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

1
Institut du Thorax, Inserm UMR 1087/CNRS UMR 6291, LabEx “Ion Channels, Science & Therapeutics”, F-44007 Nantes, France
2
Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France
3
Department of Molecular Chemistry, Univ. Grenoble Alpes, CNRS, 570 rue de la chimie, CS 40700, 38000 Grenoble, France
4
Faculty of Medicine and Health Sciences, Department of Basic and Applied Medical Sciences, 9000 Gent, Belgium
5
Toxicology and Pharmacology, University of Leuven, Campus Gasthuisberg, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium
6
Université Côte d’Azur, CNRS UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 6560 Valbonne, France
7
Institute of Legal Medicine, University of Frankfurt, Kennedyallee 104, 60488 Frankfurt, Germany
8
Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Australia
*
Author to whom correspondence should be addressed.
Contributed equally to this work.
Toxins 2019, 11(6), 367; https://doi.org/10.3390/toxins11060367
Received: 16 May 2019 / Revised: 11 June 2019 / Accepted: 16 June 2019 / Published: 21 June 2019
(This article belongs to the Special Issue Arthropod Venom Components and Their Potential Usage)
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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. View Full-Text
Keywords: spider toxin; directed disulfide bond formation; Nav channel activity; Nav1.7; pain target; automated patch-clamp spider toxin; directed disulfide bond formation; Nav channel activity; Nav1.7; pain target; automated patch-clamp
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Nicolas, S.; Zoukimian, C.; Bosmans, F.; Montnach, J.; Diochot, S.; Cuypers, E.; De Waard, S.; Béroud, R.; Mebs, D.; Craik, D.; Boturyn, D.; Lazdunski, M.; Tytgat, J.; De Waard, M. Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1. Toxins 2019, 11, 367.

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