Abstract
Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar multi-domain structure at the molecular level. In nature, BoNTs are encoded by DNA in producing clostridial bacteria and, as such, are amenable to recombinant production through insertion of the coding DNA into other bacterial species. This, in turn, creates possibilities for protein engineering. Here, we review the production of BoNTs by the natural host and also recombinant production approaches utilised in the field. Applications of recombinant BoNT-production include the generation of BoNT-derived domain fragments, the creation of novel BoNTs with improved performance and enhanced therapeutic potential, as well as the advancement of BoNT vaccines. In this article, we discuss site directed mutagenesis, used to affect the biological properties of BoNTs, including approaches to alter their binding to neurons and to alter the specificity and kinetics of substrate cleavage. We also discuss the target secretion inhibitor (TSI) platform, in which the neuronal binding domain of BoNTs is substituted with an alternative cellular ligand to re-target the toxins to non-neuronal systems. Understanding and harnessing the potential of the biological diversity of natural BoNTs, together with the ability to engineer novel mutations and further changes to the protein structure, will provide the basis for increasing the scope of future BoNT-based therapeutics.
Highlights
Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics
The current preparations of BoNT approved for clinical use in the US and Europe correspond to two different active molecules—BoNT type A (BoNT/A) and
Previous insights into the molecular basisa of the BoNT type B (BoNT/B)–Syt interaction by X-ray mutagenesis screen, allowed identification of a series of point mutations that can be introduced into the crystallography [77,78,79], combined with a bacterial adenylate cyclase two-hybrid saturation receptor-binding of BoNT/B
Summary
Botulinum neurotoxin (BoNT) has been used as a versatile and multipurpose therapeutic for the last 30 years [1,2,3]. New BoNT types have been described, such as type FA (previously classified as H), and several of them are being explored as new therapeutic agents [5,6,7]. BoNTs are produced by several bacteria of the genus Clostridia [8], and more than 40 different BoNT protein sequences have been described to date [9]. BoNT selectively target the neuromuscular junction and, in particular, exerts its effect at the level of nerve innervation of the muscle. It is the muscle paralysis in intoxicated individuals that causes harm in the form of botulism, and in the therapeutic setting brings relief to movement disorders such as dystonia and spasticity [14]. Our increasing understanding of the molecular structure of BoNTs and the relationships between each domain and their function is opening up both the ability to produce natural BoNTs in a recombinant manner [18] and the ability to modify BoNTs using protein engineering [19]
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