2. Academic Validation
  3. A BRET biosensor for measuring uncompetitive engagement of PRMT5 complexes in cells

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

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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