Intracellular target selectivity is a key aspect of pharmacological mechanism. Although a number of acellular techniques have been developed to quantitatively measure kinase binding or enzymatic inhibition, currently no biophysical approaches exist that offer quantitative analysis of target occupancy or drug affinity in live cells. Here we report the application of an energy transfer technique (NanoBRET) that enables the first quantitative approach to broadly profile kinase occupancy under thermodynamic equilibrium with the test compound inside live cells. This approach enabled a mechanistic interrogation of the potency offsets observed between cellular and acellular analysis of kinase engagement, and uncovered improved intracellular selectivity profiles for certain clinically-relevant multi-kinase inhibitors. The energy transfer technique can also be performed in real time, enabling assessments of compound residence time. Applications of NanoBRET for the measurements of target engagement and residence time for ATP-competitive and non-competitive inhibitors will be presented.
Sr Research Scientist & Group Leader
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