Archives of Toxicology

, Volume 93, Issue 1, pp 189–206 | Cite as

Novel neurotoxic peptides from Protopalythoa variabilis virtually interact with voltage-gated sodium channel and display anti-epilepsy and neuroprotective activities in zebrafish

  • Qiwen Liao
  • Shengnan Li
  • Shirley Weng In Siu
  • Jean-Étienne R. L. Morlighem
  • Clarence Tsun Ting Wong
  • Xiufen Wang
  • Gandhi Rádis-BaptistaEmail author
  • Simon Ming-Yuen LeeEmail author


We previously reported a novel toxic peptide identified from the anthozoan Protopalythoa variabilis transcriptome which is homologous to a novel structural type of sodium channel toxin isolated from a parental species (Palythoa caribaeorum). The peptide was named, according to its homologous, as Pp V-shape α-helical peptide (PpVα) in the present study. Through molecular docking and dynamics simulation, linear and hairpin folded PpVα peptides were shown to be potential voltage-gated sodium channel blockers. Nowadays, sodium channel blockers have been the mainstream of the pharmacological management of epileptic seizures. Also, sodium channel blockers could promote neuronal survival by reducing sodium influx and reducing the likelihood of calcium importation resulting in suppressing microglial activation and protecting dopaminergic neurons from degeneration. The folded PpVα peptide could decrease pentylenetetrazol (PTZ)-induced c-fos and npas4a expression level leading to reverse PTZ-induced locomotor hyperactivity in zebrafish model. In vitro, the folded PpVα peptide protected PC12 cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via activating heme oxygenase-1 (HO-1) and attenuating inducible nitric oxide synthase (iNOS) expression. In vivo, PpVα peptide suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish and, importantly, prevented the 6-OHDA-induced excessive ROS generation and subsequent dopaminergic neurons loss. This study indicates that the single S–S bond folded PpVα peptide arises as a new structural template to develop sodium channel blockers and provides an insight on the peptide discovery from cnidarian transcriptome to potentially manage epilepsy and neurodegenerative disorders.


Neuroactive peptides Voltage-gated sodium ion channel blocker Protein docking Anti-epilepsy Neuroprotection Disulfide bond 



Research at University of Macau was supported by grants from the Science and Technology Development Fund (FDCT) of Macao SAR (Ref. no. 069/2015/A2 and no. 134/2014/A3) and Research Committee, University of Macau (MYRG2016-00133-ICMS-QRCM, MYRG2015-00182-ICMS-QRCM, and MYRG2016-00129-ICMS-QRCM). Research work at the Institute for Marine Sciences, Federal University of Ceará, was supported by the Brazilian National Council for Scientific and Technological Development—CNPq, under the auspices of the Marine Biotechnology Network Initiative (Grant no. 408835/2013-3 to G.R.-B.), the Ministry of Science, Technology, Innovation & Communication (MCTI-C) of the Federal Government of Brazil. J.-E.RLM was a former doctoral fellowship recipient from the Coordination for the improvement of Higher Education Personnel (CAPES, the Ministry of Education, Brazil).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

204_2018_2334_MOESM1_ESM.docx (980 kb)
Supplementary material 1 (DOCX 979 KB)


  1. Agrawal N, Alonso A, Ragsdale DS (2003) Increased persistent sodium currents in rat entorhinal cortex layer V neurons in a post-status epilepticus model of temporal lobe epilepsy. Epilepsia 44(12):1601–1604Google Scholar
  2. Anichtchik OV et al (2004) Neurochemical and behavioural changes in zebrafish Danio rerio after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. J Neurochem 88(2):443–453Google Scholar
  3. Athauda D, Foltynie T (2015) The ongoing pursuit of neuroprotective therapies in Parkinson disease. Nat Rev Neurol 11(1):25–40Google Scholar
  4. Averaimo S et al (2010) Chloride intracellular channel 1 (CLIC1): sensor and effector during oxidative stress. FEBS Lett 584(10):2076–2084Google Scholar
  5. Bagal SK et al (2015) Voltage gated sodium channels as drug discovery targets. Channels (Austin) 9(6):360–366Google Scholar
  6. Baraban SC et al (2005) Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131(3):759–768Google Scholar
  7. Baxendale S et al (2012) Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures. Dis Model Mech 5(6):773–784Google Scholar
  8. Berghmans S et al (2007) Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants. Epilepsy Res 75(1):18–28Google Scholar
  9. Blumenfeld H et al (2009) Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis. Epilepsia 50(1):44–55Google Scholar
  10. Brawek B, Garaschuk O (2014) Network-wide dysregulation of calcium homeostasis in Alzheimer’s disease. Cell Tissue Res 357(2):427–438Google Scholar
  11. Calabresi P et al (2003) Antiepileptic drugs as a possible neuroprotective strategy in brain ischemia. Ann Neurol 53(6):693–702Google Scholar
  12. Camproux AC, Gautier R, Tuffery P (2004) A hidden markov model derived structural alphabet for proteins. J Mol Biol 339(3):591–605Google Scholar
  13. Carstens BB et al (2016) Structure-activity studies of cysteine-rich alpha-conotoxins that inhibit high-voltage-activated calcium channels via GABA(B) receptor activation reveal a minimal functional motif. Angew Chem Int Ed Engl 55(15):4692–4696Google Scholar
  14. Catterall WA, Goldin AL, Waxman SG (2005) International union of pharmacology. XLVII. Nomenclature and structure–function relationships of voltage-gated sodium channels. Pharmacol Rev 57(4):397–409Google Scholar
  15. Chang BS, Lowenstein DH (2003) Epilepsy. N Engl J Med 349(13):1257–1266Google Scholar
  16. Chen R, Li L, Weng Z (2003) ZDOCK: an initial-stage protein-docking algorithm. Proteins 52(1):80–87Google Scholar
  17. Choudhury ME et al (2011) Zonisamide-induced long-lasting recovery of dopaminergic neurons from MPTP-toxicity. Brain Res 1384:170–178Google Scholar
  18. Choudhury ME et al (2012) Zonisamide up-regulated the mRNAs encoding astrocytic anti-oxidative and neurotrophic factors. Eur J Pharmacol 689(1–3):72–80Google Scholar
  19. Cui G et al (2013) A novel Danshensu derivative confers cardioprotection via PI3K/Akt and Nrf2 pathways. Int J Cardiol 168(2):1349–1359Google Scholar
  20. de Lera Ruiz M, Kraus RL (2015) Voltage-gated sodium channels: structure, function, pharmacology, and clinical indications. J Med Chem 58(18):7093–7118Google Scholar
  21. Dutton JL et al (2002) A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity. J Biol Chem 277(50):48849–48857Google Scholar
  22. Escayg A et al (2000) Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. Nat Genet 24(4):343–345Google Scholar
  23. Faber CG et al (2012) Gain of function Nanu1.7 mutations in idiopathic small fiber neuropathy. Ann Neurol 71(1):26–39Google Scholar
  24. Focken T et al (2016) Discovery of aryl sulfonamides as isoform-selective inhibitors of NaV1.7 with efficacy in rodent pain models. ACS Med Chem Lett 7(3):277–282Google Scholar
  25. Ghahremanpour MM et al (2014) MemBuilder: a web-based graphical interface to build heterogeneously mixed membrane bilayers for the GROMACS biomolecular simulation program. Bioinformatics 30(3):439–441Google Scholar
  26. Gordon D et al (2007) The differential preference of scorpion alpha-toxins for insect or mammalian sodium channels: implications for improved insect control. Toxicon 49(4):452–472Google Scholar
  27. Grau CM, Greene LA (2012) Use of PC12 cells and rat superior cervical ganglion sympathetic neurons as models for neuroprotective assays relevant to Parkinson’s disease. Methods Mol Biol 846:201–211Google Scholar
  28. Greene LA (1978) Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free medium. J Cell Biol 78(3):747–755Google Scholar
  29. Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73(7):2424–2428Google Scholar
  30. Heimer P et al (2018) Conformational mu-conotoxin PIIIA isomers revisited: impact of cysteine pairing on disulfide-bond assignment and structure elucidation. Anal Chem 90(5):3321–3327Google Scholar
  31. Hsieh CP (2008) Redox modulation of A-type K+ currents in pain-sensing dorsal root ganglion neurons. Biochem Biophys Res Commun 370(3):445–449Google Scholar
  32. Hsieh CJ et al (2014) Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the ROS/p38 MAPK pathway and epigenetic regulation. Free Radic Biol Med 67:159–170Google Scholar
  33. Huang C et al (2016) The Transcriptome of the Zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa) predicts a basal repertoire of toxin-like and venom-auxiliary polypeptides. Genome Biol Evol 8(9):3045–3064Google Scholar
  34. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38Google Scholar
  35. Iwata A et al (2004) Traumatic axonal injury induces proteolytic cleavage of the voltage-gated sodium channels modulated by tetrodotoxin and protease inhibitors. J Neurosci 24(19):4605–4613Google Scholar
  36. Jo S et al (2008) CHARMM-GUI: a web-based graphical user interface for CHARMM. J Comput Chem 29(11):1859–1865Google Scholar
  37. Kalia J et al (2015) From foe to friend: using animal toxins to investigate ion channel function. J Mol Biol 427(1):158–175Google Scholar
  38. Kearney JA et al (2001) A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities. Neuroscience 102(2):307–317Google Scholar
  39. Kinarivala N et al (2017) Passage variation of PC12 cells results in inconsistent susceptibility to externally induced apoptosis. ACS Chem Neurosci 8(1):82–88Google Scholar
  40. Klein JP et al (2004) Dysregulation of sodium channel expression in cortical neurons in a rodent model of absence epilepsy. Brain Res 1000(1–2):102–109Google Scholar
  41. Lazcano-Pérez F et al (2014) A purified palythoa venom fraction delays sodium current inactivation in sympathetic neurons. Toxicon 82:112–116Google Scholar
  42. Lazcano-Pérez F et al (2016) Activity of Palythoa caribaeorum venom on voltage-gated ion channels in mammalian superior cervical ganglion neurons. Toxins (Basel) 8(5):135Google Scholar
  43. Lenkey N et al (2010) Classification of drugs based on properties of sodium channel inhibition: a comparative automated patch-clamp study. PLoS One 5(12):e15568Google Scholar
  44. Liu PW, Bean BP (2014) Kv2 channel regulation of action potential repolarization and firing patterns in superior cervical ganglion neurons and hippocampal CA1 pyramidal neurons. J Neurosci 34(14):4991–5002Google Scholar
  45. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25(4):402–408Google Scholar
  46. Long SM et al (2014) Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish. Mar Drugs 12(6):3307–3322Google Scholar
  47. Lossin C et al (2002) Molecular basis of an inherited epilepsy. Neuron 34(6):877–884Google Scholar
  48. Lukacs P et al (2018) Non-blocking modulation contributes to sodium channel inhibition by a covalently attached photoreactive riluzole analog. Sci Rep 8(1):8110Google Scholar
  49. Malin SA, Nerbonne JM (2000) Elimination of the fast transient in superior cervical ganglion neurons with expression of KV4.2W362F: molecular dissection of IA. J Neurosci 20(14):5191–5199Google Scholar
  50. Mandel G et al (1988) Selective induction of brain type II Na+ channels by nerve growth factor. Proc Natl Acad Sci USA 85(3):924–928Google Scholar
  51. Mantegazza M et al (2010) Voltage-gated sodium channels as therapeutic targets in epilepsy and other neurological disorders. Lancet Neurol 9(4):413–424Google Scholar
  52. Maupetit J, Derreumaux P, Tufféry P (2010) A fast method for large-scale de novo peptide and miniprotein structure prediction. J Comput Chem 31(4):726–738Google Scholar
  53. Monge-Fuentes V et al (2015) Neuroactive compounds obtained from arthropod venoms as new therapeutic platforms for the treatment of neurological disorders. J Venom Anim Toxins Incl Trop Dis 21:31Google Scholar
  54. Mussulini BH et al (2013) Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization. PLoS One 8(1):e54515Google Scholar
  55. Nagoshi N, Nakashima H, Fehlings MG (2015) Riluzole as a neuroprotective drug for spinal cord injury: from bench to bedside. Molecules 20(5):7775–7789Google Scholar
  56. Naziroglu M, Dikici DM, Dursun S (2012) Role of oxidative stress and Ca(2)(+) signaling on molecular pathways of neuropathic pain in diabetes: focus on TRP channels. Neurochem Res 37(10):2065–2075Google Scholar
  57. Pardo LA, Stuhmer W (2014) The roles of K(+) channels in cancer. Nat Rev Cancer 14(1):39–48Google Scholar
  58. Payandeh J, Hackos DH (2018) Selective ligands and drug discovery targeting the voltage-gated sodium channel Nav1.7. Handb Exp Pharmacol 246:271–306Google Scholar
  59. Perfeito R, Cunha-Oliveira T, Rego AC (2012) Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease—resemblance to the effect of amphetamine drugs of abuse. Free Radic Biol Med 53(9):1791–1806Google Scholar
  60. Persson AK et al (2013) Sodium channels contribute to degeneration of dorsal root ganglion neurites induced by mitochondrial dysfunction in an in vitro model of axonal injury. J Neurosci 33(49):19250–19261Google Scholar
  61. Pierce B, Tong W, Weng Z (2005) M-ZDOCK: a grid-based approach for Cn symmetric multimer docking. Bioinformatics 21(8):1472–1478Google Scholar
  62. Pierce BG, Hourai Y, Weng Z (2011) Accelerating protein docking in ZDOCK using an advanced 3D convolution library. PLoS One 6(9):e24657Google Scholar
  63. Pineda SS et al (2014) Spider venomics: implications for drug discovery. Future Med Chem 6(15):1699–1714Google Scholar
  64. Pronk S et al (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29(7):845–854Google Scholar
  65. Rink E, Wullimann MF (2001) The teleostean (zebrafish) dopaminergic system ascending to the subpallium (striatum) is located in the basal diencephalon (posterior tuberculum). Brain Res 889(1–2):316–330Google Scholar
  66. Sadeghian M et al (2016) Neuroprotection by safinamide in the 6-hydroxydopamine model of Parkinson’s disease. Neuropathol Appl Neurobiol 42(5):423–435Google Scholar
  67. Scallet AC et al (2004) Electroencephalographic, behavioral, and c-fos responses to acute domoic acid exposure. Neurotoxicol Teratol 26(2):331–342Google Scholar
  68. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675Google Scholar
  69. Sesti F, Liu S, Cai SQ (2010) Oxidation of potassium channels by ROS: a general mechanism of aging and neurodegeneration? Trends Cell Biol 20(1):45–51Google Scholar
  70. Shafer TJ, Atchison WD (1991) Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: a model for neurotoxicological studies. Neurotoxicology 12(3):473–492Google Scholar
  71. Shao J et al (2016) MicroRNA-30b regulates expression of the sodium channel Nav1.7 in nerve injury-induced neuropathic pain in the rat. Mol Pain 12:1744806916671523Google Scholar
  72. Shen Y et al (2014) Improved PEP-FOLD approach for peptide and miniprotein structure prediction. J Chem Theory Comput 10(10):4745–4758Google Scholar
  73. Siddiqui MA et al (2008) Influence of cytotoxic doses of 4-hydroxynonenal on selected neurotransmitter receptors in PC-12 cells. Toxicol In Vitro 22(7):1681–1688Google Scholar
  74. Smith JJ, Blumenthal KM (2007) Site-3 sea anemone toxins: molecular probes of gating mechanisms in voltage-dependent sodium channels. Toxicon 49(2):159–170Google Scholar
  75. Sousa SR, Vetter I, Lewis RJ (2013) Venom peptides as a rich source of cav2.2 channel blockers. Toxins (Basel) 5(2):286–314Google Scholar
  76. Striessnig J et al (2014) L-type Ca(2+) channels in heart and brain. Wiley Interdiscip Rev Membr Transp Signal 3(2):15–38Google Scholar
  77. Stys PK (2005) General mechanisms of axonal damage and its prevention. J Neurol Sci 233(1–2):3–13Google Scholar
  78. Tietze AA et al (2012) Structurally diverse mu-conotoxin PIIIA isomers block sodium channel NaV 1.4. Angew Chem Int Ed Engl 51(17):4058–4061Google Scholar
  79. Toledo-Aral JJ et al (1997) Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons. Proc Natl Acad Sci USA 94(4):1527–1532Google Scholar
  80. Van Der Spoel D et al (2005) GROMACS: fast, flexible, and free. J Comput Chem 26(16):1701–1718Google Scholar
  81. Vreugdenhil M et al (2004) Persistent sodium current in subicular neurons isolated from patients with temporal lobe epilepsy. Eur J Neurosci 19(10):2769–2778Google Scholar
  82. Wang W et al (2011) Are voltage-gated sodium channels on the dorsal root ganglion involved in the development of neuropathic pain? Mol Pain 7:16Google Scholar
  83. Waxman SG (2008) Mechanisms of disease: sodium channels and neuroprotection in multiple sclerosis-current status. Nat Clin Pract Neurol 4(3):159–169Google Scholar
  84. Wei H et al (2000) β-Amyloid peptide-induced death of PC 12 cells and cerebellar granule cell neurons is inhibited by long-term lithium treatment. Eur J Pharmacol 392(3):117–123Google Scholar
  85. Weidinger A et al (2015) Vicious inducible nitric oxide synthase-mitochondrial reactive oxygen species cycle accelerates inflammatory response and causes liver injury in rats. Antioxid Redox Signal 22(7):572–586Google Scholar
  86. Westerfield M (2000) A guide for the laboratory use of zebrafish (Danio rerio) Eugene. University of Oregon Press, Eugene, p 1.1Google Scholar
  87. Wilson JR, Fehlings MG (2014) Riluzole for acute traumatic spinal cord injury: a promising neuroprotective treatment strategy. World Neurosurg 81(5–6):825–829Google Scholar
  88. Winter MJ et al (2008) Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J Pharmacol Toxicol Methods 57(3):176–187Google Scholar
  89. Wu EL et al (2014) CHARMM-GUI membrane builder toward realistic biological membrane simulations. J Comput Chem 35(27):1997–2004Google Scholar
  90. Xu R et al (2007) Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function. Neuroscience 148(1):164–174Google Scholar
  91. Yurekli VA et al (2013) Zonisamide attenuates MPP+-induced oxidative toxicity through modulation of Ca2+ signaling and caspase-3 activity in neuronal PC12 cells. Cell Mol Neurobiol 33(2):205–212Google Scholar
  92. Zhang ZJ et al (2012) Ethanolic extract of fructus Alpinia oxyphylla protects against 6-hydroxydopamine-induced damage of PC12 cells in vitro and dopaminergic neurons in zebrafish. Cell Mol Neurobiol 32(1):27–40Google Scholar
  93. Zhang LQ et al (2015) Schisantherin A protects against 6-OHDA-induced dopaminergic neuron damage in zebrafish and cytotoxicity in SH-SY5Y cells through the ROS/NO and AKT/GSK3beta pathways. J Ethnopharmacol 170:8–15Google Scholar
  94. Zhang Y et al (2016) Disulfide bridges: bringing together frustrated structure in a bioactive peptide. Biophys J 110(8):1744–1752Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Qiwen Liao
    • 1
  • Shengnan Li
    • 1
  • Shirley Weng In Siu
    • 2
  • Jean-Étienne R. L. Morlighem
    • 3
  • Clarence Tsun Ting Wong
    • 4
  • Xiufen Wang
    • 1
  • Gandhi Rádis-Baptista
    • 3
    Email author
  • Simon Ming-Yuen Lee
    • 1
    Email author
  1. 1.State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical SciencesUniversity of MacauMacauChina
  2. 2.Department of Computer and Information Science, Faculty of Science and TechnologyUniversity of MacauMacauChina
  3. 3.Laboratory of Biochemistry and Biotechnology, Institute for Marine SciencesFederal University of CearáFortalezaBrazil
  4. 4.Department of ChemistryThe Chinese University of Hong KongShatinChina

Personalised recommendations