An engineered cysteine sensor optimized for high-throughput screening identifies regulators of intracellular thiol levels.

Dysregulation of cysteine-dependent processes is implicated in many diseases, including cancer. Despite the importance of cysteine in crucial cellular functions, including protein synthesis, redox balance, and glutathione production, a lack of efficient assays to measure cellular cysteine has limited efforts to identify agents that affect physiological cysteine levels. We employed circular permutation to engineer a fluorescent sensor that changes conformation upon cysteine binding. Biochemical experiments showed that this sensor is selective for cysteine, operating in the 10 μM-10 mM range. To demonstrate the sensor's applicability, we performed high-throughput screens for compounds that reduce cellular cysteine. Liquid chromatography of cell extracts validated the effect of two hit compounds, and mechanistic investigations showed that one was dependent on the anticancer target, xCT. Future application of this sensor in cell biology and drug discovery will advance understanding of cysteine metabolism and drive the development of therapeutics that restore cysteine homeostasis.

• Publication year

  2025

• Venue

  Cell Chemical Biology

• Publication date

  2025-11-01

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.chembiol.2025.10.006PMID 41218609
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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