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

  • Nat Commun. 2025 Dec 3;16(1):10129. doi: 10.1038/s41467-025-65558-6.
Elisabeth M Rothweiler  #  1  2 ,  Ani Michaud  #  3 ,  Jakub Stefaniak  1  2 ,  Usha Singh  1  2 ,  Brynwood B Mikulsky  3 ,  James D Vasta  3 ,  Michael T Beck  3 ,  Jennifer Wilkinson  3 ,  Jennifer A Ward  1  2 ,  Catherine M Rogers  1  2 ,  Esra Balıkçı  1  2 ,  Jeppe Tranberg-Jensen  1  2 ,  Jesper S Hansen  1  2 ,  Peter Loppnau  4 ,  Adrian Whitty  5 ,  Paul E Brennan  1  2  6 ,  Peter J Tonge  7  8 ,  Matthew B Robers  9 ,  Kilian V M Huber  10  11
Affiliations
  • 1. Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford, UK.
  • 2. Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford, UK.
  • 3. Promega Corporation, Madison, WI, USA.
  • 4. Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
  • 5. Department of Chemistry, Boston University, Boston, MA, USA.
  • 6. Alzheimer's Research UK Oxford Drug Discovery Institute, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford, UK.
  • 7. Center for Advanced Study of Drug Action, Department of Chemistry, Stony Brook University, Stony Brook, NY, USA. [email protected].
  • 8. Department of Biomedical Genetics, University of Rochester, Rochester, NY, USA. [email protected].
  • 9. Promega Corporation, Madison, WI, USA. [email protected].
  • 10. Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford, UK. [email protected].
  • 11. Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford, UK. [email protected].
  • # Contributed equally.
PMID: 41339334 DOI: 10.1038/s41467-025-65558-6
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.

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