Cell death can be executed by regulated apoptotic and nonapoptotic pathways, including the iron-dependent process of ferroptosis. Small molecules are essential tools for studying the regulation of cell death. Using time-lapse imaging and a library of 1,833 bioactive compounds, we assembled a large compendium of kinetic cell death modulatory profiles for inducers of apoptosis and ferroptosis. From this dataset we identify dozens of ferroptosis suppressors, including numerous compounds that appear to act via cryptic off-target antioxidant or iron chelating activities. We show that the FDA-approved drug bazedoxifene acts as a potent radical trapping antioxidant inhibitor of ferroptosis both in vitro and in vivo. ATP-competitive mechanistic target of rapamycin (mTOR) inhibitors, by contrast, are on-target ferroptosis inhibitors. Further investigation revealed both mTOR-dependent and mTOR-independent mechanisms that link amino acid metabolism to ferroptosis sensitivity. These results highlight kinetic modulatory profiling as a useful tool to investigate cell death regulation.
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We thank J. Cao, A. Tarangelo, Z. Inde and E. Kolebrander Ho for experimental assistance, and B. Stockwell, L. Li, M. Bassik, J. Ye and M. Cyert for reagents. Certain constructs were obtained from Addgene. This work was supported by awards from the NSERC (grant no. RGPIN-06741-2016) to D.A.P., and the National Institutes of Health (grant no. 5T32GM007276) to D.A.A. (grant no. 1R01GM133883) to J.L.W. and (grant nos. 4R00CA166517-03 and 1R01GM122923) to S.J.D.
S.J.D. is a member of the scientific advisory board for Ferro Therapeutics, has consulted for Toray Industries and AbbVie Inc., and is an inventor on patents related to ferroptosis.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Chemical structures. b, Cell death determined using STACK. c, NRF1 (NFE2L1) protein levels. Blot is representative of three independent experiments. d, Cell death determined using STACK. e, Quantification of the timing of population cell death onset (DO) from the lethal fraction curves in d. f, Cell death in two different cell lines determined using STACK. Results in b, d and e are mean ± SD from three independent experiments, while in f results are from two or three independent experiments.
a, Cell death data extracted from the compendium for erastin treatment. Each dot represents a single modulator compound, tested once at 5 µM, organized together by major target class. Lower nAUC values indicate greater death suppression. MEKi: mitogen activated protein kinase kinase 1/2 inhibitors (n = 14); AdRm: adrenergic receptor modulators (n = 55); ER/PRm: estrogen/progesterone receptor modulators (n = 29); VEGFRi: vascular endothelial growth factor receptor inhibitors (n = 20); K+ Cm: potassium channel modulators (n = 19). The vertical dotted line indicates the mean lethality of the control erastin + DMSO conditions. b, Cell death determined by counting SYTOX Green positive (SG+) objects. The experiment was performed twice on different days and data represents mean ± SD.
a, Overview of cell free compound profiling for radical trapping and Fe2+-binding activity. b, Cell death at 48 h in HT-1080N cells treated with erastin2 (1 µM) and candidate radical trapping compounds (50 µM, n = 100) plotted against predicted hydrophilicity (LogS), predicted lipophilicity (LogP) and the %DPPH inhibition values from the cell-free assay. Dotted lines indicate a lethal fraction of 0.2. Spearman correlation values are reported with the 95% confidence interval (C.I.). Exact P values (two-tailed) are reported where computable.
a, Cell death quantified as the number of SYTOX Green positive (SG+) objects (that is dead cells) over time. Data are from two independent experiments. b,c, Cell death quantified by SG+ object counting. Data are from three or four independent experiments. d, Outline of the STY-BODIPY kinetic competition assay. Egg-phosphatidylcholine (1 mM) and STY-BODIPY (10 µM) are incubated with 0.2 mM di-tert-undecyl hyponitrite (DTUN), in addition to a radical trapping antioxidant (RTA-H).
a, Representative images of DAPI-stained adult C. elegans under the different treatment conditions indicated below each image. The gonads of fertile worms are indicated (arrows). DGLA: dihomo-γ-linolenic acid (125 µM); Baz: bazedoxifene (150 µM). Scale bar = 100 µm. Imaging was repeated twice and representative animals from one experiment are shown. b, Polyunsaturated fatty acid levels as a function of total lipids determined in worms using gas chromatography/mass spectrometry. Results are from two independent experiments on separate populations of worms.
a, Cell death over time determined using STACK. Cells were infected with control (scrambled) shRNA or shRNAs targeting RPTOR or RICTOR for 72 h prior to compound treatment. INK128 was used as a positive control. Results are mean ± SD from three independent experiments. b, Expression and phosphorylation of proteins in the mTOR pathway following infection of HT-1080 cells as in a. Blot is representative of three independent experiments.
a, Cell death determined using STACK in three different cell lines. Results are mean ± SD from three independent experiments. b, 4E-BP1 protein phosphorylation and total levels. Blot is representative of three independent experiments. c, Total glutathione (GSH + GSSG) levels measured using Ellman’s reagent (DNTB). d, System xc- activity inferred from glutamate release over 2 h from HT-1080 and U-2 OS cells treated as indicated. e, Cell death determined using STACK. Results in c-e are from three independent experiments.
a, Phosphorylation and levels of mTOR pathway effectors in U-2 OS cells. Blot is representative of three independent experiments. b, Analysis of Cancer Therapeutics Response Portal (CTRP) dataset for ferroptosis-inducing compounds. c, Cell death determined using STACK. Erastin2 was used at 2 µM in all cell lines except Caki-1N (1 µM), ML162 was used at 4 µM in all cell lines except Caki-1N (2 µM). CHX: cycloheximide, BSO: buthionine sulfoximine. Results are mean ± SD from three independent experiments.
a, Phosphorylation and levels of mTOR pathway effectors. Blot is representative of two independent experiments. b, Fold-change in amino acids levels in HT-1080 cells determined using liquid chromatography coupled to mass spectrometry.
a, Dose-dependent effect of arginine (Arg) resupplementation on erastin2-induced cell death determined using STACK. Erastin2 was used at 2 µM (U-2 OSN) or 4 µM (A549N). b, Cell death as determined using STACK in cells grown in complete medium (CM), switched to -Arg medium at the time of compound addition, or 24 h before compound addition. c, Detection of puromycylated peptides. Results are representative of three independent experiments. d, Quantification of results from three puromycylation experiments, as in c. e, SYTOX Green positive (SG+) dead cell counts normalized to initial cell confluence. f, Confirmation of ATF4 knockdown at the protein level. Blot is representative of two independent experiments. Results in a,b and e represent mean ± SD from three independent experiments.
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Conlon, M., Poltorack, C.D., Forcina, G.C. et al. A compendium of kinetic modulatory profiles identifies ferroptosis regulators. Nat Chem Biol 17, 665–674 (2021). https://doi.org/10.1038/s41589-021-00751-4
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