Abstract
Membrane dynamics are important to the integrity and function of mitochondria. Defective mitochondrial fusion underlies the pathogenesis of multiple diseases. The ability to target fusion highlights the potential to fight life-threatening conditions. Here we report a small molecule agonist, S89, that specifically promotes mitochondrial fusion by targeting endogenous MFN1. S89 interacts directly with a loop region in the helix bundle 2 domain of MFN1 to stimulate GTP hydrolysis and vesicle fusion. GTP loading or competition by S89 dislodges the loop from the GTPase domain and unlocks the molecule. S89 restores mitochondrial and cellular defects caused by mitochondrial DNA mutations, oxidative stress inducer paraquat, ferroptosis inducer RSL3 or CMT2A-causing mutations by boosting endogenous MFN1. Strikingly, S89 effectively eliminates ischemia/reperfusion (I/R)-induced mitochondrial damage and protects mouse heart from I/R injury. These results reveal the priming mechanism for MFNs and provide a therapeutic strategy for mitochondrial diseases when additional mitochondrial fusion is beneficial.
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All data generated or analyzed during this study are included in this article and its supplementary information files. Source data are provided with this paper.
Change history
22 February 2023
A Correction to this paper has been published: https://doi.org/10.1038/s41589-023-01294-6
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Acknowledgements
We thank Z. Song at Wuhan University for providing the ρ°206 cell lines; X. Li and Z. Lv for helping with the scanning electron microscope imaging (Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences); W. Yao (State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences) for assistance with the MST experiments; and A. Prater for proofreading. Our confocal microscopy work was performed at the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences. Q.C. is supported by grants from the Ministry of Science and Technology of China (2019YFA0508601) and the National Natural Science Foundation of China (31790404 and 91849201). J.H. is supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB39000000), the Project for Young Scientists in Basic Research (YSBR-075) of the Chinese Academy of Sciences and the National Key R&D Program of China (2021YFA1300800). X.J.H. is supported by a grant from the National Natural Science Foundation of China (U1812403). L.L. is supported by the National Natural Science Foundation of China (91854105). Y.C. is supported by the Science and Social Development of Anshun City (ASKS(2020)07). S.G. is supported by grants from the National Key R&D Program of China (2018YFA0508300) and the National Natural Science Foundation of China (31722016 and 81772977).
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Contributions
X.J.H. and Q.C. designed the small molecule screen. J.H. and Y.J.G. designed the biochemical and biophysical experiments. J.H., Q.C., B.R.S. and Y.J.G. designed the cell biology experiments. Y.J.G., H.Z. and B.R.S. performed most of the experiments, with help from other authors. Y.C. and H.Y.L. designed the compound library and synthesized the compound, under the supervision of X.J.H. Y.Z. and D.K. designed and performed the I/R experiments. H.L., Z.X.W. and Y.Y. provided CMT2A patient samples. S.G., J.F.Y. and Y.T.Z. prepared and analyzed the MFN KO HeLa cells. J.H. and Q.C. wrote the manuscript, with input from all authors.
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Extended data
Extended Data Fig. 1 Cellular effects of S89 treatment.
(a) Schematic representation of the topology of MFN1. HB, helix-bundle; G, GTPase; TM, transmembrane. (b) Structural steps for obtaining natural compound spiramine C/D and derived small molecule S89. (c) MFN2 KO MEFs treated with S89 were stained with MitoTracker Red and monitored at an interval of 5 min after washing of S89. Scale bar, 5 µm. (d) Immunoblots of MFN1/2 in MFN1/2 KO HeLa cells. Tubulin was used as a loading control. (e) Representative confocal images of mitochondria in MFN1/2 KO HeLa cells treated with S89. The cells were immunostained with anti-Tom20 antibody. Scale bar, 5 µm. (f) Quantification of cells with different morphological category in (e). At least 300 cells were counted per condition. Data are mean ± SEM for three replicates. ns, not significant; ****P < 0.0001, two-way ANOVA followed by multiple comparisons test. (g) Representative confocal images of MFN DKO MEFs transfected with the indicated plasmids and treated with S89 before staining with anti-HA (green) and anti-Tom20 (red) antibodies. Scale bar, 5 µm. (h) Representative confocal images of PI staining and quantification of dead cells after exposure to S89. Data are mean ± SEM of three independent experiments. Scale bar, 20 µm. (i) Left: immunoblots show knockdown efficiency of Drp1 by siRNA (#3 best) in MFN2 KO MEFs. GAPDH was used as a loading control. Right: quantification of cells with the indicated mitochondria after Drp1 knockdown and treatment of S89. (j) Representative confocal images and quantification of wild-type (WT) or ATL-DKO COS-7 cells treated with S89 before immunostaining with anti-Tom20 antibody. Scale bar, 5 µm. For i and j, cells with morphology indicative of mitochondria were counted in 10 microscopic fields (n = 10). At least 300 cells were quantified per condition for three replicates. Data are mean ± SD; ****P < 0.0001, two-way ANOVA followed by multiple comparisons test.
Extended Data Fig. 2 S89 specifically targets MFN1.
(a) Immunoblots of the indicated proteins in U-2 OS cells co-treated with S89 and cycloheximide (CHX) or MG132. Actin served as a loading control. (b) Immunoprecipitation of ubiquitin by anti-MFN1 or anti-MFN2 antibody in WT MEFs treated or not with S89 or S3. (c) Representative confocal images and quantification of WT or MFN2 KO MEFs with the indicated mitochondrial morphology. The MEFs were treated or not with S89 or S27 and then immunostained with anti-Tom20 antibody. Scale bar, 5 µm. At least 300 cells were counted per condition. Data are mean ± SEM of three independent experiments. ns, not significant; ****P < 0.0001, two-way ANOVA followed by multiple comparisons test. (d) Representative confocal images of HeLa cells with stable expression of GFP-LC3 and treated with S89 or hypoxia for 24 h (positive control). The cells were immunostained with anti-Tom20 antibody to visualize the mitochondria and LC3 puncta (clue of autophagy). Scale bar, 5 µm. Right: quantification of cells with LC3 puncta (autophagy) or LC3 puncta colocalized with mitochondria (mitophagy). Data are mean ± SEM of three independent experiments. ns, not significant; ****P < 0.0001, unpaired two-sided Student’s t test.
Extended Data Fig. 3 Interactions of S89 with MFN fragments.
(a) Analytical ultracentrifugation shows the size of wild-type (WT) MFN1-MGD (theoretical MW 47 kD, concentration: 0.4 mM) in the presence of the indicated nucleotides (2 mM) or S89 (20 µM). The estimated molecular masses are given above the peaks in kilodaltons. Data are representative of three independent experiments with similar results. (b) SDS-PAGE and Coomassie blue staining of purified MFN1 and MFN2. (c) Structural comparison of MFN1 (PDB code 5GOF for the MGD) and IniA (PDB code 6J72). The domains of the two proteins are colored in the same pattern. Predicted secondary structural elements of MFN1-HB2 are shown in the inlet with key regions highlighted. (d, e) Biolayer interferometry (BLI) to test S89 binding to MFN1 peptides L1, L2, or other alpha helices (α7, α8, α9, α10, α11a). All peptides were biotinylated in vitro. Data are representative of three independent experiments. (f) BLI of S89 binding to purified MFN1-MGD. (g) HPLC of small molecule S89 or S3 binding to MFN1 or its FF/AA mutant-expressing lysate from HEK293T cells. Left: Western blot using anti-HA antibody confirms equal loading of the indicated proteins.
Extended Data Fig. 4 S89 binds to L1-2 of MFN1-HB2.
(a, b) BLI assay of the indicated concentrations of MFN1-MGD binding to the indicated loop peptides (a) or helix peptides (b). Data are representative of three independent experiments. (c) Sequence of MFN1-L1, which is divided into three slightly overlapping peptides: L1-1, L1-2and L1-3. (d) BLI assay of MFN1-MGD binding to the L1-1, L1-2 or L1-3 peptides. (e) BLI assay of S89 binding to MFN1 peptides L1-1, L1-2 or L1-3. (f) The structure of IniA (PDB code 6J72). A loop goes through a hydrophobic groove in the bottom of the G domain. Two hydrophobic residues, F463 and M465, are shown in the loop. (g) BLI assay of S89 binding to mutated MFN1 L1 peptide (L1 IFFL/AAAA). (h) BLI assay of MFN1-MGD binding to wild-type or mutated MFN1 L1-2 peptide. (i) SDS-PAGE and Coomassie blue staining of purified MFN1 and MFN1 FF/AA mutant proteins.
Extended Data Fig. 5 In vitro functional validation of S89 with MFN1.
(a) Sequence alignment of MFN1-L1 and MFN2-L1 in the indicated species. The conservative hydrophobic amino acids are indicated by asterisks in red. (b) BLI assay of S89 binding to MFN2 L1 peptide. (c) BLI assay of MFN1 MGD or MFN2 MGD binding to the indicated peptides (MFN1-MGD + MFN1-L1-2: KD = 4.478 × 10−7 M; MFN2-MGD + MFN1-L1-2: KD = 5.37 × 10−8 M). (d) MST assay of MFN MGD binding to the indicated peptides (MFN1 MGD-MFN2-L1-2: KD = 2.13 µM; MFN2 MGD-MFN1 L1-2: KD = 0.49 µM). (e) BLI assay of MFN1 MGD binding with L1-2 peptide in the presence of GTPγS (2.5 mM) or S89 (100 µM). (f-g) SDS-PAGE and Coomassie blue staining of purified MFN1 MGD with the YBBR-tag inserted in the GTPase (G site) or HB1 site (f). The proteins were then labeled with CoA-Cy3 fluorescent dyes using Sfp phosphopantetheinyl transferase and separated by Coomassie staining (g, left) and fluorescent imaging (g, right). Asterisks mark nonspecific bands. (h) GTPase activity of MFN1-MGD or G site/HB1 site with YBBR-tagged MFN1-MGD. Data are representative of two independent experiments. (i) Fluorescence intensity of FRET donor (FITC-labeled L1-2 peptide), acceptor (CoA-Cy3 fluorescent dye-labeled MFN1 MGD), or a mix of both donor and acceptor. (j) Automatic photobleaching of donor or acceptor used for FRET with or without GTP. (k) FRET assay of MFN1 MGD (G site or HB1 site, CoA-Cy3 fluorescent dye-labeled) with FITC-labeled L1-2 peptide in the presence of the indicated nucleotides (GTP, ATP, GTPγS). Data are representative of three independent experiments with similar results. (l) Flotation assay of reconstituted MFN1. The top and bottom fractions were analyzed by SDS-PAGE and Coomassie blue staining. (m) Representative confocal imaging and quantification of membrane tethering of MFN1 in the presence of GTP with or without S89. Lipids are rhodamine-labeled. Scale bar, 50 µm. The tethered liposome area (more than 140 tethered liposomes counted per condition) was quantified. Data are presented as mean ± SEM of three independent experiments, ****P < 0.0001, unpaired two-sided Student’s t test. (n) Lipid mixing assay of MFN1 in the presence of the indicated nucleotides. Data are representative of three independent experiments.
Extended Data Fig. 6 Mitochondrial damage in MELAS cells or under oxidative stress.
(a) Representative confocal images of ρº206_A cells and ρº206_B cells treated with or without S89. The cells were immunostained with anti-Tom20 antibody and MitoTracker Orange. The boxed regions are enlarged and shown on the right. Scale bar, 5 µm. (b) Representative confocal images of U-2 OS cells treated with the indicated concentration of paraquat (PQ). The boxed regions are enlarged and shown. Scale bar, 5 µm. (c) S89 restores relative ATP generation in PQ-treated U-2 OS cells. (d–f) (Refer to Fig. 3h) Representative confocal images of U-2 OS cells co-treated with indicated gradient concentrations of PQ and S89 (2 µM). The mitochondria were labeled with anti-Tom20 antibody (green) and the mitochondrial membrane potential assessed by MitoTracker Orange (red). The boxed regions are enlarged and shown on the right (d). Scale bar, 5 µm. Quantification data are shown in (e) and (f). (g) (Refer to Fig. 3i,j) Representative confocal images of U-2 OS cells treated with PQ and S89 at the indicated time. Cells were immunostained with anti-TM20 (green) and marked by MitoTracker Orange (red). Scale bar, 5 µm. More than 300 cells were counted per condition. (h) Immunoblots of the indicated mitochondrial remodeling proteins of U-2 OS cells upon S89 and PQ treatment. Atp5a and actin serve as loading controls. For c, data are mean ± SEM of three independent experiments. ****P < 0.0001, unpaired two-sided Student’s t test. For e and f, cells with fragmented or depolarized mitochondria were counted in 10 microscopic fields (n = 10).
Extended Data Fig. 7 S89 responds to mitochondrial damage in PQ and RSL3-induced ferroptosis cell models.
(a) Representative confocal images of MFN1 KO MEFs stably rescued by lentivirus expressing HA-MFN1 and the indicated mutants. Cells were immunostained with anti-HA (red) and anti-Tom20 (green) antibodies. Scale bar, 5 µm. (b) Quantification of the fluorescence ratio of Tom20 and HA-MFN1 or its mutants with fused mitochondria network in a. (c) Representative confocal images of MFN1 KO MEFs stably rescued by lentivirus expressing HA-MFN1 and its F524A mutant, which were treated with 800 µM PQ and 2 µM S89 for 12 h. Cells were immunostained with anti-HA (red) and anti-Tom20 (green) antibodies. Scale bar, 5 µm. (d) Representative microscopy images of U-2 OS cells co-treated with S89 and the indicated concentrations (5, 10, or 15 µM) of RSL3. Cells were stained with PI to detect cell death. Scale bar, 20 µm. (e) Representative confocal images of U-2 OS cells co-treated with S89 and the indicated concentrations (5, 10, or 15 µM) of RSL3. Cells were immunostained with anti-Tom20 (green) and MitoTracker Orange (red). The boxed areas are magnified in the lower panel. Scale bar, 5 µm. (f) Time series for RSL3-induced mitochondrial fragmentation. U-2 OS cells were treated with RSL3 for the indicated time and immunostained with anti-Tom20 antibody. Significant mitochondrial fragmentation is shown at 4 h. Quantification data are shown in the lower panel. Scale bar, 5 µm. (g) Quantification of time series for RSL3-induced ferroptosis in U-2 OS cells. Considerable cell death is shown at 12 h. (h) U-2 OS cells were treated with RSL3 (10 µM, 24 h), and S89 was added for the indicated incubation time. Cells were stained with PI to detect cell death. Scale bar, 20 µm. For b, at least 130 cells were quantified per condition for three independent replicates. Data are mean ± SD; ****P < 0.0001, unpaired two-sided Student’s t test. For f and g, data are mean ± SEM for three replicates; ns, not significant; ****P < 0.0001, unpaired two-sided Student’s t test.
Extended Data Fig. 8 Generation of CMT2A patient-derived cells.
(a) Sequencing of MFN2 in CMT2A patients (R94W, T105M). Familial normal controls are shown for comparison. (b) Established CMT2A (R94W, T105M) and their familial normal control iPSC lines present a typical round-shaped colony on Matrigel. Scale bar, 100 µm. (c) Four established iPSC lines present a normal karyotype after long-term culture. (d) Quantitative PCR of the relative gene expression of pluripotency genes (POU5F1 and NANOG). Mesenchymal stem cells (MSCs) were used as a negative control and human embryonic stem cells (hESCs) as a positive control. (e) Flow cytometry profiles showing pluripotent surface markers (SSEA4 and Tra-1-81) of four established iPSCs. (f) H&E staining of teratoma sections identifying the three germ layers. Ectoderm: neural tissue; mesoderm: cartilage; and endoderm: intestinal epithelium. Scale bar, 200 µm. (g) Schematic diagram of the process of iPSC reprogramming and differentiation into motor neurons. (h) Representative images of iPSC-derived motor neurons immunostained with tubulin III and MAP2. Scale bar, 50 µm. (i) Representative images of iPSC-derived motor neurons immunostained with motor neuron markers HB9 and ChAT. Scale bar, 100 µm. (j) Representative images of iPSC-derived motor neurons from CMT2A (R94W, T105M) patients and their familial normal control (30-day differentiation) with or without S89 treatment. Scale bar, 100 µm.
Extended Data Fig. 9 Cardiac tissue damage after I/R injury can be relieved by S89.
(a) Representative images of heart sectioning for gross assessment of the infarct size 24 h after I/R surgery. Five sections, from the apex to the site of ligation, were obtained from the hearts of animals with the indicated treatments, followed by Evans Blue and TTC double staining. Scale bar, 1 cm. (b) Quantification of infarct sizes. n = 6 per group, mean ± SEM; ns, not significant; ***P < 0.001, unpaired two-sided Student’s t test. (c) Representative echocardiographic images of the left ventricular wall motion in mice with the indicated treatments. (d, e) Quantification of cardiac function by determining the left ventricular ejection fraction (LV E-F, d) or left ventricular fractional shortening (LV F-S, e). Sham: n = 8, Saline: n = 10, DMSO: n = 11, DMSO + Matrigel: n = 9, and S89 + Matrigel: n = 10. Data are mean ± SEM; ns, not significant; ****P < 0.0001, unpaired two-sided Student’s t test. (f) Top: Masson’s trichrome staining of midpapillary regions of the isolated hearts. Collagen-rich scar tissues are shown in blue and viable myocardium in red. Bottom: representative EM images showing mitochondrial morphology. Scale bar, 1 µm. (g) Quantification of scar areas. Sham: n = 9, Saline: n = 11, DMSO: n = 9, DMSO + Matrigel: n = 9, S89 + Matrigel: n = 10. Data are mean ± SEM; ns, not significant; ****P < 0.0001, two-sided Student’s t test. (h, i) Quantification of the mitochondrial area (h) and mitochondrial length (i). More than 100 mitochondria were counted per condition. (j) Immunoblots of the indicated mitochondrial proteins of lysates from the myocardium remote to the infarction zone 2 weeks after I/R. (k) Quantification of the band intensity in j normalized to actin. n = 6 per group, mean ± SEM; ns, not significant; ****P < 0.0001, two-sided Student’s t test.
Extended Data Fig. 10 Blood analysis of the I/R mice treated with S89.
(a) Quantification of body weight in mice. (b–i) Quantification of blood biochemical parameters. ALT: alanine aminotransferase, ALP: alkaline phosphatase, CREA-S: serum creatinine, UA: uric acid, TCL: total cholesterol, Glu: glucose, IL-1β: interleukin-1β, IL-6: interleukin-6. For a–i, n = 4 per group, mean ± SEM; ns, not significant; unpaired two-sided Student’s t test.
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Supplementary Information
Supplementary Table 1 and information for synthesis of the small molecule.
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Guo, Y., Zhang, H., Yan, C. et al. Small molecule agonist of mitochondrial fusion repairs mitochondrial dysfunction. Nat Chem Biol 19, 468–477 (2023). https://doi.org/10.1038/s41589-022-01224-y
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DOI: https://doi.org/10.1038/s41589-022-01224-y
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