A BRET biosensor for measuring uncompetitive engagement of PRMT5 complexes in cells
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
- Esra Balıkçı (ORCID: https://orcid.org/0009-0000-9160-5790)
- P. Loppnau (ORCID: https://orcid.org/0009-0004-0002-355X)
- Peter J. Tonge (ORCID: https://orcid.org/0000-0003-1606-3471)
- Ani Michaud (ORCID: https://orcid.org/0000-0001-9039-3212)
- James D. Vasta (ORCID: https://orcid.org/0000-0001-8511-2113)
- Catherine Rogers (ORCID: https://orcid.org/0000-0001-7005-4459)
- Matthew B. Robers (ORCID: https://orcid.org/0000-0001-8505-5227)
- Jennifer Ward (ORCID: https://orcid.org/0000-0002-8735-5263)
- Jakub Stefaniak (ORCID: https://orcid.org/0000-0001-9151-4485)
- K. Huber (ORCID: https://orcid.org/0000-0002-1103-5300)
- Paul E. Brennan (ORCID: https://orcid.org/0000-0002-8950-7646)
- Adrian Whitty (ORCID: https://orcid.org/0000-0002-0331-4369)
- Jesper S. Hansen (ORCID: https://orcid.org/0000-0003-1638-1285)
- Elisabeth M. Rothweiler (ORCID: https://orcid.org/0000-0003-3303-2044)
- Michael Beck (ORCID: https://orcid.org/0000-0003-2523-6266)
- Jeppe Tranberg-Jensen
- Usha Singh (ORCID: https://orcid.org/0000-0003-4177-0848)
- Jennifer Wilkinson (ORCID: https://orcid.org/0009-0002-1994-715X)
- Brynwood B. Mikulsky
Institutions
- Boston University (US)
- University of Toronto (CA)
- Promega (United States) (US)
- Discovery Centre (CA)
- Nuffield Orthopaedic Centre (GB)
- Structural Genomics Consortium (CA)
- University of Oxford (GB)
- Alzheimer’s Research UK (GB)
- Jenner Institute (GB)
- University of Rochester (US)
- Stony Brook University (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-12-03
- DOI
- https://doi.org/10.1038/s41467-025-65558-6
- Citations
- 2
- Primary Topic
- Cancer-related gene regulation
- Type
- article
- Field-Weighted Citation Impact
- 1.61
Funders
- Bayer
- McGill University
- Diamond Light Source
- European Federation of Pharmaceutical Industries and Associations
- European Commission
- National Institutes of Health
- Medical Research Council
- Engineering and Physical Sciences Research Council