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.

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.