A Live Cell BRET Probe to Quantify PRMT5-MTA Uncompetitive Target Engagement

The PRMT5 methyltransferase plays a global role in cell physiology and is an established therapeutic target in cancer. In 10% of human cancers, deletion of the MTAP gene results in accumulation of MTA, exposing a synthetic lethality and opportunity for precision medicine by selective targeting of PRMT5 in this context. Reported small molecule PRMT5 inhibitors engage either cosubstrate (SAM) or peptide-substrate pockets through diverse mechanisms. A subset of chemotypes demonstrate uncompetitive engagement with SAM or its inhibitory metabolic precursor, MTA. Here we describe the development of a cell-permeable BRET probe CBH-002 that acts as a dynamic biosensor of the intracellular SAM/MTA pool. Using this probe, we evaluate a range of diverse PRMT5 inhibitors to mechanistically characterize and quantify uncompetitive target engagement and ternary complex formation at PRMT5-SAM and -MTA complexes in live cells, enabling direct insights into drug mechanism-of-action and metabolite-dependent response to inhibitors.