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

Sébastien Nicolas,Sylvie Diochot,Eva Cuypers,Jan Tytgat,Stephan De Waard,Didier Boturyn,Michel Lazdunski,Dietrich Mebs,Claude Zoukimian,Frank Bosmans,Rémy Béroud,Jérôme Montnach,Michel De Waard,David Craik

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.

Highlights

Voltage-gated sodium (Nav ) channels are critical for the generation and propagation of action potentials [1,2,3,4]
The peptide contains 34 amino acid residues, six cysteine residues bridged in an inhibitory cysteine-knot (ICK) architecture fold and is amidated at the C-terminus
The random oxidative folding strategy seems to work, the yields remain largely unsatisfactory to envision any kind of folding strategy seems to work, the yields remain largely unsatisfactory to envision any kind of clinical future for this peptide or even to perform a complete Ala scan of the peptide to identify the clinical future for this peptide or even to perform a complete Ala scan of the peptide to identify the pharmacophore of PhlTx1

Summary

Introduction

Voltage-gated sodium (Nav ) channels are critical for the generation and propagation of action potentials [1,2,3,4]. They are composed of a pore-forming α-subunit and can be associated with β-subunits [5]. Nav 1.5, 1.8 and 1.9 are TTX-resistant (TTX-r), whereas the other α-subunits are TTX-sensitive (TTX-s) These channels differ by their distribution with Nav 1.1, 1.2 and 1.3 principally found in the central nervous system, whereas Nav 1.6, 1.7, 1.8 and 1.9 are predominantly, but not exclusively, expressed in the peripheral nervous system. Nav 1.4 is predominantly found within the skeletal muscle, whereas Nav 1.5 is present in cardiac muscle

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Discussion

Conclusion