New Modified Recombinant Botulinum Neurotoxin Type F with Enhanced Potency.

David Burgin,Imran Mir,Gavin Hackett,Daniel Kwan,Fraser Hornby,Cindy Périer,Jacquie Maignel,Mark Elliott,Matthew Beard,Sai Man Liu

doi:10.3390/toxins13120834

Abstract

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.

Highlights

Botulinum neurotoxins (BoNTs) are highly potent neurotoxic proteins produced by some species of the genus Clostridia including C. botulinum, C. butyrricum, C. baratii andC. argentinensis
The BoNT/F7 and BoNT/F1 gene sequences were codon-optimised for expression domain (R1111K); (4) mrBoNT/F7-1 in which the BoNT/F7 activation loop is replaced by in E. coli and used to make five constructs: (1) rBoNT/F7; that of BoNT/F1
Replaced by that of BoNT/F1; (5) mrBoNT/F7-1 (0) where (0) denotes endopeptidase inactive due to two point mutations in the light chain (E227Q and H230Y) (Figure 1)

Summary

Introduction

Botulinum neurotoxins (BoNTs) are highly potent neurotoxic proteins produced by some species of the genus Clostridia including C. botulinum, C. butyrricum, C. baratii andC. argentinensis. BoNTs are classified into seven different serotypes based on their serological typing and are designated as BoNTs A, B, C, D, E, F and G. These families can be further subdivided into sub-serotypes based on the specific amino acid variation. BoNTs are synthesised as large single chain precursors of approximately 150 kDa. The neurotoxin molecule is divided into two sub-units, an endopeptidase active light chain (LC, ~50 kDa) and a heavy chain (HC, ~100 kDa). Each serotype targets specific SNARE proteins: serotypes: A and E cleave synaptosomal-associated protein 25 (SNAP-25) at different sites, C is capable of cleaving both SNAP-25 and Syntaxin, and BoNTs B, D, F and G cleave vesicle-associated membrane proteins 1, 2, and 3

Methods

Results

Conclusion