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A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations

Nature Chemistry volume 9, pages 667–675 (2017)Cite this article

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Abstract

Perturbations in cellular chloride concentrations can affect cellular pH and autophagy and lead to the onset of apoptosis. With this in mind, synthetic ion transporters have been used to disturb cellular ion homeostasis and thereby induce cell death; however, it is not clear whether synthetic ion transporters can also be used to disrupt autophagy. Here, we show that squaramide-based ion transporters enhance the transport of chloride anions in liposomal models and promote sodium chloride influx into the cytosol. Liposomal and cellular transport activity of the squaramides is shown to correlate with cell death activity, which is attributed to caspase-dependent apoptosis. One ion transporter was also shown to cause additional changes in lysosomal pH, which leads to impairment of lysosomal enzyme activity and disruption of autophagic processes. This disruption is independent of the initiation of apoptosis by the ion transporter. This study provides the first experimental evidence that synthetic ion transporters can disrupt both autophagy and induce apoptosis.

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Figure 1: Structure of anion transporters and their activity.
Figure 2: Ion transport studies using liposomal model membranes.
Figure 3: Synthetic transporters induce apoptosis.
Figure 4: Synthetic transporters induce caspase-dependent apoptosis.
Figure 5: Effect of synthetic transporters on autophagy.
Figure 6: Effect of apoptosis induction promoted by 3 is independent of its ability to disrupt autophagy.

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References

  1. Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues. Cell 147, 728–741 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Ohsumi, Y. Molecular dissection of autophagy: two ubiquitin-like systems. Nat. Rev. Mol. Cell Biol. 2, 211–216 (2001).

    CAS  PubMed  Google Scholar 

  3. Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004).

    CAS  PubMed  Google Scholar 

  4. White, E. Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 12, 401–410 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Baek, K.-H., Park, J. & Shin, I. Autophagy-regulating small molecules and their therapeutic applications. Chem. Soc. Rev. 41, 3245–3263 (2012).

    CAS  PubMed  Google Scholar 

  6. Newmeyer, D. D. & Ferguson-Miller, S. Mitochondria: releasing power for life and unleashing the machineries of death. Cell 112, 481–490 (2003).

    CAS  PubMed  Google Scholar 

  7. Igney, F. H. & Krammer, P. H. Death and anti-death: tumour resistance to apoptosis. Nat. Rev. Cancer 2, 277–288 (2002).

    CAS  PubMed  Google Scholar 

  8. Williams, D. R., Ko, S.-K., Park, S., Lee, M.-R. & Shin, I. An apoptosis-inducing small molecule that binds to heat shock protein 70. Angew. Chem. Int. Ed. 47, 7466–7469 (2008).

    CAS  Google Scholar 

  9. Ko, S.-K. et al. A small molecule inhibitor of ATPase activity of HSP70 induces apoptosis and has antitumor activities. Chem. Biol. 22, 391–403 (2015).

    CAS  PubMed  Google Scholar 

  10. Ko, S.-K. et al. Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells. Nat. Chem. 6, 885–892 (2014).

    CAS  PubMed  Google Scholar 

  11. Stauber, T. & Jentsch, T. J. Chloride in vesicular trafficking and function. Annu. Rev. Physiol. 75, 453–477 (2013).

    CAS  PubMed  Google Scholar 

  12. Gould, D. Human physiology: From cells to systems, 3rd edition. J. Adv. Nurs. 28, 680–682 (1998).

    Google Scholar 

  13. Yu, S. P., Canzoniero, L. M. T. & Choi, D. W. Ion homeostasis and apoptosis. Curr. Opin. Cell Biol. 13, 405–411 (2001).

    CAS  PubMed  Google Scholar 

  14. Hosogi, S., Kusuzaki, K., Inui, T., Wang, X. & Marunaka, Y. Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell. J. Cell. Mol. Med. 18, 1124–1133 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Anke, D. et al. CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat. Cell Biol. 8, 933–944 (2006).

    Google Scholar 

  16. Sessler, J. L. et al. Synthesis, anion-binding properties, and in vitro anticancer activity of prodigiosin analogues. Angew. Chem. Int. Ed. 117, 6143–6146 (2005).

    Google Scholar 

  17. Ohkuma, S. et al. Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl− symport. Biochem. J. 334, 731–741 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Melvin, M. S. et al. Double-strand DNA cleavage by copper·prodigiosin. J. Am. Chem. Soc. 122, 6333–6334 (2000).

    CAS  Google Scholar 

  19. Li, H. et al. Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia. Nat. Chem. 8, 24–32 (2016).

    CAS  PubMed  Google Scholar 

  20. Busschaert, N. et al. Squaramides as potent transmembrane anion transporters. Angew. Chem. Int. Ed. 51, 4426–4430 (2012).

    CAS  Google Scholar 

  21. Jin, C. et al. Squaramide-based tripodal receptors for selective recognition of sulfate anion. Chem. Commun. 49, 2025–2027 (2013).

    CAS  Google Scholar 

  22. Busschaert, N. et al. Towards predictable transmembrane transport: QSAR analysis of anion binding and transport. Chem. Sci. 4, 3036–3045 (2013).

    CAS  Google Scholar 

  23. Busschaert, N. et al. Structure–activity relationships in tripodal transmembrane anion transporters: the effect of fluorination. J. Am. Chem. Soc. 133, 14136–14148 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Moore, S. J. et al. Chloride, carboxylate and carbonate transport by ortho-phenylenediamine-based bisureas. Chem. Sci. 4, 103–117 (2013).

    CAS  Google Scholar 

  25. Clement, N. R. & Gould, J. M. Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles. Biochemistry 20, 1534–1538 (1981).

    CAS  PubMed  Google Scholar 

  26. Busschaert, N. et al. Thiosquaramides: pH switchable anion transporters. Chem. Sci. 5, 3617–3626 (2014).

    CAS  PubMed  Google Scholar 

  27. Valkenier, H., Haynes, C. J. E., Herniman, J., Gale, P. A. & Davis, A. P. Lipophilic balance – a new design principle for transmembrane anion carriers. Chem. Sci. 5, 1128–1134 (2014).

    CAS  Google Scholar 

  28. Li, Z., Deng, L.-Q., Chen, J.-X., Zhou, C.-Q. & Chen, W.-H. Does lipophilicity affect the effectiveness of a transmembrane anion transporter? Insight from squaramido-functionalized bis(choloyl) conjugates. Org. Biomol. Chem. 13, 11761–11769 (2015).

    CAS  PubMed  Google Scholar 

  29. Hagelin, H., Murray, J. S., Politzer, P., Brinck, T. & Berthelot, M. Family-independent relationships between computed molecular surface quantities and solute hydrogen bond acidity/basicity and solute-induced methanol O–H infrared frequency shifts. Can. J. Chem. 73, 483–488 (1995).

    CAS  Google Scholar 

  30. Behr, J. P., Kirch, M. & Lehn, J. M. Carrier-mediated transport through bulk liquid membranes: dependence of transport rates and selectivity on carrier properties in a diffusion-limited process. J. Am. Chem. Soc. 107, 241–246 (1985).

    CAS  Google Scholar 

  31. Vargas Jentzsch, A. et al. Ditopic ion transport systems: anion–π interactions and halogen bonds at work. Angew. Chem. Int. Ed. 50, 11675–11678 (2011).

    CAS  Google Scholar 

  32. Wu, X. et al. Nonprotonophoric electrogenic chloride transport mediated by valinomycin-like carriers. Chem 1, 127–146 (2016).

    CAS  Google Scholar 

  33. Howe, E. N. W. et al. pH-regulated nonelectrogenic anion transport by phenylthiosemicarbazones. J. Am. Chem. Soc. 138, 8301–8308 (2016).

    CAS  PubMed  Google Scholar 

  34. Läuger, P. Mechanisms of biological ion transport—carriers, channels, and pumps in artificial lipid membranes. Angew. Chem. Int. Ed. 24, 905–923 (1985).

    Google Scholar 

  35. Mollenhauer, H. H., James Morré, D. & Rowe, L. D. Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochim. Biophys. Acta 1031, 225–246 (1990).

    CAS  PubMed  Google Scholar 

  36. Tsukimoto, M., Harada, H., Ikari, A. & Takagi, K. Involvement of chloride in apoptotic cell death induced by activation of ATP-sensitive P2 × 7 purinoceptor. J. Biol. Chem. 280, 2653–2658 (2005).

    CAS  PubMed  Google Scholar 

  37. Bortner, C. D. & Cidlowski, J. A. Uncoupling cell shrinkage from apoptosis reveals that Na+ influx is required for volume loss during programmed cell death. J. Biol. Chem. 278, 39176–39184 (2003).

    CAS  PubMed  Google Scholar 

  38. Saha, T., Hossain, M. S., Saha, D., Lahiri, M. & Talukdar, P. Chloride-mediated apoptosis-inducing activity of bis(sulfonamide) anionophores. J. Am. Chem. Soc. 138, 7558–7567 (2016).

    CAS  PubMed  Google Scholar 

  39. Waterhouse, N. J. et al. Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J. Cell Biol. 153, 319–328 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Tzung, S.-P. et al. Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3. Nat. Cell Biol. 3, 183–191 (2001).

    CAS  PubMed  Google Scholar 

  41. Elmore, S. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 (1997).

    CAS  PubMed  Google Scholar 

  43. Joza, N. et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410, 549–554 (2001).

    CAS  PubMed  Google Scholar 

  44. Cho, H. J. et al. A small molecule that binds to an ATPase domain of Hsc70 promotes membrane trafficking of mutant cystic fibrosis transmembrane conductance regulator. J. Am. Chem. Soc. 133, 20267–20276 (2011).

    CAS  PubMed  Google Scholar 

  45. Perelman, A. et al. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death Dis. 3, e430 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Yang, Y.-P. et al. Application and interpretation of current autophagy inhibitors and activators. Acta Pharmacol. Sin. 34, 625–635 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Xie, Z. & Klionsky, D. J. Autophagosome formation: core machinery and adaptations. Nat. Cell Biol. 9, 1102–1109 (2007).

    CAS  PubMed  Google Scholar 

  48. Graves, A. R., Curran, P. K., Smith, C. L. & Mindell, J. A. The Cl−/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453, 788–792 (2008).

    CAS  PubMed  Google Scholar 

  49. DiCiccio, J. E. & Steinberg, B. E. Lysosomal pH and analysis of the counter ion pathways that support acidification. J. Gen. Physiol. 137, 385–390 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Creasy, B. M., Hartmann, C. B., White, F. K. H. & McCoy, K. L. New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations. Cytometry Part A 71A, 114–123 (2007).

    Google Scholar 

  51. Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007).

    CAS  PubMed  Google Scholar 

  52. Chen, Y., McMillan-Ward, E., Kong, J., Israels, S. J. & Gibson, S. B. Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ. 15, 171–182 (2007).

    PubMed  Google Scholar 

  53. Jahreiss, L., Menzies, F. M. & Rubinsztein, D. C. The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes. Traffic 9, 574–587 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported financially by the National Creative Research Initiative (grant no. 2010-0018272 to I.S.) and Basic Science Research programmes (grant no. NRF-2012R1A1A1040142 to W.N.) in Korea. P.A.G. acknowledges the EPSRC for postdoctoral fellowships (EP/J009687/1 to N.B. and E.N.W.H.) and the Royal Society and the Wolfson Foundation for a Research Merit Award. The work in Austin was supported by the National Institutes of Health (grant no. GM103790 to J.L.S.). The theoretical studies were supported by projects P2020-PTDC/QEQ-SUP/4283/2014, CICECO – Aveiro Institute of Materials (UID/CTM/50011/2013) and iBiMED – Institute of Biomedicine (UID/BIM/04501/2013), financed by National Funds through the FCT/MEC and co-financed by QREN-FEDER through COMPETE under the PT2020 Partnership Agreement. I.M. acknowledges the FCT for PhD scholarship SFRH/BD/87520/2012. The authors thank H.J. Clarke for resynthesizing compound 5.

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Author notes

  1. Nathalie Busschaert, Ethan N. W. Howe, Jennifer R. Hiscock & Philip A. Gale

    Present address: Present addresses: Silver Center for Arts and Science, New York University, Department of Chemistry, 100 Washington Square East, New York, New York 10003, USA (N.B.); School of Chemistry, University of Sydney, NSW 2006, Australia (E.N.W.H., P.A.G.); School of Physical Sciences, University of Kent, Park Wood Road, Canterbury, Kent CT2 7NH, UK (J.R.H.),

  2. Nathalie Busschaert and Seong-Hyun Park: These authors contributed equally to this work

Authors and Affiliations

  1. Chemistry, University of Southampton, Southampton, SO17 1BJ, UK

    Nathalie Busschaert, Ethan N. W. Howe, Jennifer R. Hiscock, Louise E. Karagiannidis & Philip A. Gale

  2. Department of Chemistry, Yonsei University, Seoul, 03722, Korea

    Seong-Hyun Park, Kyung-Hwa Baek, Yoon Pyo Choi & Injae Shin

  3. College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, 21983, Korea

    Jinhong Park & Wan Namkung

  4. Department of Chemistry, Department of Medical Sciences, CICECO – Aveiro Institute of Materials, iBiMED – Institute of Biomedicine, University of Aveiro, Aveiro, 3810-193, Portugal

    Igor Marques & Vítor Félix

  5. Department of Chemistry, University of Texas at Austin, Austin, 78712-1224, Texas, USA

    Jonathan L. Sessler

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Contributions

I.S. and N.B. designed the study and wrote the manuscript. J.L.S. and P.A.G. helped with writing the manuscript. I.S., J.L.S and P.A.G. supervised the project. S.-H.P., K.-H.B. and Y.P.C. performed biological studies. N.B. designed, synthesized and characterized the compounds and performed the ion-transport studies in liposomes and the anion binding studies. E.N.W.H., J.R.H. and L.E.K. helped with some of the liposomal studies. I.M. and V.F. carried out DFT calculations and Vs,max calculations. J.P. and W.N. carried out ion-transport activity studies in cells.

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Correspondence to Jonathan L. Sessler, Philip A. Gale or Injae Shin.

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Supplementary information

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Supplementary information (PDF 5271 kb)

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Crystallographic data for compound 5a. (CIF 565 kb)

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Crystallographic data for compound 5b. (CIF 1015 kb)

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Crystallographic data for compound 8. (CIF 752 kb)

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Busschaert, N., Park, SH., Baek, KH. et al. A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations. Nature Chem 9, 667–675 (2017). https://doi.org/10.1038/nchem.2706

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