Abstract
We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.
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Data availability
The data that support the findings of this study are available from the corresponding authors upon request.
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Acknowledgements
We thank V. Kaushik for assistance with intact protein mass spectrometry experiments; M. Palte for assistance with preparing phosphatidylcholine hydroperoxide; B. Budnik and R. Robinson for assistance with proteomics. This work was supported in part by the National Institute of General Medical Sciences (R01GM038627 and R35GM127045 awarded to S.L.S.) and through a collaboration between the Broad Institute and Bayer AG.
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J.K.E. conceived and designed experiments. J.K.E., L.F., K.E.L., S.G. and C.M. performed chemical synthesis and compound characterization. J.K.E., R.A.R., M.J.R., L.L.C. and V.S.V. maintained cell cultures and performed viability, cellular thermal shift and western blotting experiments. D.M. and A.H. designed the cloning approach and expressed, purified and characterized recombinant wild-type GPX4 protein. V.B., A.H., D.M. and J.K.E. performed cellular and biochemical mass spectrometry binding assays. M.N. performed metabolite-ID studies. R.C.H., K.Z., A.K., S. Chen and B.B. contributed tools and reagents for protein characterization experiments. P.A.C. contributed to analysis of cell viability data. R.A.R. and S. Christian performed cellular lipid peroxidation assays. R.N. performed formulation work. A.L.E. performed in vivo experiments. J.K.E., V.S.V. and S.L.S. initiated the project and wrote the manuscript. V.S.V. and S.L.S. directed the project.
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S.L.S. serves on the Board of Directors of the Genomics Institute of the Novartis Research Foundation (“GNF”); is a shareholder and serves on the Board of Directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics; is a shareholder and advises Kojin Therapeutics, Kisbee Therapeutics, Decibel Therapeutics and Eikonizo Therapeutics; serves on the Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma Foundation, Exo Therapeutics, and F-Prime Capital Partners; and is a Novartis Faculty Scholar. P.A.C. is an advisor to Pfizer, Inc. D.M., A.H., K.Z., M.N., V.B., R.C.H., S.G., S. Christian, R.N. and A.L.E. are employed by Bayer AG.
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Eaton, J.K., Furst, L., Ruberto, R.A. et al. Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles. Nat Chem Biol 16, 497–506 (2020). https://doi.org/10.1038/s41589-020-0501-5
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DOI: https://doi.org/10.1038/s41589-020-0501-5
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