A compendium of kinetic modulatory profiles identifies ferroptosis regulators

Abstract

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.

Extended Data Fig. 1. Examination of compound interactions on cell death.

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 (D_O) 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.

Extended Data Fig. 2. Investigating ferroptosis inhibitors.

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.

Extended Data Fig. 3. Cell free RTA and iron chelator profiling.

a, Overview of cell free compound profiling for radical trapping and Fe²⁺-binding activity. b, Cell death at 48 h in HT-1080^N 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 value (two-tailed) are reported where computable.

Extended Data Fig. 4. Bazedoxifene suppresses ferroptosis in mammalian cells.

a, Cell death quantified as the number of SYTOX Green positive (SG⁺) objects (i.e. dead cells) over time. Data are mean ± SD 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).

Extended Data Fig. 5. Bazedoxifene prevents ferroptosis in C. elegans.

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.

Extended Data Fig. 6. mTOR regulates ferroptosis sensitivity.

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 for 48 h. Blot is representative of three independent experiments.

Extended Data Fig. 7. mTOR inhibitors suppress ferroptosis.

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 x_c⁻ 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

Extended Data Fig. 8. mTOR and protein synthesis regulate ferroptosis.

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-1^N (1 μM), ML162 was used at 4 μM in all cell lines except Caki-1^N (2 μM). CHX: cycloheximide, BSO: buthionine sulfoximine. Results are mean ± SD from three independent experiments.

Extended Data Fig. 9

a, Phosphorylation and levels of mTOR pathway effectors. Blot is representative of three independent experiments. b, Fold-change in amino acids levels in HT-1080 cells determined using liquid chromatography coupled to mass spectrometry.

Extended Data Fig. 10. Arginine uptake regulates ferroptosis.

a, Dose-dependent effect of arginine (Arg) resupplementation on erastin2-induced cell death determined using STACK. Erastin2 was used at 2 μM (U-2 OS^N) or 4 μM (A549^N). 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.

Figure 1.. A kinetic modulatory profile.

a, Overview of the kinetic modulatory profiling analysis and compendium generation. b, Deviation z-scores between the expected and observed effect of each compound combination in the compendium are plotted as a heat map. Grey cells indicate no data available. c, Three heatmap sub-clusters of interest. Orange arrows indicate kinase inhibitors that bind tubulin as off-targets. d, Cell death in HT-1080^N cells as determined using STACK. Cpt: camptothecin, Era2: erastin2. e, Time of population cell death onset (D_O) for treatments shown in d. NL: not lethal. f, Cell death in HT-1080^N cells as determined using STACK. Btz: bortezomib. g, D_O values for treatments shown in f. Note that the DMSO and Cpt data shown in d-g are from the same experiment. Results in d,f are mean ± SD from three independent experiments. Results in e,g are mean ± 95% confidence interval, derived from the curves in d and f, respectively.

Figure 2.. Analysis of ferroptosis suppressor clusters.

a, A cluster of ferroptosis suppressors (green bar in Fig. 1b) contains many known radical scavengers. b, Structures for select compounds found within the cluster in a. c, Effect of select compounds from the cluster in a (black arrows) on cell death in an S. cerevisiae model of ferroptosis. VX-765 and LGK-974 were randomly selected negative control compounds. d, A distinct cluster of ferroptosis suppressors containing known iron chelators. e, Structures for select compounds found within the cluster in d. f, Effect of select compounds from the cluster in d on cell death in an S. cerevisiae model of ferroptosis. TGX-221 and cediranib were randomly selected negative control compounds. Results in c and f are from three independent experiments.

Figure 3.. Widespread bioactive compound antioxidant and iron chelating activity.

a, Cell death in cells co-treated with erastin2 and 100 different compounds that scored positively in the cell-free DPPH assay as determined using STACK. The 43 compounds that suppressed cell death by > 80% are listed, color coded by potency. Ph.: phenethyl ester; Diphos.: diphosphate. b, Structures of bazedoxifene and raloxifene. c, Lipid ROS detected using C11 BODIPY 581/591 (C11) in HT-1080 cells following 11 h treatment. Scale bar = 20 μm. Representative images from two independent experiments are shown. d, Kinetic competition arising in the inhibited co-autoxidation of egg-phosphatidylcholine (1 mM) and STY-BODIPY (10 μM) initiated with 0.2 mM di-tert-undecyl hyponitrite (DTUN). Inhibitors were used at 2 μM. Average inhibition rate constants (k_inh) determined assuming a reaction stoichiometry (n) of unity. e, Outline of a C. elegans model of ferroptosis. f, Sterile animals ± DGLA (0.13 mM) ± inhibitors (150 μM). Each datapoint represents the mean for one independent experiment (n ≈ 50 worms/experiment).

Figure 4.. mTOR inhibition suppresses ferroptosis.

a, A cluster of mTOR and dual mTOR/PI3K inhibitors from the larger compendium (purple bar in Fig. 1c). sx_c⁻i: system x_c⁻ inhibitors. b, Lipid ROS detected using C11 BODIPY 581/591 in cells treated for 22 h prior to imaging. Fer-1: ferrostatin-1. Scale bar = 20 μm. Imaging was performed three times and representative images from one experiment are shown. c, GSH and GSSG abundance in HT-1080 cells treated as indicated for 10 h. d, Cell death over time determined using STACK. e, Expression of 4E-BP1^4A under the control of a doxycycline (Dox)-inducible rtTA promoter. Blot is representative of two independent experiments. f, Cell death in U-2 OS rtTA-4E-BP1^4A cells determined by counts of SYTOX Green positive (SG⁺) objects. All compounds were added at the same time. g, Cell death over time in U-2 OS rtTA-4E-BP1^4A cells determined by counts of SG⁺ objects. Erastin2 was used at 250 nM. h, Total glutathione levels measured using Ellman’s reagent (DNTB). Dox and BSO (1 mM) pretreatment were carried out for 24 prior to the addition of DMSO or erastin2 (2 μM). In c, f and h results from three or four independent experiments are shown. Results in d and g are mean ± SD from three independent experiments.

Figure 5.. Arginine deprivation inhibits ferroptosis.

a, Cell death following withdrawal of leucine (Leu) or arginine (Arg) determined using STACK. b, Representative images from ± Arg experiment shown in a. Scale bar = 25 μm. Representative images from one of three experiments are shown. c, Lipid ROS detected using C11 BODIPY 581/591 (C11) in U-2 OS cells treated for 24 h prior to imaging. Fer-1: ferrostatin-1. Scale bar = 20 μm. Imaging was performed twice and representative images from one experiment are shown. d, Cell death in -Arg medium resupplemented with 365 μM L-arginine (L-Arg), D-arginine (D-Arg), or citrulline, determined using STACK. e, Erastin2-induced cell death in cell lines grown in DMEM medium with dialyzed FBS ± Arg (356 μM) determined using STACK. Erastin2 was used at 1 μM (HT-1080^N, Caki-1^N, H23N^Cas9,N), 2 μM (T98G^N, H1299^N, U-2 OS^N, A375^N) or 4 μM (A549^N) to achieve equal basal cell killing. f, Cell death at 48 h determined using STACK. In a, d-f results from three independent experiments are shown.

Figure 6.. Amino acid deprivation-induced ferroptosis suppression correlates with proliferative arrest.

a, Immunoblots from cells deprived of Arg for the indicated times. Blot is representative of three independent experiments. b, Immunoblotting for ATF4 protein levels ± Arg-containing medium ± the GCN2 inhibitor GCN2iB (1 μM). Blot is representative of two independent experiments. c, Expression of the transsulfuration pathway genes under the same conditions as in b determined by RT-qPCR. d, Cell death determined using STACK. GCN2iB was used at 1 μM. e, Cell death over time determined using STACK and expressed as the normalized AUC (nAUC) versus proliferation under conditions of single amino acid withdrawal plus erastin2 (2 μM) or cystine co-withdrawal. Cell proliferation was quantified as the ratio of live cell (mKate2⁺) counts at 48 h versus 0 h in medium containing vehicle (DMSO) control and cystine. Results in c and d are from three independent experiments. Results in e represent mean ± SD from three independent experiments.