Matt Robers, Promega

We will describe the application of an energy transfer technique (NanoBRET) that enables the first live cell approach to broadly profile compound fractional occupancy and residence time against a variety of target classes including kinases, integral membrane proteins, and E3 ligases.

Using this technique, broad spectrum evaluation of compound engagement can be measured against over 300 full length human kinases in live cells.   For this kinase diversity set, target engagement potencies correlate strongly with potencies using more traditional pathway analysis readouts, thus providing a platform to establish structure-activity relationships (SARs) for kinase chemical probes or lead drug molecules. As this approach can also be performed in real time, it is feasible to profile intracellular compound binding kinetics and quantify target residence time in a simplified format. When target engagement analysis is performed under equilibrium and non-equilibrium conditions, surprising kinetic selectivity profiles are observed for certain clinically-relevant kinase inhibitors. These results support evaluation of intracellular compound residence time, in addition to potency, as a critical metric to evaluate target selectivity.

We have further advanced the NanoBRET platform for interrogation of PROTAC pharmacology in live cells. We will present a luminescent platform that can be combined for mechanistic interrogation of intracellular PROTAC permeability, E3 ligase occupancy, and real-time target degradation. Together, these approaches can allow for improved translation of binding pharmacology with functional pharmacology in living cells.