Abstract

Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F0F1 ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

All important data generated or analysed during this study are included in this Article. Additional supplementary data are available from the corresponding author upon request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Stupp, R. et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987–996 (2005).

  2. 2.

    Wen, P. Y. & Kesari, S. Malignant gliomas in adults. N. Engl. J. Med. 359, 492–507 (2008).

  3. 3.

    Chen, J. et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522–526 (2012).

  4. 4.

    Viale, A. & Draetta, G. F. Metabolic features of cancer treatment resistance. Recent Results Cancer Res. 207, 135–156 (2016).

  5. 5.

    Viale, A. et al. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature 514, 628–632 (2014).

  6. 6.

    Parada, L. F., Dirks, P. B. & Wechsler-Reya, R. J. Brain tumor stem cells remain in play. J. Clin. Oncol. 35, 2428–2431 (2017).

  7. 7.

    Caro, P. et al. Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma. Cancer Cell 22, 547–560 (2012).

  8. 8.

    Cole, A. et al. Inhibition of the mitochondrial protease ClpP as a therapeutic strategy for human acute myeloid leukemia. Cancer Cell 27, 864–876 (2015).

  9. 9.

    Bosc, C., Selak, M. A. & Sarry, J. E. Resistance is futile: targeting mitochondrial energetics and metabolism to overcome drug resistance in cancer treatment. Cell Metab. 26, 705–707 (2017).

  10. 10.

    Zorov, D. B., Juhaszova, M. & Sollott, S. J. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol. Rev. 94, 909–950 (2014).

  11. 11.

    Biasutto, L., Azzolini, M., Szabò, I. & Zoratti, M. The mitochondrial permeability transition pore in AD 2016: an update. Biochim. Biophys. Acta 1863, 2515–2530 (2016).

  12. 12.

    Libby, G. et al. New users of metformin are at low risk of incident cancer: a cohort study among people with type 2 diabetes. Diabetes Care 32, 1620–1625 (2009).

  13. 13.

    Naguib, A. et al. Mitochondrial complex I inhibitors expose a vulnerability for selective killing of Pten-null cells. Cell Reports 23, 58–67 (2018).

  14. 14.

    Molina, J. R. et al. An inhibitor of oxidative phosphorylation exploits cancer vulnerability. Nat. Med. 24, 1036–1046 (2018

  15. 15.

    Wheaton, W. W. et al. Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. eLife 3, e02242 (2014).

  16. 16.

    Liu, X., Romero, I. L., Litchfield, L. M., Lengyel, E. & Locasale, J. W. Metformin targets central carbon metabolism and reveals mitochondrial requirements in human cancers. Cell Metab. 24, 728–739 (2016).

  17. 17.

    Lord, S. R. et al. Integrated pharmacodynamic analysis identifies two metabolic adaption pathways to metformin in breast cancer. Cell Metab. 28, 679–688.e4 (2018).

  18. 18.

    Heerdt, B. G., Houston, M. A. & Augenlicht, L. H. The intrinsic mitochondrial membrane potential of colonic carcinoma cells is linked to the probability of tumor progression. Cancer Res. 65, 9861–9867 (2005).

  19. 19.

    Heerdt, B. G., Houston, M. A. & Augenlicht, L. H. Growth properties of colonic tumor cells are a function of the intrinsic mitochondrial membrane potential. Cancer Res. 66, 1591–1596 (2006).

  20. 20.

    Modica-Napolitano, J. S. & Aprille, J. R. Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells. Adv. Drug Deliv. Rev. 49, 63–70 (2001).

  21. 21.

    Bernal, S. D., Lampidis, T. J., Summerhayes, I. C. & Chen, L. B. Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro. Science 218, 1117–1119 (1982).

  22. 22.

    Jekimovs, C. et al. Chemotherapeutic compounds targeting the DNA double-strand break repair pathways: the good, the bad, and the promising. Front. Oncol. 4, 86 (2014).

  23. 23.

    Senese, S. et al. Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development. Cell Death Dis. 5, e1462 (2014).

  24. 24.

    Kwon, C. H. et al. Pten haploinsufficiency accelerates formation of high-grade astrocytomas. Cancer Res. 68, 3286–3294 (2008).

  25. 25.

    Zhu, Y. et al. Early inactivation of p53 tumor suppressor gene cooperating with NF1 loss induces malignant astrocytoma. Cancer Cell 8, 119–130 (2005).

  26. 26.

    Ye, J. et al. The GCN2–ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation. EMBO J. 29, 2082–2096 (2010).

  27. 27.

    Milani, M. et al. The role of ATF4 stabilization and autophagy in resistance of breast cancer cells treated with Bortezomib. Cancer Res. 69, 4415–4423 (2009).

  28. 28.

    Quirós, P. M. et al. Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J. Cell Biol. 216, 2027–2045 (2017).

  29. 29.

    Yu, F. X., Chai, T. F., He, H., Hagen, T. & Luo, Y. Thioredoxin-interacting protein (Txnip) gene expression: sensing oxidative phosphorylation status and glycolytic rate. J. Biol. Chem. 285, 25822–25830 (2010).

  30. 30.

    Parikh, H. et al. TXNIP regulates peripheral glucose metabolism in humans. PLoS Med. 4, e158 (2007).

  31. 31.

    Perry, S. W., Norman, J. P., Barbieri, J., Brown, E. B. & Gelbard, H. A. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques 50, 98–115 (2011).

  32. 32.

    Dasgupta, B. & Chhipa, R. R. Evolving lessons on the complex role of AMPK in normal physiology and cancer. Trends Pharmacol. Sci. 37, 192–206 (2016).

  33. 33.

    De Brabander, J. K. et al. Substituted benzimidazolium, pyrido-imidazolium, or pyrazino-imidazolium compounds as chemotherapeutic agents. International application no. PCT/US2016/065751, pub. no. WO/2017/100525 (2017).

  34. 34.

    Bonora, M. & Pinton, P. The mitochondrial permeability transition pore and cancer: molecular mechanisms involved in cell death. Front. Oncol. 4, 302 (2014).

  35. 35.

    Zhou, W., Marinelli, F., Nief, C. & Faraldo-Gómez, J. D. Atomistic simulations indicate the c-subunit ring of the F1Fo ATP synthase is not the mitochondrial permeability transition pore. eLife 6, e23781 (2017).

  36. 36.

    Baines, C. P. & Gutiérrez-Aguilar, M. The still uncertain identity of the channel-forming unit(s) of the mitochondrial permeability transition pore. Cell Calcium 73, 121–130 (2018).

  37. 37.

    Basso, E. et al. Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. J. Biol. Chem. 280, 18558–18561 (2005).

  38. 38.

    Dubinsky, L., Krom, B. P. & Meijler, M. M. Diazirine based photoaffinity labeling. Bioorg. Med. Chem. 20, 554–570 (2012).

  39. 39.

    Bonora, M. et al. Molecular mechanisms of cell death: central implication of ATP synthase in mitochondrial permeability transition. Oncogene 34, 1608 (2015).

  40. 40.

    Brenner, C. & Grimm, S. The permeability transition pore complex in cancer cell death. Oncogene 25, 4744–4756 (2006).

  41. 41.

    Galluzzi, L. et al. Molecular mechanisms of cisplatin resistance. Oncogene 31, 1869–1883 (2012).

  42. 42.

    Mo, W. et al. CXCR4/CXCL12 mediate autocrine cell-cycle progression in NF1-associated malignant peripheral nerve sheath tumors. Cell 152, 1077–1090 (2013).

  43. 43.

    Weinberg, S. E. & Chandel, N. S. Targeting mitochondria metabolism for cancer therapy. Nat. Chem. Biol. 11, 9–15 (2015).

  44. 44.

    Vander Heiden, M. G. & DeBerardinis, R. J. Understanding the intersections between metabolism and cancer biology. Cell 168, 657–669 (2017).

  45. 45.

    Li, F. et al. Myc stimulates nuclearly encoded mitochondrial genes and mitochondrial biogenesis. Mol. Cell. Biol. 25, 6225–6234 (2005).

  46. 46.

    Weinberg, F. et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc. Natl Acad. Sci. USA 107, 8788–8793 (2010).

  47. 47.

    Evans, J. M., Donnelly, L. A., Emslie-Smith, A. M., Alessi, D. R. & Morris, A. D. Metformin and reduced risk of cancer in diabetic patients. Br. Med. J. 330, 1304–1305 (2005).

  48. 48.

    Dilman, V. M. & Anisimov, V. N. Effect of treatment with phenformin, diphenylhydantoin or L-dopa on life span and tumour incidence in C3H/Sn mice. Gerontology 26, 241–246 (1980).

  49. 49.

    Lissanu Deribe, Y. et al. Mutations in the SWI/SNF complex induce a targetable dependence on oxidative phosphorylation in lung cancer. Nat. Med. 24, 1047–1057 (2018).

  50. 50.

    Forrest, M. D. Why cancer cells have a more hyperpolarised mitochondrial membrane potential and emergent prospects for therapy. Preprint at https://www.biorxiv.org/content/early/2015/08/21/025197 (2015).

Download references

Acknowledgements

We thank Y.-J. Li, T. Shipman and S. Bapat for technical assistance; D. Sabatini (Whitehead Institute) for supplying U937, NCI-H82, Cal-62 and NCI-H524 cell lines, N. Rosen (MSKCC) for supplying Mel30, Colo205, NCI-H2030, HCT116, SK-MEL113 and A375 cell lines, A. Messing (University of Wisconsin-Madison) for supplying primary astrocytes, and C. Brennan (MSKCC) for supplying ts603 IDH1 mutant human GBM cells. S.K.L. was a recipient of the Basic Research Fellowship from American Brain Tumor Association (in memory of Theodore Sapper). L.F.P. received funding from NCI (R35: CA210100 and R01: CA131313) and support from ‘Mr. William H. and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research’ and the Center for Experimental Therapeutics at the Memorial Sloan Kettering Cancer Center. L.F.P. and J.K.D.B. received funding from CPRIT (RP100782, RP120262 and RP150242). J.K.D.B. acknowledges support from the Robert A. Welch Foundation (I-1422).

Reviewer information

Nature thanks Scott Gilbertson and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • S. Kyun Lim

    Present address: Vivid Biosciences, Boston, MA, USA

Affiliations

  1. Brain Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Yufeng Shi
    • , Swathi V. Iyer
    • , Zilai Wang
    • , Xuanhua Xie
    • , Daochun Sun
    • , Yu-Jung Chen
    • , Viviane Tabar
    • , Philip Gutin
    •  & Luis F. Parada
  2. Cancer Biology & Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Yufeng Shi
    • , Swathi V. Iyer
    • , Zilai Wang
    • , Xuanhua Xie
    • , Daochun Sun
    • , Yu-Jung Chen
    •  & Luis F. Parada
  3. Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX, USA

    • S. Kyun Lim
    • , Qiren Liang
    • , Hua-Yu Wang
    • , Noelle Williams
    •  & Jef K. De Brabander
  4. Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA

    • S. Kyun Lim
  5. Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Yu-Jung Chen
  6. Department of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Viviane Tabar
    • , Philip Gutin
    •  & Luis F. Parada
  7. Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Luis F. Parada

Authors

  1. Search for Yufeng Shi in:

  2. Search for S. Kyun Lim in:

  3. Search for Qiren Liang in:

  4. Search for Swathi V. Iyer in:

  5. Search for Hua-Yu Wang in:

  6. Search for Zilai Wang in:

  7. Search for Xuanhua Xie in:

  8. Search for Daochun Sun in:

  9. Search for Yu-Jung Chen in:

  10. Search for Viviane Tabar in:

  11. Search for Philip Gutin in:

  12. Search for Noelle Williams in:

  13. Search for Jef K. De Brabander in:

  14. Search for Luis F. Parada in:

Contributions

S.K.L. performed and analysed the HTS compound screen. Y.S. and S.K.L. identified Gboxin targets and Y.S. identified the mechanism for resistance. Y.S., S.V.I. and Z.W. performed mouse experiments. Y.S., S.K.L. and Y.-J.C. performed the Gboxin sensitivity test for cell lines. X.X. generated mouse GBM cell cultures. D.S. generated malignant peripheral nerve sheath tumour cell cultures. J.K.D.B. designed, supervised and, with Q.L. and H.-Y.W., generated Gboxin chemical analogues. P.G. and V.T. provided surgical specimens for generating PDX models. N.W. performed PK and metabolic studies. Y.S. and S.K.L. prepared all figures and tables. L.F.P. conceptualized and supervised the study, and wrote the manuscript with Y.S. and J.K.D.B.

Competing interests

L.F.P. served as consultant for Bio-Thera Pharmaceuticals (2013–2018). L.F.P., J.K.D.B., S.K.L., Q.L., H.-Y.W. and Y.S. are coauthors on the patent application ‘Substituted benzimidazolium, pyrido-imidazolium, or pyrazino-imidazolium compounds as chemotherapeutic agents’ (international application no. PCT/US2016/065751, pub. no. WO/2017/100525 (2017)).

Corresponding author

Correspondence to Luis F. Parada.

Extended data figures and tables

  1. Extended Data Fig. 1 Gboxin, isolated from a 200,000-compound screen, specifically inhibits GBM growth (HTS cells) but not that of MEFs or astrocytes.

    a, Flow chart of the primary and secondary compound screens performed with HTS, MEF, astrocyte and neural stem and progenitor cells (NSCs). b, Representative live cell images show Gboxin-specific toxicity to HTS cells. Cells were treated with DMSO, Gboxin (1 μM) or cycloheximide (CHX, 1 μM) for three days. n = 4. c, Cell viability assays for subventricular-zone-derived primary neural stem and progenitor cells and HTS cells treated with increasing doses of Gboxin indicate a therapeutic window for HTS cells. Mean ± s.d., n = 3. d, Cell viability assays show irreversible growth inhibition in HTS cells as early as 6 h after Gboxin (1 μM) exposure. Cells were exposed to Gboxin for the indicated time periods, followed by culturing in Gboxin-free medium. Assay was performed 96 h after initial compound treatment. Mean ± s.d. n = 3. e, Gboxin induces specific transcription alterations in HTS cells. mRNA-specific quantitative PCR with reverse transcription (RT–qPCR) analyses in MEFs and HTS cells treated with DMSO or Gboxin for 12 h. n = 2. f, mRNA-specific RT–qPCR assays in HTS cells, MEFs and astrocytes treated with DMSO or Gboxin (1 μM) for 12 h demonstrate HTS-specific upregulation and downregulation of gene expression. Mean ± s.d., n = 3. g, Representative western blot analyses with astrocytes treated with DMSO or Gboxin (1 μM) for 6 h indicate no effect of Gboxin on expression of ATF4 and phospho-S6. n = 3. h, Representative western blot analyses using HTS cells exposed to DMSO or Gboxin (1 μM) detect ATF4 upregulation within 3 h of Gboxin treatment. n = 2. i, HTS cell cycle progression analysed by flow cytometry of cells treated with DMSO or Gboxin (1 μM) for 24 h indicates an increase in the ratio of G1 and G0 to S phase cells. j, Representative western blot analyses for proteins involved in apoptosis and survival with HTS cells treated with DMSO or Gboxin (1 μM) for 3 days. n = 3. k, Extended Gboxin exposure causes reduction in mitochondrial membrane potential in HTS cells. Representative images for TMRE staining show HTS-cell-specific neutralization of mitochondrial membrane potential after an 18-h incubation with Gboxin. n = 3. l, Quantification for k. Mean ± s.d. Source data

  2. Extended Data Fig. 2 Gboxin-mediated OXPHOS inhibition is reversible in wild-type MEFs and astrocytes.

    ad, Graphs show OCR as measured by Seahorse analyser. a, OCR inhibition of HTS cells by three different compounds (CMP) (added after 24 min): Gboxin (blue), oligomycin A (red) and antimycin A (green). FCCP and rotenone were added after 104 min and 128 min, respectively. Mean ± s.e.m., n = 3. b, Gboxin causes acute OCR inhibition in primary neonatal astrocytes. Gboxin (2 μM, red line) was added after 18 min, followed by oligomycin A (added after 60 min), FCCP (added after 78 min) and a mixture of rotenone and antimycin A (96 min); n = 2. Mean is shown. c, MEFs (green and purple)—but not HTS cells (blue and red)—regain a normal OCR in the presence of Gboxin. Cells were pretreated with DMSO (blue and green) or Gboxin (red and purple) for 30 h (time = 0; red arrowhead). Following the addition of DMSO (blue and green) or antimycin A (red and purple) after 16 min, oligomycin A, FCCP and rotenone were added after 80 min, 104 min and 128 min, respectively. Mean ± s.e.m., n = 3. d, Astrocytes overcome Gboxin-mediated inhibition of OCR. Cells were pretreated with DMSO (blue line) or Gboxin (red line) for 30 h (time = 0; red arrowhead), followed by the addition of DMSO (blue) or antimycin A (red) after 12 min, oligomycin A (after 60 min), FCCP (after 78 min) and a mixture of rotenone and antimycin A (96 min). Mean is shown, n = 2. e, Representative western blot analyses with astrocytes indicate that—unlike known OXPHOS inhibitors—Gboxin treatment does not induce ATF4 expression after a 12-h exposure. n = 3. Source data

  3. Extended Data Fig. 3 B-Gboxin interacts with OXPHOS proteins.

    a, Structure of B-Gboxin, a Gboxin analogue with covalently linked biotin moiety. b, B-Gboxin toxicity in HTS cells at an IC50 value higher than that for Gboxin (150 nM versus 1,530 nM). Mean ± s.d., n = 3. c, B-Gboxin inhibits OCR in HTS cells. OCR was measured under basal conditions and following addition of DMSO (blue) or B-Gboxin (red line, 10 μM) after 18 min, oligomycin A (after 48 min), FCCP (after 66 min) and a mixture of rotenone and antimycin A (after 96 min). Mean is shown, n = 2. d, B-Gboxin mediates induction of ATF4 and suppression of phospho-S6 expression in HTS cells. Cells were treated with DMSO, Gboxin (1 μM) or B-Gboxin (10 μM) for 12 h. n = 2. e, B-Gboxin associates with multiple components of the OXPHOS chain. B-Gboxin pull-down followed by mass spectrometry analyses, performed using purified mitochondria from HTS cells treated with Gboxin, B-Gboxin or Gboxin followed by B-Gboxin. The numbers in parentheses show the total known subunits for the indicated OXPHOS complexes. f, g, Representative western blot analyses validate interactions between B-Gboxin and OXPHOS proteins. f, Western blot following biotin pull-down of B-Gboxin verifies an interaction with complex V ATP5A1, which can be competed for by Gboxin. HTS cells were treated with Gboxin, B-Gboxin or Gboxin followed by B-Gboxin. n = 3. g, B-Gboxin–OXPHOS protein interactions can be detected by pull-down within 10 min. HTS cells were treated with B-Gboxin for the indicated time periods. n = 2. hk, Evidence of covalent interaction between B-Gboxin and OXPHOS proteins. See also ‘Supplementary Results’ in Supplementary Information. h, Input, western blots for OXPHOS proteins ATP5B or SDHA of HTS cells incubated with Gboxin or B-Gboxin. Pull-down, no signal with Gboxin. B-Gboxin pull-downs were not disrupted by pretreatment of the sample with high concentrations of salt (NaCl, 300 mM), urea (3 M) or sodium dodecyl sulfate (SDS; 0.2% and 0.5%). n = 2. i, Incubation with Gboxin after preincubation with B-Gboxin cannot displace B-Gboxin interactions with OXPHOS proteins; however, preincubation with Gboxin followed by B-Gboxin can displace these interactions. Pull-down assays using HTS cells  treated as indicated. P, preincubated. n = 2. j, Western blot analysis for OXPHOS proteins and B-Gboxin binding proteins following immunoprecipitation (IP) assays for corresponding OXPHOS proteins, as indicated. IB, immunoblot. n = 2. k, Western blot images for B-Gboxin interaction with cell-lysate proteins as a function of increasing pH. HTS cell lysates were incubated with B-Gboxin, and pH was adjusted using sodium hydroxide. n = 3. Source data

  4. Extended Data Fig. 4 Gboxin interacts with OXPHOS proteins in a manner that is dependent on the mitochondrial membrane potential, which leads to inhibition of complex V.

    a, Depolarization of mitochondrial inner-membrane potential dissipates Gboxin association with OXPHOS proteins in HTS cells, whereas an increase in membrane potential enhances MEF and astrocyte interactions. HTS cells, MEFs or astrocytes were pretreated with different doses of OXPHOS inhibitors for 10 min, followed by incubating with B-Gboxin for 1 h. FCCP and rotenone depolarize and decrease mitochondrial inner-membrane potential, respectively. Oligomycin A mediates an increase in membrane potential. n = 2. b, RT–qPCR analyses for gene-expression change in HTS cells treated with antimycin A, rotenone, oligomycin A or Gboxin. Fold change in gene expression was compared to DMSO-treated cells, showing enhanced downregulation of Survivin, the aurora kinases Aurka and Aurkb, and Plk1 in cells treated with oligomycin A or Gboxin. n = 2. c, Cell viability assays for cells treated with increasing doses of Gboxin or oligomycin A in the presence or absence of CsA. HTS cells are unresponsive to CsA mPTP blockade. Mean ± s.d., n = 3. d, MEF mPTP inhibition (CsA) elicits ATF4 cell-stress response to Gboxin. Representative western blot for ATF4 expression in MEFs treated with Gboxin (1 μM) in the presence or absence of CsA for 6 h. n = 2. e, Cell viability assays for MEFs transfected with control siRNA (‘Con si’) or CypD siRNA (‘CypD si’) exposed to increasing doses of Gboxin in the presence or absence of CsA (1 μM). Inset shows western blot image for efficiency of CypD knockdown. Mean ± s.d., n = 3. Source data

  5. Extended Data Fig. 5 C-Gboxin, a functional Gboxin analogue that is suitable for live-cell ultraviolet crosslink conjugation and click chemistry.

    a, Schematic for C-Gboxin (C-Gb) detection in live cells after ultraviolet crosslinking and fluorophore click chemistry. b, C-Gboxin structure. c, Cell viability assays show that C-Gboxin inhibits HTS cells (IC50 of about 350 nM) and not MEFs (IC50 > 5 μM) treated with increasing doses of C-Gboxin. Mean ± s.d., n = 3. d, C-Gboxin spontaneously accumulates in the mitochondria of HTS GBM cells. n = 3. Source data

  6. Extended Data Fig. 6 Gboxin exerts toxicity on primary mouse GBM cells and inhibits OCR in sampled human cancer cell lines.

    a, Gboxin inhibition of three primary mouse GBM cell cultures (no. 2396, no. 1661 and no. 1663), established from GBM with mutations in Nf1, Trp53 and Pten, treated with increasing doses of oligomycin A or Gboxin in the presence or absence of CsA (1 μM). Mean ± s.d., n = 3. bh, OCR Seahorse measurements in sampled cell lines (Colo205, A375, SK-MEL113, Cal-62, Daoy, U937 and NCI-H82) under basal conditions and following addition of DMSO or Gboxin (blue or red lines respectively; added after 18 min for Colo205 cells and 12 min for the others), oligomycin A (added after 60 min), FCCP (added after 78 min) and a mixture of rotenone and antimycin A (added after 96 min). Mean is shown, n = 2. i, Viability assay for primary mouse malignant peripheral nerve sheath tumour (MPNST) cells carrying Trp53 and Nf1 mutations indicates Gboxin resistance and induced sensitivity by inhibition of mPTP. Thus, Gboxin sensitivity is not directly linked to Nf1 and Trp53 driver mutations. Mean ± s.d., n = 3. Source data

  7. Extended Data Fig. 7 S-Gboxin is metabolically stable and has pharmacokinetic properties suitable for in vivo studies.

    a, Gboxin S9 half-life. n = 1. b, Gboxin plasma half-life. n = 1. c, S-Gboxin structure. d, S-Gboxin S9 half-life. n = 1. e, S-Gboxin plasma half-life. n = 1. f. S-Gboxin plasma pharmacokinetics data. Mean ± s.d., n = 3. g, S-Gboxin tumour pharmacokinetics data. Mean ± s.d., n = 3–6 at each time point. Plasma (f) and tumour (g) pharmacokinetics data indicate S-Gboxin is suitable for in vivo studies. h, Representative western blots show that S-Gboxin—similar to its original compound Gboxin—upregulates ATF4 and suppresses phospho-S6 expression in HTS cells. Cells were treated with DMSO, Gboxin (1 μM) or S-Gboxin (1 μM) for 12 h. n = 2. Source data

  8. Extended Data Fig. 8 S-Gboxin inhibits mouse and human GBM growth in vivo.

    ac, One hundred thousand HTS cells were subcutaneously injected into the flanks of nude mice, which were treated by intraperitoneal injection daily with vehicle control or S-Gboxin, beginning two weeks after allograft. a, Tumour growth by volume (W × L × H) was assessed every two days. Insets are representative images for tumours treated with vehicle control or S-Gboxin, collected after mice were killed. Vehicle, n = 8; S-Gboxin, n = 11. Mean ± s.d., two-way ANOVA. b, Representative H&E and immunohistochemical staining for Ki67, GFAP and OLIG2, indicating reduced cellularity, proliferation and expression of glioma markers after treatment with S-Gboxin. Scale bar, 50 μm. n = 2. c, Quantification of tumour cellular density (left) and Ki67-positive cells (right) in mouse flank tumours treated with vehicle control or S-Gboxin. Each graph is based on five representative images from two different tumours. Hpf, high-power field. Mean ± s.d., paired t-test, two-tailed. d, Molecular analysis shows S-Gboxin inhibition of phospho-S6 and transient induction of tumour cell apoptosis, as manifested by upregulated cleaved caspase 3 (cleaved-CAS3) and downregulated survivin expression at three and five days after treatment. n = 2. eh, Two hundred thousand primary PDX cells (ts1156) mixed with matrigel were subcutaneously injected into flanks of nude mice, which were treated by intraperitoneal injection daily with vehicle control or S-Gboxin, beginning three days after xenograft implantation. e, Graph represents tumour growth measured by tumour volume (W × L × H), assessed every two days. Vehicle, n = 7; S-Gboxin, n = 8. Mean ± s.e.m., two-way ANOVA. f, Quantification of tumour size on the day mice were killed. Vehicle, n = 7; S-Gboxin, n = 8. Unpaired t-test, two-tailed. g, Representative H&E staining images for PDX tumours treated with vehicle control or S-Gboxin show reduced cellularity after S-Gboxin treatment. Scale bar, 25 μm. n = 2. h, Images show reduced size of PDX tumours treated with S-Gboxin, collected after mice were killed. ik, Intracranial transplantation of primary mouse GBM cells (no. 1663), followed by installation of subcutaneous minipumps with intracranial catheters after two weeks, for local delivery of vehicle control or S-Gboxin (2.16 μg per day). n = 5 for each group. i, Representative images of tumour-bearing brains after treatment with vehicle control or S-Gboxin. Note the marked haemorrhage of tumours (dotted red lines) from vehicle-treated brains. j, Representative H&E and immunohistochemical staining of Ki67 and glioma markers (GFAP and OLIG2), indicating reduced cellularity, proliferation and glioma markers in brains treated with S-Gboxin. k, Quantification of tumour cellular density (left) and Ki67-positive cells (right) in tumours treated with DMSO or S-Gboxin. For each group, five representative images from two different tumours were used in quantification. Mean ± s.d., paired t-test, two-tailed. l, Intracranial treatment with S-Gboxin reduces nestin expression in tumours, but not in adjacent normal subventricular-zone tissue. Representative immunofluorescence staining for nestin, and DAPI staining, for intracranial tumours from i. n = 2. m, Immunohistochemical staining for Ki67 and glioma markers (GFAP and OLIG2), for intracranial PDX tumours shown in Fig. 6e, f, indicates reduced proliferation and expression of glioma markers in brains treated with S-Gboxin. Top panel shows patient tumour 1 (PDX-170620) and bottom panel shows patient tumour 2 (PDX-170404). Scale bar, 25 μm. n = 2. n, Mice treated with S-Gboxin at 10 mg kg−1 day−1 for a 32-day period do not exhibit weight variation, compared with vehicle-treated mice. Vehicle, n = 20; S-Gboxin, n = 20. Two-way ANOVA. Source data

  9. Extended Data Fig. 9 Primary explants from residual S-Gboxin-treated tumours retain Gboxin/S-Gboxin sensitivity in culture.

    a, b, Cell viability assays show primary tumour cultures from tumours treated with vehicle control (a) or S-Gboxin (b), as in Fig. 6a. Mean ± s.d., n = 3. Residual tumour cells treated with S-Gboxin remain sensitive to Gboxin and S-Gboxin, and have a blunted mPTP response. Source data

  10. Extended Data Fig. 10 Model for Gboxin-mediated OXPHOS inhibition in GBM cells.

    Top, high pH in mitochondrial matrix of GBM cells, and high inner mitochondrial membrane (IMM) potential, leads to the persistent accumulation of positively charged Gboxin, owing to blunted mPTP activity. At a high local concentration, Gboxin associates with multiple OXPHOS proteins and inhibits complex V activity, causing cell death. Bottom, in wild-type Gboxin-resistant cells, mPTP function stabilizes mitochondrial membrane potential and maintains a lower pH. This limits mitochondrial Gboxin accumulation, and thus limits OXPHOS inhibition and sustains cell survival. mPTP (green cylinder) is depicted adjacent to F0F1 ATPase complex V. The precise nature and contribution of complex V to mPTP remains unresolved.

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Results, Methods and additional references.

  2. Reporting Summary

  3. Supplementary Figures

    Uncropped blots for Figs. 1–5 and Extended Data Figs. 1–4, 7 & 8.

  4. Supplementary Table

    Supplementary Table 1: Microassay results showing transcriptional alternations in HTS cells treated with 1 μM Gboxin for 6, 12 and 24 hours.

  5. Supplementary Table

    Supplementary Table 2: Mass spectrometry analysis of eluate from pull-down samples from Gboxin, B-Gboxin, and Gboxin/B-Gboxin treated HTS cells showing Gboxin-specfic interacting proteins.

  6. Supplementary Table

    Supplementary Table 3: Mass spectrometry analysis of eluate from pull-down samples using mitochondria purified from Gboxin, B-Gboxin, and Gboxin/B-Gboxin treated HTS cells showing Gboxin-specific interacting proteins.

  7. Supplementary Table

    Supplementary Table 4: Patient information.

Source data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41586-019-0993-x

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.