Metal coordination and peripheral substitution modulate the activity of cyclic tetrapyrroles on αS aggregation: a structural and cell-based study

  • Nazareno González
  • Iñaki Gentile
  • Hugo A. Garro
  • Susana Delgado-Ocaña
  • Carla F. Ramunno
  • Fiamma A. Buratti
  • Christian Griesinger
  • Claudio O. FernándezEmail author
Original Paper
Part of the following topical collections:
  1. Metal Ions and Degenerative Diseases


The discovery of aggregation inhibitors and the elucidation of their mechanism of action are key in the quest to mitigate the toxic consequences of amyloid formation. We have previously characterized the antiamyloidogenic mechanism of action of sodium phtalocyanine tetrasulfonate ([Na4(H2PcTS)]) on α-Synuclein (αS), demonstrating that specific aromatic interactions are fundamental for the inhibition of amyloid assembly. Here we studied the influence that metal preferential affinity and peripheral substituents may have on the activity of tetrapyrrolic compounds on αS aggregation. For the first time, our laboratory has extended the studies in the field of the bioinorganic chemistry and biophysics to cellular biology, using a well-established cell-based model to study αS aggregation. The interaction scenario described in our work revealed that both N- and C-terminal regions of αS represent binding interfaces for the studied compounds, a behavior that is mainly driven by the presence of negatively or positively charged substituents located at the periphery of the macrocycle. Binding modes of the tetrapyrrole ligands to αS are determined by the planarity and hydrophobicity of the aromatic ring system in the tetrapyrrolic molecule and/or the preferential affinity of the metal ion conjugated at the center of the macrocyclic ring. The different capability of phthalocyanines and meso-tetra (N-methyl-4-pyridyl) porphine tetrachloride ([H2PrTPCl4]) to modulate αS aggregation in vitro was reproduced in cell-based models of αS aggregation, demonstrating unequivocally that the modulation exerted by these compounds on amyloid assembly is a direct consequence of their interaction with the target protein.


Misfolding Amyloid Neurodegeneration Inhibitors 



C.O.F. thanks Universidad Nacional de Rosario (UNR) and ANPCyT- FONCyT (PICT 2014-3704 and PICT 2017-4665) for financial support. C.O.F. and C.G. thank the Max Planck Society (P10390) for support. C.O.F thanks Dietmar Riedel and Gudrum Heim for helpful assistance during the transmission electron microscopy measurements. N.G. and I.G. thanks UNR for fellowships. F.B. thanks CONICET for fellowship.

Supplementary material

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Supplementary material 1 (PDF 584 kb)


  1. 1.
    Gandhi S, Wood NW (2010) Genome-wide association studies: the key to unlocking neurodegeneration? Nat Neurosci 13:789–794. CrossRefGoogle Scholar
  2. 2.
    de Lau LML, Breteler MMB (2006) Epidemiology of Parkinson’s disease. Lancet Neurol 5:525–535. CrossRefGoogle Scholar
  3. 3.
    Henchcliffe C, Beal MF (2008) Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis. Nat Clin Pract Neurol 4:600–609. CrossRefGoogle Scholar
  4. 4.
    Spillantini MG, Crowther RA, Jakes R et al (1998) Filamentous alpha-synuclein inclusions link multiple system atrophy with Parkinson’s disease and dementia with Lewy bodies. Neurosci Lett 251:205–208. CrossRefGoogle Scholar
  5. 5.
    Papapetropoulos S, Adi N, Ellul J et al (2007) A prospective study of familial versus sporadic Parkinson’s disease. Neurodegener Dis 4:424–427. CrossRefGoogle Scholar
  6. 6.
    Dawson TM, Dawson VL (2003) Rare genetic mutations shed light on the pathogenesis of Parkinson disease. J Clin Invest 111:145–151. CrossRefGoogle Scholar
  7. 7.
    McCann H, Stevens CH, Cartwright H, Halliday GM (2014) α-Synucleinopathy phenotypes. Parkinsonism Relat Disord 20(Suppl 1):S62–S67. CrossRefGoogle Scholar
  8. 8.
    Spillantini MG, Crowther RA, Jakes R et al (1998) alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with lewy bodies. Proc Natl Acad Sci USA 95:6469–6473. CrossRefGoogle Scholar
  9. 9.
    Volles MJ, Lansbury PT (2003) Zeroing in on the pathogenic form of alpha-synuclein and its mechanism of neurotoxicity in Parkinson’s disease. Biochemistry 42:7871–7878. CrossRefGoogle Scholar
  10. 10.
    Cookson MR, van der Brug M (2008) Cell systems and the toxic mechanism(s) of alpha-synuclein. Exp Neurol 209:5–11. CrossRefGoogle Scholar
  11. 11.
    Eisbach SE, Outeiro TF (2013) Alpha-synuclein and intracellular trafficking: impact on the spreading of Parkinson’s disease pathology. J Mol Med 91:693–703. CrossRefGoogle Scholar
  12. 12.
    Roberts HL, Brown DR (2015) Seeking a mechanism for the toxicity of oligomeric α-synuclein. Biomolecules 5:282–305. CrossRefGoogle Scholar
  13. 13.
    Winner B, Jappelli R, Maji SK et al (2011) In vivo demonstration that alpha-synuclein oligomers are toxic. Proc Natl Acad Sci USA 108:4194–4199. CrossRefGoogle Scholar
  14. 14.
    Karpinar DP, Balija MBG, Kügler S et al (2009) Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson’s disease models. EMBO J 28:3256–3268. CrossRefGoogle Scholar
  15. 15.
    Lázaro DF, Rodrigues EF, Langohr R et al (2014) Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation. PLoS Genet 10:e1004741. CrossRefGoogle Scholar
  16. 16.
    Pokrzywa M, Pawełek K, Kucia WE et al (2017) Effects of small-molecule amyloid modulators on a Drosophila model of Parkinson’s disease. PLoS One 12:e0184117. CrossRefGoogle Scholar
  17. 17.
    Kurnik M, Sahin C, Andersen CB et al (2018) Potent α-synuclein aggregation inhibitors, identified by high-throughput screening, mainly target the monomeric state. Cell Chem Biol 25:1389–1402.e9. CrossRefGoogle Scholar
  18. 18.
    Tonda-Turo C, Herva M, Chiono V et al (2018) Influence of drug-carrier polymers on alpha-synucleinopathies: a neglected aspect in new therapies development. Biomed Res Int 2018:4518060. CrossRefGoogle Scholar
  19. 19.
    Trigo-Damas I, Del Rey NL-G, Blesa J (2018) Novel models for Parkinson’s disease and their impact on future drug discovery. Expert Opin Drug Discov 13:229–239. CrossRefGoogle Scholar
  20. 20.
    Maroteaux L, Campanelli JT, Scheller RH (1988) Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J Neurosci 8:2804–2815CrossRefGoogle Scholar
  21. 21.
    Burré J, Sharma M, Südhof TC (2014) α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc Natl Acad Sci USA 111:E4274–E4283. CrossRefGoogle Scholar
  22. 22.
    Stefanis L (2012) α-Synuclein in Parkinson’s disease. Cold Spring Harb Perspect Med 2:a009399. CrossRefGoogle Scholar
  23. 23.
    Sidhu A, Wersinger C, Vernier P (2004) Does alpha-synuclein modulate dopaminergic synaptic content and tone at the synapse? FASEB J 18:637–647. CrossRefGoogle Scholar
  24. 24.
    Yavich L, Tanila H, Vepsäläinen S, Jäkälä P (2004) Role of alpha-synuclein in presynaptic dopamine recruitment. J Neurosci 24:11165–11170. CrossRefGoogle Scholar
  25. 25.
    Breydo L, Wu JW, Uversky VN (2012) Α-synuclein misfolding and Parkinson’s disease. Biochim Biophys Acta 1822:261–285. CrossRefGoogle Scholar
  26. 26.
    Dedmon MM, Lindorff-Larsen K, Christodoulou J et al (2005) Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J Am Chem Soc 127:476–477. CrossRefGoogle Scholar
  27. 27.
    Bertoncini CW, Jung Y-S, Fernandez CO et al (2005) Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein. Proc Natl Acad Sci USA 102:1430–1435. CrossRefGoogle Scholar
  28. 28.
    Cho M-K, Nodet G, Kim H-Y et al (2009) Structural characterization of alpha-synuclein in an aggregation prone state. Protein Sci 18:1840–1846. CrossRefGoogle Scholar
  29. 29.
    Chiti F, Dobson CM (2017) Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. Annu Rev Biochem 86:27–68. CrossRefGoogle Scholar
  30. 30.
    Moriarty GM, Janowska MK, Kang L, Baum J (2013) Exploring the accessible conformations of N-terminal acetylated α-synuclein. FEBS Lett 587:1128–1138. CrossRefGoogle Scholar
  31. 31.
    Kang L, Moriarty GM, Woods LA et al (2012) N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer. Protein Sci 21:911–917. CrossRefGoogle Scholar
  32. 32.
    Liu T, Bitan G (2012) Modulating self-assembly of amyloidogenic proteins as a therapeutic approach for neurodegenerative diseases: strategies and mechanisms. ChemMedChem 7:359–374. CrossRefGoogle Scholar
  33. 33.
    Villemagne VL, Doré V, Bourgeat P et al (2017) Aβ-amyloid and Tau Imaging in Dementia. Semin Nucl Med 47:75–88. CrossRefGoogle Scholar
  34. 34.
    Arja K, Sjölander D, Åslund A et al (2013) Enhanced fluorescent assignment of protein aggregates by an oligothiophene-porphyrin-based amyloid ligand. Macromol Rapid Commun 34:723–730. CrossRefGoogle Scholar
  35. 35.
    Lamberto GR, Binolfi A, Orcellet ML et al (2009) Structural and mechanistic basis behind the inhibitory interaction of PcTS on alpha-synuclein amyloid fibril formation. Proc Natl Acad Sci USA 106:21057–21062. CrossRefGoogle Scholar
  36. 36.
    Valiente-Gabioud AA, Riedel D, Outeiro TF et al (2018) Binding modes of phthalocyanines to amyloid β peptide and their effects on amyloid fibril formation. Biophys J 114:1036–1045. CrossRefGoogle Scholar
  37. 37.
    Lamberto GR, Torres-Monserrat V, Bertoncini CW et al (2011) Toward the discovery of effective polycyclic inhibitors of alpha-synuclein amyloid assembly. J Biol Chem 286:32036–32044. CrossRefGoogle Scholar
  38. 38.
    Bulic B, Pickhardt M, Khlistunova I et al (2007) Rhodanine-based tau aggregation inhibitors in cell models of tauopathy. Angew Chem Int Ed Engl 46:9215–9219. CrossRefGoogle Scholar
  39. 39.
    Schenk D, Basi GS, Pangalos MN (2012) Treatment strategies targeting amyloid β-protein. Cold Spring Harb Perspect Med 2:a006387. CrossRefGoogle Scholar
  40. 40.
    Masuda M, Suzuki N, Taniguchi S et al (2006) Small molecule inhibitors of alpha-synuclein filament assembly. Biochemistry 45:6085–6094. CrossRefGoogle Scholar
  41. 41.
    Caughey B, Caughey WS, Kocisko DA et al (2006) Prions and transmissible spongiform encephalopathy (TSE) chemotherapeutics: a common mechanism for anti-TSE compounds? Acc Chem Res 39:646–653. CrossRefGoogle Scholar
  42. 42.
    Ehrnhoefer DE, Bieschke J, Boeddrich A et al (2008) EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat Struct Mol Biol 15:558–566. CrossRefGoogle Scholar
  43. 43.
    Caughey WS, Priola SA, Kocisko DA et al (2007) Cyclic tetrapyrrole sulfonation, metals, and oligomerization in antiprion activity. Antimicrob Agents Chemother 51:3887–3894. CrossRefGoogle Scholar
  44. 44.
    Wagner J, Ryazanov S, Leonov A et al (2013) Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease. Acta Neuropathol 125:795–813. CrossRefGoogle Scholar
  45. 45.
    Levin J, Schmidt F, Boehm C et al (2014) The oligomer modulator anle138b inhibits disease progression in a Parkinson mouse model even with treatment started after disease onset. Acta Neuropathol 127:779–780. CrossRefGoogle Scholar
  46. 46.
    Scherzer-Attali R, Shaltiel-Karyo R, Adalist YH et al (2012) Generic inhibition of amyloidogenic proteins by two naphthoquinone-tryptophan hybrid molecules. Proteins 80:1962–1973. Google Scholar
  47. 47.
    Frydman-Marom A, Shaltiel-Karyo R, Moshe S, Gazit E (2011) The generic amyloid formation inhibition effect of a designed small aromatic β-breaking peptide. Amyloid 18:119–127. CrossRefGoogle Scholar
  48. 48.
    Frydman-Marom A, Rechter M, Shefler I et al (2009) Cognitive-performance recovery of Alzheimer’s disease model mice by modulation of early soluble amyloidal assemblies. Angew Chem Int Ed Engl 48:1981–1986. CrossRefGoogle Scholar
  49. 49.
    González-Lizárraga F, Socías SB, Ávila CL et al (2017) Repurposing doxycycline for synucleinopathies: remodelling of α-synuclein oligomers towards non-toxic parallel beta-sheet structured species. Sci Rep 7:41755. CrossRefGoogle Scholar
  50. 50.
    Pujols J, Peña-Díaz S, Lázaro DF et al (2018) Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons. Proc Natl Acad Sci USA 115:10481–10486. CrossRefGoogle Scholar
  51. 51.
    Valdinocci D, Grant GD, Dickson TC, Pountney DL (2018) Epothilone D inhibits microglia-mediated spread of alpha-synuclein aggregates. Mol Cell Neurosci 89:80–94. CrossRefGoogle Scholar
  52. 52.
    Schwab K, Frahm S, Horsley D et al (2017) A protein aggregation inhibitor, leuco-methylthioninium bis(hydromethanesulfonate), decreases α-synuclein inclusions in a transgenic mouse model of synucleinopathy. Front Mol Neurosci 10:447. CrossRefGoogle Scholar
  53. 53.
    Palazzi L, Bruzzone E, Bisello G et al (2018) Oleuropein aglycone stabilizes the monomeric α-synuclein and favours the growth of non-toxic aggregates. Sci Rep 8:8337. CrossRefGoogle Scholar
  54. 54.
    Jha NN, Kumar R, Panigrahi R et al (2017) Comparison of α-synuclein fibril inhibition by four different amyloid inhibitors. ACS Chem Neurosci 8:2722–2733. CrossRefGoogle Scholar
  55. 55.
    Reiner AM, Schmidt F, Ryazanov S et al (2018) Photophysics of diphenyl-pyrazole compounds in solutions and α-synuclein aggregates. Biochim Biophys Acta Gen Subj 1862:800–807. CrossRefGoogle Scholar
  56. 56.
    Valiente-Gabioud AA, Miotto MC, Chesta ME et al (2016) Phthalocyanines as molecular scaffolds to block disease-associated protein aggregation. Acc Chem Res 49:801–808. CrossRefGoogle Scholar
  57. 57.
    Fonseca-Ornelas L, Eisbach SE, Paulat M et al (2014) Small molecule-mediated stabilization of vesicle-associated helical α-synuclein inhibits pathogenic misfolding and aggregation. Nat Commun 5:5857. CrossRefGoogle Scholar
  58. 58.
    Akoury E, Gajda M, Pickhardt M et al (2013) Inhibition of tau filament formation by conformational modulation. J Am Chem Soc 135:2853–2862. CrossRefGoogle Scholar
  59. 59.
    Chakraborty R, Sahoo S, Halder N et al (2019) Conformational-switch based strategy triggered by [18] π heteroannulenes toward reduction of alpha synuclein oligomer toxicity. ACS Chem Neurosci 10:573–587. CrossRefGoogle Scholar
  60. 60.
    Caughey B, Lansbury PT (2003) Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu Rev Neurosci 26:267–298. CrossRefGoogle Scholar
  61. 61.
    Caughey WS, Raymond LD, Horiuchi M, Caughey B (1998) Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines. Proc Natl Acad Sci USA 95:12117–12122. CrossRefGoogle Scholar
  62. 62.
    Priola SA, Raines A, Caughey WS (2000) Porphyrin and phthalocyanine antiscrapie compounds. Science 287:1503–1506CrossRefGoogle Scholar
  63. 63.
    Johnson M, Geeves MA, Mulvihill DP (2013) Production of amino-terminally acetylated recombinant proteins in E. coli. Methods Mol Biol 981:193–200. CrossRefGoogle Scholar
  64. 64.
    Hoyer W, Cherny D, Subramaniam V, Jovin TM (2004) Impact of the acidic C-terminal region comprising amino acids 109-140 on alpha-synuclein aggregation in vitro. Biochemistry 43:16233–16242. CrossRefGoogle Scholar
  65. 65.
    Cavanagh J, Fairbrother W, Palmer A III, Skelton N (1995) Protein NMR spectroscopy: principles and practice. Academic Press. ISBN: 9780080515298Google Scholar
  66. 66.
    Valiente-Gabioud AA, Torres-Monserrat V, Molina-Rubino L et al (2012) Structural basis behind the interaction of Zn2+ with the protein α-synuclein and the Aβ peptide: a comparative analysis. J Inorg Biochem 117:334–341. CrossRefGoogle Scholar

Copyright information

© Society for Biological Inorganic Chemistry (SBIC) 2019

Authors and Affiliations

  • Nazareno González
    • 1
  • Iñaki Gentile
    • 1
  • Hugo A. Garro
    • 1
    • 2
  • Susana Delgado-Ocaña
    • 1
  • Carla F. Ramunno
    • 1
  • Fiamma A. Buratti
    • 1
  • Christian Griesinger
    • 3
  • Claudio O. Fernández
    • 1
    • 3
    Email author
  1. 1.Max Planck Laboratory for Structural Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR, UNR-MPIbpC) and Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR, UNR-CONICET)Universidad Nacional de RosarioRosarioArgentina
  2. 2.Facultad de Química, Bioquímica y FarmaciaUniversidad Nacional de San LuisSan LuisArgentina
  3. 3.Department of NMR-based Structural BiologyMax Planck Institute for Biophysical ChemistryGöttingenGermany

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