USP8/GPX4-based live-cell quantitative FRET imaging for ferroptosis drug screening

  • Biochim Biophys Acta Mol Cell Res. 2026 Jun;1873(5):120159. doi: 10.1016/j.bbamcr.2026.120159.
Xinghong Cai  1 ,  Ziru Wu  1 ,  Zhiyu Xiao  1 ,  Guiping Zhuang  1 ,  Zhengfei Zhuang  2
Affiliations
  • 1. MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China; Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China.
  • 2. MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China; Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China. Electronic address: [email protected].
PMID: 42140526 DOI: 10.1016/j.bbamcr.2026.120159
Abstract

Targeting the functional interface between USP8 and GPX4 to disrupt Ferroptosis defense represents a novel therapeutic strategy in Cancer. Currently, there is a lack of drug screening models targeting the USP8/GPX4 interaction. This study utilized human breast Cancer cell lines as a research model and established a drug screening platform capable of real-time, quantitative detection of USP8/GPX4 interactions in live cells based on fluorescence resonance energy transfer (FRET) imaging technology. We first confirmed that USP8 promotes malignant growth in breast Cancer cells. Using the USP8 Inhibitor DUB-IN-2 as a positive control alongside key Ferroptosis markers, we employed FRET technology to quantitatively assess the interaction efficiency (ED) between USP8 and GPX4. Screening of candidate compounds revealed that treatment with ML162, RSL3, and sorafenib resulted in maximum FRET efficiencies (EDmax) of 0.492, 0.483, and 0.503, respectively-significantly lower than that of control cells (0.548)-indicating effective disruption of USP8/GPX4 binding. This study successfully establishes a novel and efficient FRET-based drug screening model for targeting Ferroptosis.

Keywords
Drug screening; FRET; Ferroptosis; GPX4; USP8.
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