Glutathione peroxidase 4 (GPX4) plays a crucial role in the ferroptosis pathway, emerging as a potential drug target in the treatment of refractory tumors. Unfortunately, the development of GPX4-targeted treatment has been very limited due to the poor selectivity and drug-like properties of current GPX4 inhibitors. Here, we report a proof-of-concept study of potent anti-GPX4 nanobodies, successfully identified through immunizing Bactrian camels and constructing a phage library. Utilizing a cell-penetrating peptide fusion strategy, these nanobodies with high affinities to GPX4 efficiently internalized in cells and formed the basis for further applications. In particular, 12E significantly inhibited cellular GPX4 and consequently induced remarkable ferroptosis in cancer cells. Furthermore, 12E could impair zebrafish dorsal organizer formation in vivo, as evidenced by a phenotype comparable to that observed in zebrafish with the gpx4b gene knocked out. The new GPX4-inhibiting nanobody described here exhibits superior proteome-wide selectivity and a vastly improved safety profile compared to existing GPX4 inhibitors. These incredible features of 12E, as an anti-GPX4 nanobody, may not only contribute to ferroptosis-related anticancer treatment but also establish a new paradigm for nanobodies in drug development for traditionally undruggable targets.
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