Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: ML210 is a selective covalent inhibitor of cellular GPX4.
Fig. 2: ML210 requires the intact-cell context to bind GPX4.
Fig. 3: ML210 requires conversion in cells to the α-nitroketoxime JKE-1674.
Fig. 4: Dehydration of JKE-1674 yields a nitrile-oxide electrophile that binds GPX4.
Fig. 5: Diverse masked nitrile oxides target GPX4.
Fig. 6: Profiling of structurally diverse GPX4 inhibitors in cellular and pharmacokinetic assays.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.


  1. 1.

    Viswanathan, V. S. et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature 547, 453–457 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Hangauer, M. J. et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 551, 247–250 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Tsoi, J. et al. Multi-stage differentiation defines melanoma subtypes with differential vulnerability to drug-induced iron-dependent oxidative stress. Cancer Cell 33, 890–904 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Thomas, J. P., Geiger, P. G., Maiorino, M., Ursini, F. & Girotti, A. W. Enzymatic reduction of phospholipid and cholesterol hydroperoxides in artificial bilayers and lipoproteins. Biochim. Biophys. Acta Lipids Lipid Metab. 1045, 252–260 (1990).

    CAS  PubMed  Google Scholar 

  6. 6.

    Kühn, H. & Borchert, A. Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes. Free Radic. Biol. Med. 33, 154–172 (2002).

    PubMed  Google Scholar 

  7. 7.

    Scheerer, P. et al. Structural basis for catalytic activity and enzyme polymerization of phospholipid. Biochemistry 46, 9041–9049 (2007).

    CAS  PubMed  Google Scholar 

  8. 8.

    Borchert, A. et al. Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1863, 1095–1107 (2018).

    CAS  Google Scholar 

  9. 9.

    Yang, W. S. et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc. Natl Acad. Sci. USA 113, E4966–E4975 (2016).

    CAS  PubMed  Google Scholar 

  10. 10.

    Shimada, K. et al. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis. Nat. Chem. Biol. 12, 497–503 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Yang, W. S. & Stockwell, B. R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 15, 234–245 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Sakamoto, K. et al. Discovery of GPX4 inhibitory peptides from random peptide T7 phage display and subsequent structural analysis. Biochem. Biophys. Res. Commun. 482, 195–201 (2017).

    CAS  PubMed  Google Scholar 

  13. 13.

    Jiang, C., Chen, R., Pandey, A., Kalita, B. & Duraiswamy, A. J. Compounds and method of use. US patent 2019/0263802 A1. 1–292 (2019).

  14. 14.

    Weïwer, M. et al. Development of small-molecule probes that selectively kill cells induced to express mutant RAS. Bioorg. Med. Chem. Lett. 22, 1822–1826 (2012).

    PubMed  Google Scholar 

  15. 15.

    Seashore-Ludlow, B. et al. Harnessing connectivity in a large-scale small-molecule sensitivity dataset. Cancer Disco. 5, 1210–1223 (2015).

    CAS  Google Scholar 

  16. 16.

    Rees, M. G. et al. Correlating chemical sensitivity and basal gene expression reveals mechanism of action. Nat. Chem. Biol. 12, 109–116 (2015).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Basu, A. et al. An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules. Cell 154, 1151–1161 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Gaschler, M. M. et al. FINO2 initiates ferroptosis through GPX4 inactivation and iron oxidation. Nat. Chem. Biol. 14, 507–515 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Dixon, S. J. et al. Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Skouta, R. et al. Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models. J. Am. Chem. Soc. 136, 4551–4556 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Zilka, O. et al. On the mechanism of cytoprotection by ferrostatin-1 and liproxstatin-1 and the role of lipid peroxidation in ferroptotic cell death. ACS Cent. Sci. 3, 232–243 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Molina, D. M. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

    CAS  Google Scholar 

  23. 23.

    Gao, J. et al. Selenium-encoded isotopic signature targeted profiling. ACS Cent. Sci. 4, 960–970 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Friedmann Angeli, J. P. et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol. 16, 1180–1191 (2014).

    CAS  PubMed  Google Scholar 

  25. 25.

    Trefzer, C. et al. Benzothiazinones: prodrugs that covalently modify the decaprenylphosphoryl-beta-d-ribose 2’-epimerase DprE1 of Mycobacterium tuberculosis. J. Am. Chem. Soc. 132, 13663–13665 (2010).

    CAS  PubMed  Google Scholar 

  26. 26.

    Patterson, S. & Wyllie, S. Nitro drugs for the treatment of trypanosomatid diseases: past, present, and future prospects. Trends Parasitol. 30, 289–298 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Yu, J. et al. Elucidation of a novel bioactivation pathway of a 3,4-unsubstituted isoxazole in human liver microsomes: formation of a glutathione adduct of a cyanoacrolein derivative after isoxazole ring opening. Drug Metab. Dispos. 39, 302–311 (2011).

    CAS  PubMed  Google Scholar 

  28. 28.

    Duranti, E., Balsamini, C., Spadoni, G. & Staccioli, L. Reaction of secondary acetylenic bromides with sodium nitrite: synthesis of 3,5-alkyl(aryl)-4-nitroisoxazoles. J. Org. Chem. 53, 2870–2872 (1988).

    CAS  Google Scholar 

  29. 29.

    Ray, S., Kreitler, D. F., Gulick, A. M. & Murkin, A. S. The nitro group as a masked electrophile in covalent enzyme inhibition. ACS Chem. Biol. 13, 1470–1473 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Curini, M. et al. Alumina promoted cyclization of α-nitro-oximes: a new entry to the synthesis of 1,2,5-oxadiazoles N-oxides (furoxans). Tetrahedron Lett. 41, 8817–8820 (2000).

    CAS  Google Scholar 

  31. 31.

    Zhao, J. Q. et al. Synthesis of furoxan derivatives: DABCO-mediated cascade sulfonylation/cyclization reaction of α-nitro-ketoximes. Tetrahedron 71, 1560–1565 (2015).

    CAS  Google Scholar 

  32. 32.

    Burakevich, J. V., Butler, R. S. & Volpp, G. P. Phenylfurazan oxide. Chemistry. J. Org. Chem. 37, 593–596 (1972).

    CAS  Google Scholar 

  33. 33.

    Kalinina, M. I. & Mosiev, I. K. Properties of furoxans monosubstituted with adamantanes. Chem. Heterocycl. Compd. 24, 217–220 (1988).

    Google Scholar 

  34. 34.

    Eaton, J. K., Ruberto, R. A., Kramm, A., Viswanathan, V. S. & Schreiber, S. L. Diacylfuroxans are masked nitrile oxides that inhibit GPX4 covalently. J. Am. Chem. Soc. 141, 20407–20415 (2019).

    CAS  PubMed  Google Scholar 

  35. 35.

    Shelton, B. R., Howe, R. & Liu, K. C. A particularly convenient preparation of benzohydroximinoyl chlorides (nitrile oxide precursors). J. Org. Chem. 45, 3916–3918 (1980).

    Google Scholar 

  36. 36.

    Egan, C., Clery, M., Hegarty, A. F. & Welch, A. J. Mechanism of reaction of isomeric nitrolic acids to nitrile oxides in aqueous solution. J. Chem. Soc. Perkin Trans. 2, 249–256 (1991).

    Google Scholar 

  37. 37.

    Matt, C., Gissot, A., Wagner, A. & Mioskowski, C. Nitrolic acids: efficient precursors of nitrile oxides under neutral conditions. Tetrahedron Lett. 41, 1191–1194 (2000).

    CAS  Google Scholar 

  38. 38.

    Matt, C., Wagner, A. & Mioskowski, C. Novel transformation of primary nitroalkanes and primary alkyl bromides to the corresponding carboxylic acids. J. Org. Chem. 62, 234–235 (1997).

    CAS  PubMed  Google Scholar 

  39. 39.

    Meyers, R. M. et al. Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779–1784 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Shah, R., Shchepinov, M. S. & Pratt, D. A. Resolving the role of lipoxygenases in the initiation and execution of ferroptosis. ACS Cent. Sci. 4, 387–396 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–797 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Backus, K. M. et al. Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Bar-Peled, L. et al. Chemical proteomics identifies druggable vulnerabilities in a genetically defined cancer. Cell 171, 696–709 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Allimuthu, D. & Adams, D. J. 2-Chloropropionamide as a low-reactivity electrophile for irreversible small-molecule probe identification. ACS Chem. Biol. 12, 2124–2131 (2017).

    CAS  PubMed  Google Scholar 

  45. 45.

    Shindo, N. et al. Selective and reversible modification of kinase cysteines with chlorofluoroacetamides. Nat. Chem. Biol. 15, 250–258 (2019).

    CAS  PubMed  Google Scholar 

  46. 46.

    Grundmann, C. & Dean, J. M. Nitrile oxides. V. Stable aromatic nitrile oxides. J. Org. Chem. 30, 2809–2812 (1965).

    CAS  Google Scholar 

  47. 47.

    Zaro, B. W., Whitby, L. R., Lum, K. M. & Cravatt, B. F. Metabolically labile fumarate esters impart kinetic selectivity to irreversible inhibitors. J. Am. Chem. Soc. 138, 15841–15844 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Martín-Gago, P. et al. Covalent protein labeling at glutamic acids. Cell Chem. Biol. 24, 589–597.e5 (2017).

    PubMed  Google Scholar 

  49. 49.

    Geu-Flores, F. et al. Glucosinolate engineering identifies a γ-glutamyl peptidase. Nat. Chem. Biol. 5, 575–577 (2009).

    CAS  PubMed  Google Scholar 

  50. 50.

    Mutlib, A.E. et al. P450-mediated metabolism of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2’-(methylsulfonyl)-[1,1’-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogues to aldoximes. Characterization of glutathione conjugates of postulated intermediates derived from aldoximes. Chem. Res. Toxicol. 15, 63–75 (2002).

  51. 51.

    Roveri, A., Maiorino, M. & Ursini, F. Enzymatic and immunological measurements of soluble and membrane-bound phospholipid-hydroperoxide glutathione peroxidase. Methods Enzymol. 233, 202–212 (1994).

    CAS  PubMed  Google Scholar 

  52. 52.

    Kato, S. et al. Preparation of 13 or 9-hydroperoxy-9Z,11E (9E,11E) or 10E,12Z (10E,12E)-octadecadienoic phosphatidylcholine hydroperoxide. J. Oleo Sci. 63, 431–437 (2014).

    CAS  PubMed  Google Scholar 

  53. 53.

    Kriska, T. & Girotti, A. W. A thin layer chromatographic method for determining the enzymatic activity of peroxidases catalyzing the two-electron reduction of lipid hydroperoxides. J. Chromatogr. B. 827, 58–64 (2005).

    CAS  Google Scholar 

  54. 54.

    Novoselov, S. V. et al. A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells. Proc. Natl Acad. Sci. USA 104, 7857–7862 (2007).

    CAS  PubMed  Google Scholar 

  55. 55.

    Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30, e9 (2002).

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Nguyen, D. et al. Discovery and characterization of the potent and highly selective(piperidin-4-yl)pyrido[3,2-d]pyrimidine based in vitro probe BAY-885 for the kinase ERK5. J. Med. Chem. 62, 928–940 (2019).

    CAS  PubMed  Google Scholar 

  57. 57.

    Werner, S. et al. Discovery and characterization of the potent and selective P2X4 inhibitor N-[4-(3-chlorophenoxy)-3-sulfamoylphenyl]-2-phenylacetamide (BAY-1797) and structure-guided amelioration of its CYP3A4 induction profile. J. Med. Chem. 62, 11194–11217 (2019).

  58. 58.

    Cee, V. J. et al. Systematic study of the glutathione (GSH) reactivity of N-arylacrylamides: 1. effects of aryl substitution. J. Med. Chem. 58, 9171–9178 (2015).

    CAS  PubMed  Google Scholar 

Download references


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.

Author information




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.

Corresponding authors

Correspondence to Vasanthi S. Viswanathan or Stuart L. Schreiber.

Ethics declarations

Competing interests

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.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1–19 and synthetic methods.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing