Scholarlink

<p>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.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年12月3日

查看全文
  • 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,
  • Kilian V. M. Huber

摘要

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.