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.

Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound–target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.

The binding affinity and kinetics of target engagement are fundamental to establishing structure–activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.