Objective

We present the first technique to broadly and quantitatively profile kinase target engagement under physiological conditions, without disruption of cellular membrane integrity.   NanoBRET enables a biophysical assessment of compound engagement and residence time for chosen intracellular targets.  A quantitative capability is achieved in living cells, via energy transfer from cell-permeable tracers reversibly engaged to selected NanoLuc-tagged target proteins.   As the specificity of the BRET signal is dictated by the placement of NanoLuc on the chosen target, a diverse set of broad-coverage tracers support an HTS-compatible method to profile the isozyme-specific affinity and binding kinetics over entire enzyme classes.  This technique has enabled a quantitative analysis of compound binding against > 200 selected full-length protein kinases, including a key panel of integral membrane receptors.  A systematic comparison of kinase target engagement in live cells vs. lysates allowed a mechanistic study on the influences of local intracellular ATP concentrations on kinase affinity profiles.   These results demonstrate the value of assessing kinase target engagement in the presence of the intracellular milieu that may influence potency and selectivity profiles. Furthermore, analysis of intracellular residence time reveals kinetic selectivity for various clinically-relevant kinase inhibitors in living cells.  O