A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins.

Domenico Azarnia Tehran,Silvia Fillo,Clifford C Shone,Oneda Leka,Florigio Lista,Marco Pirazzini,Ornella Rossetto,Andrea Mattarei,Thomas Binz,Giulia Zanetti,Cristina Paradisi,Cesare Montecucco

doi:10.1038/srep17513

Abstract

Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins.

Highlights

Action envisages a first interaction with polysialogangliosides, which mediate the toxin binding to the plasma membrane
Similar to what found for Botulinum neurotoxins (BoNTs)/A, we found that EGA substantially prevents the action of this potent neurotoxin (Fig. 2c, right panel), and this inhibition is dependent on the amount of the chemical, as estimated in western blot with an antibody specific for the intact form of VAMP2 (Fig. 3e, bottom panel and Fig. 3f)
We found that EGA, used at the concentration which displayed the maximum efficacy in protecting cerebellar granular neurons (CGNs), does not interfere with the binding and the endocytosis of both BoNT/A and BoNT/B, as assessed by the internalization of their respective derivatives which show the same pattern regardless of drug presence (Fig. 4a and Figure S2a)

Summary

Introduction

Action envisages a first interaction with polysialogangliosides, which mediate the toxin binding to the plasma membrane. All known novel BoNTs are classified as subtypes, and indicated with an Arabic number added to the parental serotype (e.g. A2, A3 etc., when their amino acid sequences differ by more than 2.4% from the parental serotype A1)[2], or as mosaic BoNTs, and indicated with a double capital letter, e.g. BoNT/DC,/ CD,/FA, when they are chimeras of the different serotypes Due to their different origin, BoNT variants exhibit different antigenicity and are neutralized to a different degree by existing serotype specific antisera[23,24]. It is possible that the therapy with humanized monoclonal antibodies raised versus a BoNT subtype may not neutralize variants of the same serotype[25,26] This situation calls for increased efforts in the identification of inhibitors effective in preventing the neuroparalytic action of BoNTs irrespectively of their serotype and subtype which could be used without knowing the particular type of BoNT involved. We report an optimized procedure for the synthesis of EGA, which involves milder reaction conditions and provides much higher overall yield than previously reported[29]

Objectives

Methods

Results

Conclusion