Abstract: Ferroptosis is a recently described form of cell death driven by iron-dependent lipid peroxidation. This type of cell death was first observed in response to treatment of tumor cells with a small-molecule chemical probe named erastin. Most subsequent advances in understanding the mechanisms governing ferroptosis involved the use of genetic screens and small-molecule probes. We describe herein the utility and limitations of chemical probes that have been used to analyze and perturb ferroptosis, as well as mechanistic studies of ferroptosis that benefitted from the use of these probes and genetic screens. We also suggest probes for ferroptosis and highlight mechanistic questions surrounding this form of cell death that will be a high priority for exploration in the future. Ferroptosis is a recently described form of cell death driven by iron-dependent lipid peroxidation. This type of cell death was first observed in response to treatment of tumor cells with a small-molecule chemical probe named erastin. Most subsequent advances in understanding the mechanisms governing ferroptosis involved the use of genetic screens and small-molecule probes. We describe herein the utility and limitations of chemical probes that have been used to analyze and perturb ferroptosis, as well as mechanistic studies of ferroptosis that benefitted from the use of these probes and genetic screens. We also suggest probes for ferroptosis and highlight mechanistic questions surrounding this form of cell death that will be a high priority for exploration in the future. Small-molecule probes are valuable tools both for interrogating and perturbing biological systems (Stockwell, 2000Stockwell B.R. Chemical genetics: ligand-based discovery of gene function.Nat. Rev. Genet. 2000; 1: 116-125Crossref PubMed Google Scholar). Such tools have been valuable in the study of different types of regulated cell death, including apoptosis and necroptosis (Gangadhar and Stockwell, 2007Gangadhar N.M. Stockwell B.R. Chemical genetic approaches to probing cell death.Curr. Opin. Chem. Biol. 2007; 11: 83-87Crossref PubMed Scopus (0) Google Scholar). Most recently, a new mode of cell death, ferroptosis, was elucidated and studied through the use of small-molecule chemical probes (Dixon et al., 2012Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). We consider here some of the features and limitations of these ferroptosis probes, and suggest potential future probes of interest. Ferroptosis is driven by the peroxidation of phospholipids that contain polyunsaturated fatty acyl tails (Stockwell et al., 2017Stockwell B.R. Friedmann Angeli J.P. Bayir H. Bush A.I. Conrad M. Dixon S.J. Fulda S. Gascon S. Hatzios S.K. Kagan V.E. et al.Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease.Cell. 2017; 171: 273-285Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). The induction of this form of cell death requires three hallmarks (see Figure 1)––(1) the presence of redox-active iron, in the form of the labile iron pool (Yang and Stockwell, 2008Yang W.S. Stockwell B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells.Chem. Biol. 2008; 15: 234-245Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), and iron-dependent peroxidation enzymes, such as lipoxygenases (Wenzel et al., 2017Wenzel S.E. Tyurina Y.Y. Zhao J. St. Croix C.M. Dar H.H. Mao G. Tyurin V.A. Anthonymuthu T.S. Kapralov A.A. Amoscato A.A. et al.PEBP1 wardens ferroptosis by enabling lipoxygenase generation of lipid death signals.Cell. 2017; 171: 628-641.e26Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Yang et al., 2016Yang W.S. Kim K.J. Gaschler M.M. Patel M. Shchepinov M.S. Stockwell B.R. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.Proc. Natl. Acad. Sci. U S A. 2016; 113: E4966-E4975Crossref PubMed Scopus (179) Google Scholar) and cytochrome P450s (Zou et al., 2020Zou Y. Li H. Graham E.T. Deik A.A. Eaton J.K. Wang W. Sandoval-Gomez G. Clish C.B. Doench J.G. Schreiber S.L. Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis.Nat. Chem. Biol. 2020; 16: 302-309Crossref PubMed Scopus (1) Google Scholar), (2) the presence of the key substrates that undergo peroxidation, namely phospholipids that have polyunsaturated fatty acyl tails with bis-allylic carbons that are prone to undergo peroxidation (Dixon et al., 2015Dixon S.J. Winter G.E. Musavi L.S. Lee E.D. Snijder B. Rebsamen M. Superti-Furga G. Stockwell B.R. Human haploid cell genetics reveals roles for lipid metabolism genes in nonapoptotic cell death.ACS Chem. Biol. 2015; 10: 1604-1609Crossref PubMed Scopus (97) Google Scholar, Yang et al., 2016Yang W.S. Kim K.J. Gaschler M.M. Patel M. Shchepinov M.S. Stockwell B.R. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.Proc. Natl. Acad. Sci. U S A. 2016; 113: E4966-E4975Crossref PubMed Scopus (179) Google Scholar), and (3) failure of the complex lipid peroxide repair network, involving pathways, such as glutathione-GPX4 (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar), GCH1-BH4 (Kraft et al., 2020Kraft V.A.N. Bezjian C.T. Pfeiffer S. Ringelstetter L. Muller C. Zandkarimi F. Merl-Pham J. Bao X. Anastasov N. Kossl J. et al.GTP cyclohydrolase 1/tetrahydrobiopterin counteract ferroptosis through lipid remodeling.ACS Cent. Sci. 2020; 6: 41-53Crossref PubMed Scopus (2) Google Scholar), and NADPH-FSP1-CoQ10 (Bersuker et al., 2019Bersuker K. Hendricks J.M. Li Z. Magtanong L. Ford B. Tang P.H. Roberts M.A. Tong B. Maimone T.J. Zoncu R. et al.The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.Nature. 2019; 575: 688-692Crossref PubMed Scopus (28) Google Scholar, Dixon and Stockwell, 2019Dixon S.J. Stockwell B.R. The hallmarks of ferroptosis.Annu. Rev. Cancer Biol. 2019; 3: 35-54Crossref Google Scholar, Doll et al., 2019Doll S. Freitas F.P. Shah R. Aldrovandi M. da Silva M.C. Ingold I. Grocin A.G. Xavier da Silva T.N. Panzilius E. Scheel C.H. et al.FSP1 is a glutathione-independent ferroptosis suppressor.Nature. 2019; 575: 693-698Crossref PubMed Scopus (30) Google Scholar, Shimada et al., 2016aShimada K. Hayano M. Pagano N.C. Stockwell B.R. Cell-line selectivity improves the predictive power of pharmacogenomic analyses and helps identify NADPH as biomarker for ferroptosis sensitivity.Cell Chem. Biol. 2016; 23: 225-235Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, Shimada et al., 2016bShimada K. Skouta R. Kaplan A. Yang W.S. Hayano M. Dixon S.J. Brown L.M. Valenzuela C.A. Wolpaw A.J. Stockwell B.R. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.Nat. Chem. Biol. 2016; 12: 497-503Crossref PubMed Scopus (103) Google Scholar). The first discovered inducer of ferroptosis, and which illuminated the phenomenon of ferroptosis itself, was a novel small molecule identified from a diverse chemical library, which was termed erastin (Dolma et al., 2003Dolma S. Lessnick S.L. Hahn W.C. Stockwell B.R. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells.Cancer Cell. 2003; 3: 285-296Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Subsequent studies revealed that erastin induces ferroptosis by inhibiting the activity of the cystine-glutamate antiporter known as system xc− (Dixon et al., 2012Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar, Dixon et al., 2014Dixon S.J. Patel D.N. Welsch M.E. Skouta R. Lee E.D. Hayano M. Thomas A.G. Gleason C.E. Tatonetti N. Slusher B.S. et al.Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis.eLife. 2014; https://doi.org/10.7554/eLife.02523Crossref PubMed Scopus (269) Google Scholar) (Table 1). In cell types that obtain cysteine in the form of cystine supplied by system xc−, inhibition of this antiporter results in depletion of the intracellular pools of both the reduced and oxidized forms of glutathione (GSH and GSSG) (Dixon et al., 2014Dixon S.J. Patel D.N. Welsch M.E. Skouta R. Lee E.D. Hayano M. Thomas A.G. Gleason C.E. Tatonetti N. Slusher B.S. et al.Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis.eLife. 2014; https://doi.org/10.7554/eLife.02523Crossref PubMed Scopus (269) Google Scholar). This depletion in glutathione impairs the activity of glutathione peroxidase 4 (GPX4), which normally suppresses the formation of phospholipid hydroperoxides; the end result is accumulation of peroxidized phospholipids, which leads to ferroptotic death (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar).Table 1Ferroptosis-Relevant Chemical ProbesCompoundPubchem CIDClassGI50 (nM)Cell LineReference (PMID)System xc− InhibitorsErastin11214940chlorophenoxyacetamide2000BJeLR12676586Piperazine erastin72710858chlorophenoxyacetamide900BJeLR24439385Imidazole ketone erastin91824786chlorophenoxyacetamide3BJeLR26231156Sorafenib216239diarylurea18,000HT-108024844246Sulfasalazine5339diazo20,000U87G21889337DPI25728915rhodanine2000BJeLR24439385RSL52863472hexahydroquinolinone10,000IBS CSC12170769318355723Glutamate23672308amino acid5,000,000N18-RE-1052576375GPX4 Inhibitors(1S, 3R)-RSL31750826chloroacetamide200BJeLR18355723DPI33689415chloroacetamide20BJeLR24439385DPI43689416chloroacetamide20BJeLR24439385DPI7/ML1623689413chloroacetamide20BJeLR24439385DPI64381125chloroacetamide200BJeLR24439385DPI84230741chloroacetamide200BJeLR24439385DPI912004949chloroacetamide200BJeLR24439386DPI122416356chloroacetamide100BJeLR24439385DPI132449454chloroacetamide800BJeLR24439385DPI18932657chloroacetamide200BJeLR24439385DPI191637653chloroacetamide200BJeLR24439385DPI156545175chloromethyltriazine400BJeLR24439385DPI17932617chloromethyltriazine80BJeLR24439385Altretamine2123chloromethyltriazine250,000U-293226186195DPI1015945537nitroisoxazole300BJeLR22297109ML21049766530nitroisoxazole70BJeLR22297109JKE-1674N/Amasked nitrile oxide30LOX-IMVIbioRxiv: doi: https://doi.org/10.1101/376764NSC144988286532furoxan1,700LOX-IMVI31841309Withaferin A265237steroidal lactone1,000IMR3229939160RTAsFerrostatin-14068248ortho-phenylenediamine45MEFs28386601Liproxstatin-1135735917spiroquinoxalinamine38MEFs28386601Butylated hydroxytoluene31,404phenol10,000BJeLR22632970α-Tocopherol86,472dihydrochromene20,000BJeLR22632970Phenoxazine67,278phenoxazine9MEFs28837769CoQ10 PathwayIdebenone36861,4-benzoquinone10,000HT-1080271595774-Chlorobenzoic acid6318benzoid acid3,000,000U2OS31634900Cerivastatin446156statin1,000HT-18027159577FIN56N/Afluorene100BJeLR27159577iFSP1N/Apyridobenzimidazole100Pfa131634899Iron Chelators and SourcesDeferoxamine2973polyamide100,000HT-108022632970Ciclopirox2749hydroxamic acid5,000HT-108022632970EndoperoxidesFINO2N/Aendoperoxide15,000BJeLR26797166Compounds that have been reported to inhibit functions relevant to ferroptosis are listed, along with Pubchem CID numbers, if known, chemical class, growth inhibitory potency (GI50) in a relevant cell line, and PMID for the relevant reference. MEFs, murine embryonic fibroblasts. Open table in a new tab Compounds that have been reported to inhibit functions relevant to ferroptosis are listed, along with Pubchem CID numbers, if known, chemical class, growth inhibitory potency (GI50) in a relevant cell line, and PMID for the relevant reference. MEFs, murine embryonic fibroblasts. Erastin was subsequently modified to create more potent and drug-like system xc− inhibitors for inducing ferroptosis: piperazine erastin (PE) is slightly more potent than erastin (Table 1), but has substantially improved water solubility and metabolic stability (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). Imidazole ketone erastin (IKE) has substantially improved potency and metabolic stability (Table 1), with moderately improved aqueous solubility (Larraufie et al., 2015Larraufie M.H. Yang W.S. Jiang E. Thomas A.G. Slusher B.S. Stockwell B.R. Incorporation of metabolically stable ketones into a small molecule probe to increase potency and water solubility.Bioorg. Med. Chem. Lett. 2015; 25: 4787-4792Crossref PubMed Google Scholar). Both PE (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar) and IKE (Zhang et al., 2019Zhang Y. Tan H. Daniels J.D. Zandkarimi F. Liu H. Brown L.M. Uchida K. O'Connor O.A. Stockwell B.R. Imidazole ketone erastin induces ferroptosis and slows tumor growth in a mouse lymphoma model.Cell Chem. Biol. 2019; 26: 623-633.e629Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) are suitable for use in cell culture and animal studies, but IKE is generally more effective due to its greater potency. Additional compounds have also been found to inhibit system xc− and induce ferroptosis, but with lower potency and less selectivity than erastin, PE, and IKE. The approved drug and multi-targeted kinase inhibitor sorafenib blocks system xc− function, likely indirectly as a result of inhibiting one of its kinase targets, but also induces a necrotic death mechanism at slightly higher concentrations (Dixon et al., 2014Dixon S.J. Patel D.N. Welsch M.E. Skouta R. Lee E.D. Hayano M. Thomas A.G. Gleason C.E. Tatonetti N. Slusher B.S. et al.Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis.eLife. 2014; https://doi.org/10.7554/eLife.02523Crossref PubMed Scopus (269) Google Scholar). Sorafenib has also been reported to inhibit necrosome assembly and necroptosis (Feldmann et al., 2017Feldmann F. Schenk B. Martens S. Vandenabeele P. Fulda S. Sorafenib inhibits therapeutic induction of necroptosis in acute leukemia cells.Oncotarget. 2017; 8: 68208-68220Crossref PubMed Google Scholar, Martens et al., 2017Martens S. Jeong M. Tonnus W. Feldmann F. Hofmans S. Goossens V. Takahashi N. Brasen J.H. Lee E.W. Van der Veken P. et al.Sorafenib tosylate inhibits directly necrosome complex formation and protects in mouse models of inflammation and tissue injury.Cell Death Dis. 2017; 8: e2904Crossref PubMed Scopus (15) Google Scholar). Therefore, sorafenib (Figure 1) can be used to inhibit system xc− and to induce ferroptosis in cell culture and in animals, but its necrotic activity, multi-targeted kinase activity, and anti-necroptotic activity also need to be considered in evaluating the mechanistic implications of sorafenib's effects. Simultaneous pharmacodynamic studies testing for markers of ferroptosis with sorafenib are one means of testing whether sorafenib induces ferroptosis under specific conditions. The approved drug sulfasalazine (Figure 1) has also been used as a system xc− inhibitor (Gout et al., 2001Gout P.W. Buckley A.R. Simms C.R. Bruchovsky N. Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x(c)- cystine transporter: a new action for an old drug.Leukemia. 2001; 15: 1633-1640Crossref PubMed Scopus (205) Google Scholar). However, sulfasalazine has very low potency, and is metabolically unstable in vivo, making it difficult to use in animal studies; it is best used as a confirmatory tool in cell culture studies. The compounds DPI2 (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar) and RSL5 (Yang and Stockwell, 2008Yang W.S. Stockwell B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells.Chem. Biol. 2008; 15: 234-245Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) have similar effects as erastin, and may also be system xc− inhibitors, although this potential mechanism has not been tested directly (Figure 1; Table 1). The amino acid and neurotransmitter glutamate has also been shown to inhibit system xc− and to induce ferroptosis in some cellular contexts (Murphy et al., 1989Murphy T.H. Miyamoto M. Sastre A. Schnaar R.L. Coyle J.T. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress.Neuron. 1989; 2: 1547-1558Abstract Full Text PDF PubMed Scopus (736) Google Scholar). Glutamate has a variety of other effects, including the induction of Ca2+-mediated excitotoxicity via NMDA receptors, implying that it is difficult to use glutamate as a chemical probe per se for inducing ferroptosis. However, glutamate-induced ferroptosis may be a relevant model for some neurological conditions (Dixon et al., 2012Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). The effects of all system xc− inhibitors should depend entirely on inhibition of cystine uptake; this can be tested experimentally by co-treating cells in culture with β-mercaptoethanol or other reducing agents that reduce extracellular cystine to cysteine, resulting in uptake through transporters independent of system xc−. The effects of erastin, for example, are entirely reversed by co-treatment with β-mercaptoethanol, demonstrating that erastin blocks cystine import, but not cysteine import (Dixon et al., 2014Dixon S.J. Patel D.N. Welsch M.E. Skouta R. Lee E.D. Hayano M. Thomas A.G. Gleason C.E. Tatonetti N. Slusher B.S. et al.Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis.eLife. 2014; https://doi.org/10.7554/eLife.02523Crossref PubMed Scopus (269) Google Scholar). Of note, erastin affinity analogs bind directly to VDAC2 and VDAC3 in a pulldown assay, and VDAC2/3 are require for erastin lethality (Yagoda et al., 2007Yagoda N. von Rechenberg M. Zaganjor E. Bauer A.J. Yang W.S. Fridman D.J. Wolpaw A.J. Smukste I. Peltier J.M. Boniface J.J. et al.RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.Nature. 2007; 447: 864-868Crossref PubMed Scopus (380) Google Scholar), but the relationship of this observation to system xc− inhibition is not clear. Shortly after erastin was discovered, another small-molecule inducer of ferroptosis was identified that acted independently of system xc− (Yang and Stockwell, 2008Yang W.S. Stockwell B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells.Chem. Biol. 2008; 15: 234-245Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). This compound, named RSL3, was found to covalently inhibit GPX4, blocking one of the key repair systems for phospholipid peroxides in cells (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). RSL3 is a potent and irreversible inhibitor of GPX4, due to its reactive chloroacetamide moiety (Yang et al., 2016Yang W.S. Kim K.J. Gaschler M.M. Patel M. Shchepinov M.S. Stockwell B.R. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.Proc. Natl. Acad. Sci. U S A. 2016; 113: E4966-E4975Crossref PubMed Scopus (179) Google Scholar), but is not generally suitable for in vivo use due to poor solubility and unfavorable absorption, distribution, metabolism, and excretion (ADME) properties. Additional chloroacetamide-containing inhibitors of GPX4 have subsequently been identified, including DPI7/ML162, DPI6, DPI8, DPI9, DPI12, DPI13, DPI15, and DPI19 (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). All of these compounds have been shown to exhibit butylated hydroxytoluene (BHT)-sensitive cell killing, and buthionine sulfoximine-enhanced cell killing in the BJ-derived cell lines, along with the induction of C11-BODIPY oxidation, all hallmarks of ferroptosis (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). DPI7/ML162, DPI12, DPI13, and DPI19 have been confirmed to inhibit GPX4 activity in BJeLR cell lysates (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). These compounds are not as well characterized as RSL3; DPI7/ML162, the best characterized among these additional chloroacetamide GPX4 inhibitors, can be used to verify the GPX4 dependence of RSL3. RSL3 and ML162 have substantially different structures, aside from the chloroacetamide moiety, and thus likely have different off-target effects; use of both compounds increases the probability that effects observed with the two probes are due to GPX4 inhibition. Three additional structural classes of GPX4 inhibitors have been reported (Figure 1). First, DPI17, DPI18 (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar), and altretamine (Woo et al., 2015Woo J.H. Shimoni Y. Yang W.S. Subramaniam P. Iyer A. Nicoletti P. Rodriguez Martinez M. Lopez G. Mattioli M. Realubit R. et al.Elucidating compound mechanism of action by network perturbation analysis.Cell. 2015; 162: 441-451Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) are chloromethyltriazines that also act as covalent GPX4 inhibitors. DPI17 and DPI18 are structurally similar compounds and have been shown, like the chloroacetamide compounds above, to exhibit cell killing activity with hallmarks of ferroptosis. DPI17 has been confirmed to inhibit GPX4 activity in BJeLR cell lysates. DPI17 and DPI18 are thus likely to be covalent GPX4 inhibitors (Yang et al., 2014Yang W.S. Sriramaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. et al.Regulation of ferroptotic cancer cell death by Gpx4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). Second, DPI10 and ML210 (Weiwer et al., 2012Weiwer M. Bittker J.A. Lewis T.A. Shimada K. Yang W.S. MacPherson L. Dandapani S. Palmer M. Stockwell B.R. Schreiber S.L. et al.Development of small-molecule probes that selectively kill cells induced to express mutant RAS.Bioorg. Med. Chem. Lett. 2012; 22: 1822-1826Crossref PubMed Scopus (43) Google Scholar) contain a nitroisoxazole moiety that has been reported to generate a nitrile oxide electrophile that reacts with GPX4 in a cellular context (Eaton et al., 2018Eaton J.K. Furst L. Ruberto R.A. Moosmayer D. Hillig R.C. Hilpmann A. Zimmermann K. Ryan M.J. Niehues M. Badock V. et al.Targeting a therapy-resistant cancer cell state using masked electrophiles as GPX4 inhibitors.bioRxiv. 2018; https://doi.org/10.1101/376764Crossref Google Scholar); the related compounds JKE-1674 and JKE-1716, and the furoxan-containing NSC144988, as well as related diacylfuroxans, also generate nitrile oxide electrophiles that inhibit GPX4 (Eaton et al., 2019Eaton 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. 2019; 141: 20407-20415Crossref PubMed Scopus (1) Google Scholar). Finally, the natural product withaferin A, which is a steroidal lactone and epoxide, has been reported to act as a GPX4 inhibitor, likely through its electrophilic groups (Hassannia et al., 2018Hassannia B. Wiernicki B. Ingold I. Qu F. Van Herck S. Tyurina Y.Y. Bayir H. Abhari B.A. Angeli J.P.F. Choi S.M. et al.Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma.J. Clin. Invest. 2018; 128: 3341-3355Crossref PubMed Scopus (34) Google Scholar). A number of small-molecule radical-trapping agents (RTAs) suppress ferroptosis through their ability to interrupt the lipid peroxidation process (Figure 1). These RTAs include the potent RTAs ferrostatin-1 (Dixon et al., 2012Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar), liproxstatin-1 (Friedmann Angeli et al., 2014Friedmann Angeli J.P. Schneider M. Proneth B. Tyurina Y.Y. Tyurin V.A. Hammond V.J. Herbach N. Aichler M. Walch A. Eggenhofer E. et al.Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.Nat. Cell Biol. 2014; 16: 1180-1191Crossref PubMed Scopus (445) Google Scholar), phenoxazine (Shah et al., 2017Shah R. Margison K. Pratt D.A. The potency of diarylamine radical-trapping antioxidants as inhibitors of ferroptosis underscores the role of autoxidation in the mechanism of cell death.ACS Chem. Biol. 2017; 12: 2538-2545Crossref PubMed Scopus (26) Google Scholar), and the less potent BHT, and α-tocopherol (Dixon et al., 2012Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar) (Table 1). Ferrostatin-1 has low metabolic stability (Hofmans et al., 2016Hofmans S. Vanden Berghe T. Devisscher L. Hassannia B. Lyssens S. Joossens J. Van Der Veken P. Vandenabeele P. Augustyns K. Novel ferroptosis inhibitors with improved potency and ADME properties.J. Med. Chem. 2016; 59: 2041-2053Crossref PubMed Scopus (0) Google Scholar), and therefore in vivo experiments with this compound should be undertaken with caution. More stable ferrostatins are needed for in vivo studies. Some improved analogs have been developed and tested in vivo (Linkermann et al., 2014Linkermann A. Skouta R. Himmerkus N. Mulay S.R. Dewitz C. De Zen F. Prokai A. Zuchtriegel G. Krombach F. Welz P.S. et al.Synchronized renal tubular cell death involves ferroptosis.Proc. Natl. Acad. Sci. U S A. 2014; 111: 16836-16841Crossref PubMed Scopus (252) Google Scholar), although additional improvements are needed. Liproxstatin-1, on the other hand, is as potent as ferrostatin-1 and has suitable properties for use in animal models (Friedmann Angeli et al., 2014Friedmann Angeli J.P. Schneider M. Proneth B. Tyurina Y.Y. Tyurin V.A. Hammond V.J. Herbach N. Aichler M. Walch A. Eggenhofer E. et al.Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.Nat. Cell Biol. 2014; 16: 1180-1191Crossref PubMed Scopus (445) Google Scholar). Both ferrostatin-1 and liproxstatin-1 have been used in numerous cell culture experiments and to date have been verified to selectively suppress ferroptosis and not other cell death processes, nor to exhibit any obvious off-target effects. Phenoxazine is also potent, but needs to be more thoroughly characterized in terms of its selectivity and in vivo suitability. BHT and α-tocopherol have moderate potencies as ferroptosis suppressors and are less likely to be selective and effective in vivo for this purpose (Table 1). Moreover, since all of these RTAs act with widely different potencies, the concentration necessary in each cell system should be examined carefully. In addition, these compounds can also suppress lipid peroxidation that does not lead to ferroptotic death, so they should be used in combination with other probes of ferroptosis to verify that their activity in any given system is due to suppressi