Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1

  • Toxins (Basel). 2019 Jun 21;11(6):367. doi: 10.3390/toxins11060367.
Sébastien Nicolas  1 ,  Claude Zoukimian  2  3 ,  Frank Bosmans  4  5 ,  Jérôme Montnach  6 ,  Sylvie Diochot  7 ,  Eva Cuypers  8 ,  Stephan De Waard  9 ,  Rémy Béroud  10 ,  Dietrich Mebs  11 ,  David Craik  12 ,  Didier Boturyn  13 ,  Michel Lazdunski  14 ,  Jan Tytgat  15 ,  Michel De Waard  16  17
Affiliations
  • 1. Institut du Thorax, Inserm UMR 1087/CNRS UMR 6291, LabEx "Ion Channels, Science & Therapeutics", F-44007 Nantes, France. [email protected].
  • 2. Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France. [email protected].
  • 3. Department of Molecular Chemistry, Univ. Grenoble Alpes, CNRS, 570 rue de la chimie, CS 40700, 38000 Grenoble, France. [email protected].
  • 4. Faculty of Medicine and Health Sciences, Department of Basic and Applied Medical Sciences, 9000 Gent, Belgium. [email protected].
  • 5. Toxicology and Pharmacology, University of Leuven, Campus Gasthuisberg, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium. [email protected].
  • 6. Institut du Thorax, Inserm UMR 1087/CNRS UMR 6291, LabEx "Ion Channels, Science & Therapeutics", F-44007 Nantes, France. [email protected].
  • 7. Université Côte d'Azur, CNRS UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 6560 Valbonne, France. [email protected].
  • 8. Toxicology and Pharmacology, University of Leuven, Campus Gasthuisberg, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium. [email protected].
  • 9. Institut du Thorax, Inserm UMR 1087/CNRS UMR 6291, LabEx "Ion Channels, Science & Therapeutics", F-44007 Nantes, France. [email protected].
  • 10. Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France. [email protected].
  • 11. Institute of Legal Medicine, University of Frankfurt, Kennedyallee 104, Frankfurt, Germany. [email protected].
  • 12. Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Australia. [email protected].
  • 13. Department of Molecular Chemistry, Univ. Grenoble Alpes, CNRS, 570 rue de la chimie, CS 40700, 38000 Grenoble, France. [email protected].
  • 14. Université Côte d'Azur, CNRS UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 6560 Valbonne, France. [email protected].
  • 15. Toxicology and Pharmacology, University of Leuven, Campus Gasthuisberg, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium. [email protected].
  • 16. Institut du Thorax, Inserm UMR 1087/CNRS UMR 6291, LabEx "Ion Channels, Science & Therapeutics", F-44007 Nantes, France. [email protected].
  • 17. Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France. [email protected].
PMID: 31234412 DOI: 10.3390/toxins11060367
Abstract

Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 Other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

Keywords
Nav channel activity; Nav1.7; automated patch-clamp; directed disulfide bond formation; pain target; spider toxin.
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