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.

Clostridium botulinum and botulism, the disease it causes, have been known for centuries. In the later part of the 20th century, the deadly toxin produced by C. botulinum and related species emerged as a new class of therapeutic for the treatment of a variety of neuromuscular conditions. This chapter reviews the current level of understanding of this complex multidomain family of protein toxins. Each of the three major domains that constitute a botulinum toxin has a specific function within the intoxication process and, thanks to the input of many researchers in the last 10–15 years, is now understood in much more detail. The potential to engineer novel biotherapeutics is discussed, while the importance of developments in detection and treatment approaches is also acknowledged.

The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3?Å and that of SXN101959 at 3.1?Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B and D. Taken together, these results demonstrate the robustness of the ‘LHn fold’ across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin and serotype D in particular, as a basis for engineering novel secretion inhibitors.

Understanding the structure and molecular basis of neurotoxin function has opened up opportunities to engineer novel therapeutic proteins that utilise the neurotoxins and neurotoxin domains. These opportunities and the status of their development are reviewed in this chapter, which brings together the findings detailed in the companion volume to this book, KA Foster (ed) Molecular Aspects of Botulinum Neurotoxin, Springer, NewYork, and shows how they can be applied for the development of innovative therapeutics and research tools.

Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. Each BoNT consists of three domains that are essential for toxicity: the binding domain, the translocation domain, and the catalytic light-chain domain. BoNT modular architecture is associated with a multistep mechanism that culminates in the intracellular proteolysis of SNARE (soluble N-ethylmaleimide-sensitive-fusion-protein attachment protein receptor) proteins, which prevents synaptic vesicle exocytosis. As the most toxic proteins known, BoNTs have been extensively studied and are used as pharmaceutical agents to treat an increasing variety of disorders. This review summarizes the level of sophistication reached in BoNT engineering and highlights the diversity of approaches taken to utilize the modularity of the toxin. Improved efficiency and applicability have been achieved by direct mutagenesis and interserotype domain rearrangement. The scope of BoNT activity has been extended to nonneuronal cells and offers the basis for novel biomolecules in the treatment of secretion disorders.

Botulinum neurotoxins are comprised of multiple identifiable protein domains. Recent advances in understanding the relationships between domain structure and neurotoxin function have provided a number of opportunities to engineer innovative therapeutic proteins that utilise the neurotoxins and neurotoxin domains. For example, recent insights into the properties of the catalytic, translocation and binding domains open up opportunities to develop botulinum neurotoxins with enhanced properties of selectivity, potency and duration of action. In parallel, the broad scope for utilisation of the individual domains is becoming clearer as significant advancements are made to exploit the unique biology of the catalytic and translocation domains. These opportunities and the status of their development will be reviewed in this chapter.

Bacterial protein toxins are causative agents of multiple healthcare issues and yet are also components of strategies leading the therapeutic fight against a range of debilitating conditions. Chapter 8 provides an insight into the biology of key exemplar toxic proteins and describes how understanding the structure of such proteins can lead to the design of new therapeutics with novel mechanisms of action. Focusing the discussion on examples of cytotoxic proteins (in particular diphtheria toxin and pseudomonas exotoxin A) and non-cytotoxins (in particular botulinum neurotoxin), the possibility for using recombinant DNA techniques to create families of novel proteins is described. After approximately 30 years of investigation into the applications of bacterial toxins to treat human disease, there is clinical evidence of the success of this strategy and a number of additional examples are moving through pre-clinical and clinical investigation. Indeed, in a number of cases, bacterial toxins, or novel agents derived from fragments of bacterial toxins, are first line therapy options.

'Engineering Toxins for 21st Century Therapies' (9-10 September 2010) was part of the Royal Society International Seminar series held at the Kavli International Centre, UK. Participants were assembled from a range of disciplines (academic, industry, regulatory, public health) to discuss the future potential of toxin-based therapies. The meeting explored how the current structural and mechanistic knowledge of toxins could be used to engineer future toxin-based therapies. To date, significant progress has been made in the design of novel recombinant biologics based on domains of natural toxins, engineered to exhibit advantageous properties. The meeting concluded, firstly that future product development vitally required the appropriate combination of creativity and innovation that can come from the academic, biotechnology and pharma sectors. Second, that continued investigation into understanding the basic science of the toxins and their targets was essential in order to develop new opportunities for the existing products and to create new products with enhanced properties. Finally, it was concluded that the clinical potential for development of novel biologics based on toxin domains was evident.

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.