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CGI1746 targets σ1R to modulate ferroptosis through mitochondria-associated membranes

Nature Chemical Biology (2024)Cite this article

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Abstract

Ferroptosis is iron-dependent oxidative cell death. Labile iron and polyunsaturated fatty acid (PUFA)-containing lipids are two critical factors for ferroptosis execution. Many processes regulating iron homeostasis and lipid synthesis are critically involved in ferroptosis. However, it remains unclear whether biological processes other than iron homeostasis and lipid synthesis are associated with ferroptosis. Using kinase inhibitor library screening, we discovered a small molecule named CGI1746 that potently blocks ferroptosis. Further studies demonstrate that CGI1746 acts through sigma-1 receptor (σ1R), a chaperone primarily located at mitochondria-associated membranes (MAMs), to inhibit ferroptosis. Suppression of σ1R protects mice from cisplatin-induced acute kidney injury hallmarked by ferroptosis. Mechanistically, CGI1746 treatment or genetic disruption of MAMs leads to defective Ca2+ transfer, mitochondrial reactive oxygen species (ROS) production and PUFA-containing triacylglycerol accumulation. Therefore, we propose a critical role for MAMs in ferroptosis execution.

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Fig. 1: CGI1746 acts as a potent ferroptosis inhibitor.
Fig. 2: ER Ca2+ storage contributes to ferroptosis.
Fig. 3: σ1R mediates inhibitory effect of CGI1746 on ferroptosis.
Fig. 4: CGI1746 blocks calcium transfer from ER to mitochondria in ferroptosis.
Fig. 5: The consequence of dysregulated calcium and lipid transfer upon MAMs deficiency in ferroptosis.
Fig. 6: Blocking σ1R protects mice from cisplatin-induced AKI.

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All original data that support the findings of this study are provided in source data and supplementary information. Source data are provided with this paper.

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Acknowledgements

We thank the BioRender platform for the illustration created with BioRender.com (graphic abstract and figures). We thank J. Zheng, C. Huang, Z. Wang and X. Sun for providing the reagents. We thank the core facilities at Westlake University for lipidomic analysis. This work was supported, in part, by grants from the National Natural Science Foundation of China (92254307, 91754205, 91957204 and M-1040) to Q.Z.; MOST (2019YFA0508602, 2023YFA0914900) to Q.Z.; the Shanghai Municipal Science and Technology Project (20JC1411100 and 19XD1402200) to Q.Z.; the National Natural Science Foundation of China (32070734 and 32370794) to J.Z.; the State Key Laboratory of Oncogenes and Related Genes (KF2126-93) to J.Z.; the National Natural Science Foundation of China (91954205) to Y.W.; and the Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases; and the innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20212000); and the Shanghai Science and Technology Commission (20JC1410100).

Author information

Author notes

  1. These authors contributed equally: Zili Zhang, Hong Zhou, Wenjia Gu.

Authors and Affiliations

  1. Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zili Zhang, Hong Zhou, Jing Zhang & Qing Zhong

  2. Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, China

    Wenjia Gu & Youjun Wang

  3. Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China

    Wenjia Gu & Youjun Wang

  4. Department of Nephrology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yuehan Wei & Shan Mou

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Contributions

Z.Z. and H.Z. started the project and performed most of the experiments. W.G. contributed with MAMs calcium transfer experiments, with guidance from Y. Wang. Y. Wei contributed with animal experiments, with guidance from S.M. Z.Z., H.Z., J.Z. and Q.Z. conceived the project, designed the experiments, analyzed the data and wrote the manuscript, with the help of all authors. All authors discussed the results and commented on the manuscript.

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Correspondence to Youjun Wang, Jing Zhang or Qing Zhong.

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Extended data

Extended Data Fig. 1 Chemical screening to identify CGI1746 as a ferroptosis inhibitor.

a. The workflow of the high-throughput chemical screening. Ferroptosis inhibitors were screened from a cell death-based assay, and then candidate ferroptosis inhibitors were tested in cell-free DPPH assay for antioxidant activity or Ferrozine assay for Fe2+ chelating activity. The illustration was created with BioRender.com. b. Cell-based chemical screening for ferroptosis inhibitors. Orange dot: CGI1746, Red dot: Ferrostatin-1. The cutoff value for cell survival rate is 60%. c. The labile iron chelation activity of 93 ferroptosis inhibitors derived from cell death screening. The labile iron chelation activity of chemicals was determined by the Ferrozine-Iron chelation assay. Yellow dot: CGI1746. The cutoff value for relative ferrozine/Fe2+ signal is 80%. d. Analysis of radical trapping capacity of 93 ferroptosis inhibitors derived from cell death screening. The antioxidant activity of chemicals was assessed in the cell-free DPPH assay. Orange dot: CGI1746.The cutoff value for relative DPPH signal is 80%. e. The characterization of 93 ferroptosis inhibitors is shown in venn diagram. f. The list of 26 chemicals that potently suppressed RSL3 induced ferroptosis with negligible cellular toxicity and no detectable activities with free radical scavenging and labile iron chelation. These chemicals are listed based on their ferroptosis inhibition efficacy from high to low.

Source data

Extended Data Fig. 2 CGI1746 acts as a ferroptosis inhibitor independent of BTK.

a. MDA-MB-468 cells were treated with RSL3 as indicated in the absence or presence of CGI1746 (10 μM) for 6 hours. b-f. HK-2 (b), PC-3 (c), Huh-7 (d), MEF (e) and Raji (f) cells were treated with RSL3 for 24 hours in the absence or presence of CGI1746 (10 μM) to evaluate cell death. g-h. Cell viability was determined in NCI-H1299 (g) and MDA-MB-468 (h) cells treated by erastin (g) or ML210 (h) for 48 (g) or 24 (h) hours with or without CGI1746 (10 μM) treatment. i. Cell death in Raji cells in the absence or presence of ibrutinib (10 μM), followed by RSL3 for 24 hours. j. Raji cells were treated with or without CGI1746 (10 μM) or ibrutinib (10 μM) for 1 hour followed by pervanadate (1 mM) treatment for 30 min. Cell lysates were subjected to western blot analysis for PLCγ2, p-PLCγ2 (Y1217), BTK and p-BTK (Y223) expression. k. Protein expression of endogenous BTK in the indicated cell lines by immunoblotting. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. Western blot data show one representative out of three independent experiments.

Source data

Extended Data Fig. 3 CGI1746 acts as a new class of ferroptosis inhibitor.

a. Immunoblotting for GPX4, SLC7A11, DHODH, TFRC and ACSL4 protein levels in MDA-MB-468 cells subjected to RSL3 treatment (1 μM) for indicated hours, with or without CGI1746 pre-treatment (10 μM). b-d. MDA-MB-468 cells were treated by 10 μM CGI1746 and/or 100 μM DFO, then labeled with FerroOrange to detect the cellular labile iron pool by confocal microscope (b and c) or flow cytometry (d). DFO was utilized as a positive control to reduce the cellular labile iron pool. The presented images are representatives (b, Scale bars, 20 μm). The labile iron levels in b were quantified in c. e. The effect of CGI1746 on chelating labile iron up to 200 μM was determined by Ferrozine–iron chelation assay, using DFO (100 μM) as a positive control. f. GSH abundance was measured by GSH-Glo™ Glutathione Assay in NCI-H1299 cells treated with erastin for 18 hours in the absence or presence of CGI1746 (10 μM). g. GPX4 degradation was analyzed using western blot in NCI-H1299 cells treated with erastin (5 μM) for indicated hours with or without CGI1746 (10 μM) pre-treatment. h. The capacity of antioxidant activity of CGI1746 in DPPH assay. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. For bar graphs c and e, data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. Micrograph data show one representative out of three replicates from one representative of three independent experiments. Flow cytometry data show one representative out of two replicates from one representative of three independent experiments. Western blot data show one representative out of three independent experiments. ****P < 0.0001 and ns, not significant. One-way ANOVA (GraphPad Prism 9.5.1).

Source data

Extended Data Fig. 4 ER Ca2+ storage contributes to ferroptosis.

a-b. Compound CGI1746 did not protect U937 cells from apoptosis (a) and necroptosis (b). c. Cell viability of NCI-H1299 cells treated by RSL3 for 9 hours with or without BAPTA-AM (5 μM) pre-treatment. d. The morphology of NCI-H1299 cells treated with RSL3 (2 μM) for 8 hours following BAPTA-AM pre-treatment (5 μM). Scale bar, 50 µm. e-f. Cell viability of PC-3 (e) and MEF (f) cells treated by RSL3 for 10 (e) and 12 (f) hours respectively with or without BAPTA-AM (10 μM) pre-treatment. g. MEF cells were treated by erastin for 12 hours with or without BAPTA-AM (10 μM) pre-treatment for cell death analysis. h. Cell survival rates of PC-3 cells incubated with RSL3 for 24 hours in the presence or absence of BAPTA-AM (10 μM). i. Cell death at 48 hours in NCI-H1299 cells treated by erastin after pre-treatment with or without BAPTA-AM (10 μM). j. The effect of BAPTA-AM (10 μM) on ML210 induced ferroptosis in NCI-H1299 cells for 6 hours. k. Cell death in MDA-MB-468 cells treated by tBOOH plus Z-VAD (20 μM) for 6 hours with or without BAPTA-AM (10 μM) pre-treatment. l. The capacity of antioxidant activity of BAPTA-AM (5 μM) in DPPH assay. m-o. The effect of EGTA-AM (20 μM) on ferroptosis in MDA-MB-468 cells (m), NCI-H1299 cells (n), MEF cells (o). p. The mRNA levels of SERCA1, SERCA2 and SERCA3 by quantitative real-time RT-PCR in NCI-H1299 cells. Two pairs of primers were used. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. For bar graphs k and p, data represent mean ± s.d. of n = 9 (k) and 3 (p) replicates from one representative of three independent experiments. Micrograph data show one representative out of three replicates from one representative of three independent experiments. One-way ANOVA (GraphPad Prism 9.5.1), ****P < 0.0001 and ns, not significant.

Source data

Extended Data Fig. 5 Suppression of Sigma-1 Receptor blocks ferroptosis.

a-b. Cell viability of MDA-MB-468 cells treated by indicated doses of RSL3 for 6 hours in the presence or absence of MINK1 antagonist DMX-5084 (10 μM, a), or Adenosine A1/A2 receptor antagonist Diphylline (10 μM, b). c-d. MDA-MB-468 cells were exposed to indicated doses of RSL3 for 8 hours with or without σ1R antagonist S1RA (50 μM, c) or BD1047 (100 μM, d). Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments.

Source data

Extended Data Fig. 6 The involvement of IP3R in ferroptosis.

a. Representative PLA images from three independent experiments. Nuclear marker DAPI is in blue and PLA signal is in red. Scale bar, 20 μm. b. Statistics of PLA dots/DAPI for PLA assay (a). Mean ± s.d., n = 12 ROIs containing 226 cells for DMSO, 216 cells for CGI1746, 184 cells for erastin, 222 cells for CGI1746+erastin. One-way ANOVA (GraphPad Prism 9.5.1; ****P < 0.0001). c-d. Cell viability analysis in NCI-H1299 cells exposed to RSL3 (c) or erastin (d) for 24 and 48 hours respectively with or without IP3R inhibitor 2-APB (100 μM). e-f. Cell viability analysis in MEF cells exposed to RSL3 (e) or erastin (f) for 24 hours with or without IP3R inhibitor 2-APB (100 μM). g. The mRNA levels of IP3R1, IP3R2 and IP3R3 by quantitative real-time RT-PCR in NCI-H1299 cells. Two pairs of primers were used to detect the expression of each isoform. h. Cell viability of NCI-H1299 cells transfected with siRNA against IP3R followed by indicated doses of erastin for 48 hours. i-j. PLC inhibition conferred NCI-H1299 and MEF cells resistance to RSL3 induced ferroptosis. Quantification of cell death in NCI-H1299 cells (i) or MEF cells (j) exposed to RSL3 for 24 hours following pre-treatment with PLC inhibitor U73122 (2 μM). Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments.

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Extended Data Fig. 7 The involvement of GRP75 and VDAC1 in ferroptosis.

a-b. GRP75 inhibition conferred cellular resistance to RSL3 (a) or erastin (b) induced ferroptosis. RSL3 or erastin were used to elicit ferroptosis for 24 and 48 hours respectively, with or without GRP75 inhibitor MKT-077 (50 μM) pre-treatment. c-d. GRP75 inhibition conferred cellular resistance to RSL3 (c) or erastin (d) induced ferroptosis. RSL3 or erastin were used to elicit ferroptosis for 24 hours with or without MKT-077 (50 μM) pre-treatment. e-f. Cell viability of NCI-H1299 cells transfected with siRNA against GRP75 followed by RSL3 (e) or erastin (f) for 48 hours. g-h. Grp75 was knocked down in MEF cells, followed by RSL3 (g) or erastin (h) for 24 hours to evaluate cell death. i. NCI-H1299 cells were transfected with siRNA specially against GRP75 for 48 hours, then subjected to 1 μM RSL3 for 8 hours. Lipid peroxidation was measured with C11-BODIPY staining. j-k. MEF cells were transfected with siRNA specially against Grp75 for 48 hours, then treated by 200 nM RSL3 (j) or 1 µM erastin (k) for 6 hours. Lipid peroxidation was measured with C11-BODIPY staining. l-m. VDAC1 was inhibited by 5 μM VBIT-4, 200 μM VBIT-12 or 250 μM DIDS, followed by RSL3 for 24 hours (l) or erastin for 48 hours (m) in NCI-H1299 cells for cell viability analysis. n-o. RSL3 (n) or erastin (o) were used for 48 hours to elicit ferroptosis in NCI-H1299 WT and VDAC1 KO cells for cell death analysis. p. RSL3 were used for 24 hours to elicit ferroptosis in MDA-MB-468 WT and VDAC1 KO cells. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. Flow cytometry data show one representative out of three replicates from one representative of three independent experiments.

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Extended Data Fig. 8 Tethering of ER and mitochondria contributes to ferroptosis.

a-b. NCI-H1299 cells were transfected with two individual siRNAs specially against MFN2 for 48 hours, then treated with increased concentrations of RSL3 (a) or erastin (b) to evaluate cell death. c. NCI-H1299 cells were transfected with siRNA specially against MFN2 for 48 hours, then subjected to 1 μM RSL3 for 8 hours to detect lipid peroxidation by C11-BODIPY. d-e. NCI-H1299 cells were transfected with two individual siRNAs specially against MFN1 for 48 hours, then treated with increased concentrations of RSL3 (d) or erastin (e) to evaluate cell death. f. NCI-H1299 cells were transfected with siRNA specially against MFN1 for 48 hours, then subjected to 1 μM RSL3 for 8 hours to detect lipid peroxidation by C11-BODIPY. g-h. NCI-H1299 cells were transfected with siRNA specially against PACS2 for 48 hours, then subjected to different concentrations of RSL3 (g) or erastin (h) treatments to measure cell viability. i-j. NCI-H1299 cells were transfected with siRNA specially against VAPB for 48 hours, then subjected to different concentrations of RSL3 (i) or erastin (j) treatments to measure cell viability. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. Flow cytometry data show one representative out of three replicates from one representative of three independent experiments.

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Extended Data Fig. 9 Mitochondrial dysfunctions upon ferroptosis.

a-b. The effect of MCU inhibitor Ru360 (20 μM, a) or DS16570511 (100 μM, b) on RSL3 induced ferroptosis at 6 hours in MDA-MB-468 cells. c. Cell death in NCI-H1299 cells incubated with RSL3 following pre-treatment with DS16570511 (100 μM). d-e. The effect of DS16570511 (100 μM) on RSL3 (d) or erastin (e) induced ferroptosis. f-g. MCU was knocked down in NCI-H1299 cells, followed by RSL3 for 24 hours (f) or erastin for 48 hours (g) to detect cell death. h. MDA-MB-468 cells were transfected with siRNAs specially against MCU for 48 hours, followed by RSL3 treatments for 6 hours before cell death analysis. i. NCI-H1299 cells were transfected with siRNA specially against MCU for 48 hours, then subjected to 500 nM RSL3 for 4 hours to detect lipid peroxidation. j. MEF cells were treated by 200 nM RSL3 or 1 µM erastin for 6 hours. Mitochondrial membrane potential was measured with TMRE staining. k. MEF cells were transfected with siRNA specially against Grp75 for 48 hours, then treated by 200 nM RSL3 for 6 hours. The mitochondrial Ca2+ concentration was measured by Rhod-2, AM (5 μM) staining (upper panel). Mitochondrial ROS was measured with MitoSOX (5 μM) staining (middle panel). Mitochondrial lipid peroxidation level was measured by MitoPerOx (100 nM) staining (lower panel). l. NCI-H1299 cells were pretreated with DMSO, Fer-1 (2 μM) or CGI1746 (5 μM), then treated with 5 μM erastin for 18 hours. The mitochondrial Ca2+ concentration was measured by Rhod-2, AM staining. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments (n = 2 for h). Flow cytometry data show one representative out of three replicates from one representative of three independent experiments.

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Zhang, Z., Zhou, H., Gu, W. et al. CGI1746 targets σ1R to modulate ferroptosis through mitochondria-associated membranes. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01512-1

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