therapeutic relevance of the human kinome deepens, a growing need has emerged for methods capable of quantifying kinase inhibitor occupancy, selectivity, and affinity within the cellular environment where engagement would naturally occur
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