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

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.