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Synthesis of Dynole 34-2, Dynole 2-24 and Dyngo 4a for investigating dynamin GTPase

Nature Protocols volume 9, pages 851–870 (2014)Cite this article

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Abstract

Dynamin is a large GTPase with roles in membrane fission during clathrin-mediated endocytosis, in actin dynamics and in cytokinesis. Defects in dynamin have been linked to human diseases. The synthesis of a dynamin modulator toolkit comprising two different inhibitor classes is described. The first series comprises Dynole 34-2, Dynole 2-24 and the inactive control Dynole 31-2. The Dynole compounds act on the dynamin G domain, are not GTP competitive and can be synthesized in 2–3 d. Knoevenagel condensation of 1-(3-(dimethylamino)propyl)-1H-indole-3-carbaldehyde (1) with cyanoamides (2 and 3) affords Dynole 31-2 and Dynole 34-2, respectively. Reductive amination of 1 with decylamine gives Dynole 2-24. The second series acts at an allosteric site in the G domain of dynamin and comprises Dyngo 4a and Dyngo Ø (inactive control). Both are synthesized in an overnight reaction via condensation of 3-hydroxy-2-naphthoic hydrazide with 2,4,5-trihydroxybenzaldehyde to afford Dyngo 4a, or with benzaldehyde to afford Dyngo Ø.

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Figure 1: Synthesis of the Dynole series of compounds.
Figure 2: Synthesis of the Dyngo series of compounds.

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References

  1. Ferguson, S.M. & De Camilli, P. Dynamin, a membrane-remodelling GTPase. Nat. Rev. Mol. Cell Biol. 13, 75–88 (2012).

    Article  CAS  Google Scholar 

  2. Chappie, J.S., Acharya, S., Leonard, M., Schmid, S.L. & Dyda, F. G domain dimerization controls dynamin's assembly-stimulated GTPase activity. Nature 465, 435–440 (2010).

    Article  CAS  Google Scholar 

  3. Chappie, J.S. et al. A pseudoatomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke. Cell 147, 209–222 (2011).

    Article  CAS  Google Scholar 

  4. Marks, B. et al. GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 410, 231–235 (2001).

    Article  CAS  Google Scholar 

  5. Praefcke, G.J.K. & McMahon, H.T. The dynamin superfamily: universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell Biol. 5, 133–147 (2004).

    Article  CAS  Google Scholar 

  6. Ferguson, K.M., Lemmon, M.A., Schlessinger, J. & Sigler, P.B. Crystal structure at 2.2 Å resolution of the pleckstrin homology domain from human dynamin. Cell 79, 199–209 (1994).

    Article  CAS  Google Scholar 

  7. Reubold, T.F. et al. Crystal structure of the GTPase domain of rat dynamin 1. Proc. Natl. Acad. Sci. USA 102, 13093–13098 (2005).

    Article  CAS  Google Scholar 

  8. Niemann, H.H., Knetsch, M.L.W., Scherer, A., Manstein, D.J. & Kull, F.J. Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms. EMBO J. 20, 5813–5821 (2001).

    Article  CAS  Google Scholar 

  9. Gao, S. et al. Structural basis of oligomerization in the stalk region of dynamin-like MxA. Nature 465, 502–506 (2010).

    Article  CAS  Google Scholar 

  10. Ford, M.G.J., Jenni, S. & Nunnari, J. The crystal structure of dynamin. Nature 477, 561–566 (2011).

    Article  CAS  Google Scholar 

  11. Faelber, K. et al. Crystal structure of nucleotide-free dynamin. Nature 477, 556–560 (2011).

    Article  CAS  Google Scholar 

  12. McMahon, H.T. & Boucrot, E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 12, 517–533 (2011).

    Article  CAS  Google Scholar 

  13. Schmid, S.L. & Frolov, V.A. Dynamin: functional design of a membrane fission catalyst. Ann. Rev. Cell Develop. Biol. 27, 79–105 (2011).

    Article  CAS  Google Scholar 

  14. Morlot, S. et al. Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction. Cell 151, 619–629 (2012).

    Article  CAS  Google Scholar 

  15. Smith, S.M., Renden, R. & von Gersdorff, H. Synaptic vesicle endocytosis: fast and slow modes of membrane retrieval. Trends Neurosci. 31, 559–568 (2008).

    Article  CAS  Google Scholar 

  16. Ferguson, S.M. et al. A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis. Science 316, 570–574 (2007).

    Article  CAS  Google Scholar 

  17. Bunnage, M.E., Piatnitski Chekler, E.L. & Jones, L.H. Target validation using chemical probes. Nat. Chem. Biol. 9, 195–199 (2013).

    Article  CAS  Google Scholar 

  18. Bain, J., McLauchlan, H., Elliott, M. & Cohen, P. The specificities of protein kinase inhibitors: an update. Biochem. J. 371, 199–204 (2003).

    Article  CAS  Google Scholar 

  19. Harper, C.B., Popoff, M.R., McCluskey, A., Robinson, P.J. & Meunier, F.A. Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors. Trends Cell Biol. 23, 90–101 (2012).

    Article  Google Scholar 

  20. Quan, A. et al. Myristyl trimethyl ammonium bromide and octadecyl trimethyl ammonium bromide are surface-active small molecule dynamin inhibitors that block endocytosis mediated by dynamin I or dynamin II. Mol. Pharm. 72, 1425–39 (2007).

    Article  CAS  Google Scholar 

  21. Hill, T. et al. Small molecule inhibitors of dynamin I GTPase activity: development of dimeric tyrphostins. J. Med. Chem. 48, 7781–7788 (2005).

    Article  CAS  Google Scholar 

  22. Odell, L.R. et al. Azido and diazarinyl analogues of bis-tyrphostin as asymmetrical inhibitors of dynamin GTPase. ChemMedChem 4, 1182–1188 (2009).

    Article  CAS  Google Scholar 

  23. Zhang, J., Lawrance, G.A., Chau, N., Robinson, P.J. & McCluskey, A. From Spanish fly to room-temperature ionic liquids (RTILs): synthesis, thermal stability and inhibition of dynamin 1 GTPase by a novel class of RTILs. New J. Chem. 32, 28–36 (2008).

    Article  CAS  Google Scholar 

  24. Hill, T.A. et al. Iminochromene inhibitors of dynamins I and II GTPase activity and endocytosis. J. Med. Chem. 53, 4094–4102 (2010).

    Article  CAS  Google Scholar 

  25. Odell, L.R. et al. The pthaladyns: GTP competitive inhibitors of dynamin I and II GTPase derived from virtual screening. J. Med. Chem. 53, 5267–5280 (2010).

    Article  CAS  Google Scholar 

  26. Harper, C.B. et al. Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J. Biol. Chem. 286, 35966–35976 (2011).

    Article  CAS  Google Scholar 

  27. McCluskey, A. et al. Building a better dynasore: the Dyngo compounds potently inhibit dynamin and endocytosis. Traffic 14, 1272–1289 (2013).

    Article  CAS  Google Scholar 

  28. Robertson, M.J. et al. The rhodadyns, a new class of small molecule inhibitors of dynamin GTPase activity. Med. Chem. Lett. 3, 352–356 (2012).

    Article  CAS  Google Scholar 

  29. McGeachie, A.B. et al. The Pyrimidyns: novel small molecule PH domain targeted pyrimidine-based dynamin inhibitors. ACS Chem. Biol. 8, 1507–1518 (2013).

    Article  CAS  Google Scholar 

  30. Hill, T.A. et al. Inhibition of dynamin mediated endocytosis by the dynoles—synthesis and functional activity of a family of indoles. J. Med. Chem. 52, 3762–3773 (2009).

    Article  CAS  Google Scholar 

  31. Gordon, C.P. et al. Development of second-generation indole-based dynamin GTPase inhibitors. J. Med. Chem. 56, 46–59 (2013).

    Article  CAS  Google Scholar 

  32. Gorobets, N.Y., Yousefi, B.H., Belaj, F. & Kappe, C.O. Rapid microwave-assisted solution phase synthesis of substituted 2-pyridone libraries. Tetrahedron 60, 8633–8644 (2004).

    Article  CAS  Google Scholar 

  33. Bhawal, B.M., Khanapure, S.P. & Biehl, E.R. Rapid, low-temperature amidation of ethyl cyano-acetate with lithium amides derived from primary and secondary amines. Syn. Commun. 20, 3235–3243 (1990).

    Article  CAS  Google Scholar 

  34. ThalesNano. H-cube continuous-flow hydrogenation reactor (http://www.thalesnano.com/products/h-cube).

  35. Palla, G., Predieri, G., Domiano, P., Vignali, C. & Turner, W. Conformational behaviour and E/Z isomerization of N-acyl and N-aroylhydrazones. Tetrahedron 42, 3649–3654 (1986).

    Article  CAS  Google Scholar 

  36. Franzen, H. & Eichler, T. Replacement of hydroxyl by the hydrazine group. J. Prakt. Chem. 78, 157–164 (1909).

    Article  CAS  Google Scholar 

  37. Sadowski, L. et al. Dynamin inhibitors impair endocytosis and mitogenic signaling of PDGF. Traffic 14, 725–736 (2013).

    Article  Google Scholar 

  38. Bain, J. et al. The selectivity of protein kinase inhibitors: a further update. Biochem. J. 408, 297–315 (2007).

    Article  CAS  Google Scholar 

  39. Park, R. et al. Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors. J. Cell Sci. 126 (part 22): 5305–5312 (2013).

    Article  CAS  Google Scholar 

  40. Daniel, J.A., Malladi, C.S., Kettle, E., McCluskey, A. & Robinson, P.J. Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening. Nat. Prot. 7, 1439–1455 (2012).

    Article  CAS  Google Scholar 

  41. Still, W.C., Kahn, M. & Mitra, A. Rapid chromatographic technique for preparative separations with moderate resolution. J. Org. Chem. 43, 2923–2925 (1978).

    Article  CAS  Google Scholar 

  42. Hill, T.A. et al. Long-chain amines and long chain ammonium salts as novel inhibitors of dynamin GTPase activity. Bioorg. Med. Chem. Lett. 14, 3275–3278 (2004).

    Article  CAS  Google Scholar 

  43. Chircop, M. et al. Inhibition of dynamin by dynole 34-2 induces cell death following cytokinesis failure in cancer cells. Mol. Can. Therap. 10, 1553–1562 (2011).

    Article  CAS  Google Scholar 

  44. Takahashi, K. et al. . Suppression of dynamin GTPase activity by sertraline leads to inhibition of dynamin-dependent endocytosis. Biochem. Biophys. Res. Commun. 391, 382–387 (2010).

    Article  CAS  Google Scholar 

  45. Joffre, C. et al. A direct role for Met endocytosis in tumorigenesis. Nat. Cell Biol. 13, 821–837 (2011).

    Article  Google Scholar 

  46. Smith, C.M. & Chircop, M. Clathrin-mediated endocytic proteins are involved in regulating mitotic progression and completion. Traffic 13, 1628–1641 (2012).

    Article  CAS  Google Scholar 

  47. Richard, J.P. et al. Intracellular curvature-generating proteins in cell-to-cell fusion. Biochemical J. 440, 185–193 (2011).

    Article  CAS  Google Scholar 

  48. Bittner, M.A., Aikman, R.L. & Holz, R.W. A nibbling mechanism for clathrin-mediated retrieval of secretory granule membrane after exocytosis. J. Biol. Chem. 288, 9177–9188 (2013).

    Article  CAS  Google Scholar 

  49. Kural, C. et al. Dynamics of intracellular clathrin/AP1- and clathrin/AP3-containing carriers. Cell Rep. 2, 1111–1119 (2012).

    Article  CAS  Google Scholar 

  50. Sorkina, T., Caltagarone, J. & Sorkin, A. Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C–dependent endocytosis. Traffic 14, 709–724 (2013).

    Article  Google Scholar 

  51. Reid, A.T. et al. Dynamin regulates specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa. J. Biol. Chem. 287, 37659–37672 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants from the National Health and Medical Research Council (Australia), The Australia Research Council, The Australian Cancer Research Foundation, The Ramaciotti Foundation, The Children's Medical Research Institute and Newcastle Innovation, Ltd.

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Author notes

  1. Mark J Robertson and Fiona M Deane: These authors contributed equally to this work.

Authors and Affiliations

  1. Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, New South Wales, Australia

    Mark J Robertson, Fiona M Deane & Adam McCluskey

  2. Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, New South Wales, Australia

    Phillip J Robinson

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Contributions

M.J.R. and F.M.D. contributed equally to the synthesis of all analogs described in this work. P.J.R. and A.M. are responsible for the concept, design and use of the dynamin inhibitors reported herein.

Corresponding authors

Correspondence to Phillip J Robinson or Adam McCluskey.

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Competing interests

We have a commercial agreement with Abcam Biochemicals (Bristol, UK) for the supply of our dynamin inhibitors. This includes many of the compounds listed in this paper. Quinodyn, Dynole, Rhodadyn, MiTMAB, Bis-T, Dyngo and Iminodyn are trademarks of Children's Medical Research Institute and Newcastle Innovation, Ltd. Most of the lead dynamin inhibitors described in this paper are available from Abcam Biochemicals (Bristol, UK).

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Robertson, M., Deane, F., Robinson, P. et al. Synthesis of Dynole 34-2, Dynole 2-24 and Dyngo 4a for investigating dynamin GTPase. Nat Protoc 9, 851–870 (2014). https://doi.org/10.1038/nprot.2014.046

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