A BRET biosensor for measuring uncompetitive engagement of PRMT5 complexes in cells

Journal article

Protein arginine methyl transferase 5 (PRMT5) plays a global role in cell physiology and is an established therapeutic target in cancer. In approximately 10-15% of human cancers, deletion of the methylthioadenosine phosphorylase (MTAP) gene results in accumulation of methylthioadenosine (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 S-adenosyl methionine (SAM) or peptide-substrate pockets through diverse mechanisms. A subset of chemotypes demonstrate uncompetitive engagement with SAM or its inhibitory metabolic precursor, MTA. Although uncompetitive engagement can be evaluated in cell-free systems, no methods exist to directly assess this in cells. Here, we describe the development of a fluorescent probe that acts as a dynamic BRET biosensor of the intracellular SAM/MTA pool that overcomes the current limitations of competitive binding analyses. Using this biosensor, we evaluate a range of diverse PRMT5 inhibitors to mechanistically characterize and quantify uncompetitive target engagement as well as ternary complex formation at PRMT5-SAM and PRMT5-MTA complexes in live cells, enabling direct insights into drug mechanism-of-action and metabolite-dependent responses of inhibitors.

Authors: Rothweiler, EM; Michaud, A; Stefaniak, J; Singh, U; Mikulsky, BB

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Nature Communications
• Volume: 16
• Issue: 1
• Article number: 10129
• Publication date: 2025-12-03
• Acceptance date: 2025-10-13
• DOI: 10.1038/s41467-025-65558-6
• EISSN: 2041-1723
• ISSN: 2041-1723
• Language: English