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

Elisabeth M. Rothweiler, Ani Michaud, Jakub Stefaniak, Usha Singh, Brynwood B. Mikulsky, James D. Vasta, Michael T. Beck, Jennifer Wilkinson, Jennifer A. Ward, Catherine M. Rogers, Esra Balıkçı, Jeppe Tranberg-Jensen, Jesper S. Hansen, Peter Loppnau, Adrian Whitty, Paul E. Brennan, Peter J. Tonge (), Matthew B. Robers () and Kilian V. M. Huber ()
Additional contact information
Elisabeth M. Rothweiler: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Ani Michaud: Promega Corporation
Jakub Stefaniak: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Usha Singh: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Brynwood B. Mikulsky: Promega Corporation
James D. Vasta: Promega Corporation
Michael T. Beck: Promega Corporation
Jennifer Wilkinson: Promega Corporation
Jennifer A. Ward: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Catherine M. Rogers: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Esra Balıkçı: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Jeppe Tranberg-Jensen: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Jesper S. Hansen: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Peter Loppnau: University of Toronto, Structural Genomics Consortium
Adrian Whitty: Boston University, Department of Chemistry
Paul E. Brennan: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine
Peter J. Tonge: Stony Brook University, Center for Advanced Study of Drug Action, Department of Chemistry
Matthew B. Robers: Promega Corporation
Kilian V. M. Huber: University of Oxford, Old Road Campus, Centre for Medicines Discovery, Nuffield Department of Medicine

Nature Communications, 2025, vol. 16, issue 1, 1-16

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-65558-6

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