Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1_MY) and E1191Q/S1199W (rBoNT/B1_QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1_MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.

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.

We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here we report purification and characterization of enzymatically active, and of inactive nontoxic, recombinant forms of BoNT/FA as tractable alternatives to purifying this neurotoxin from native Clostridium botulinum. BoNT/FA cleaves the same intracellular target proteins as BoNT/F1 and other F serotype BoNTs; the intracellular targets are vesicle associated membrane proteins (VAMP) 1, 2 and 3. BoNT/FA cleaves the same site in VAMP-2 as BoNT/F5, which is different from the cleavage site of other F serotype BoNTs. BoNT/FA has slower enzyme kinetics than BoNT/F1 in a cell-free protease assay and is less potent at inhibiting ex vivo nerve-stimulated skeletal muscle contraction. In contrast, BoNT/FA is more potent at inhibiting neurotransmitter release from cultured neurons.

Clinically used botulinum neurotoxins (BoNTs) are natural products of Clostridium botulinum. A novel, recombinant BoNT type A1 (rBoNT/A1; IPN10260) has been synthesized using the native amino acid sequence expressed in Escherichia coli and has previously been characterized in vitro and ex vivo. Here, we aimed to characterize rBoNT/A1 in vivo and evaluate its effects on skeletal muscle. The properties of rBoNT/A1 following single, intramuscular administration were evaluated in the mouse and rat digit abduction score (DAS) assays and compared with those of natural BoNT/A1 (nBoNT/A1). rBoNT/A1‐injected tibialis anterior was assessed in the in situ muscle force test in rats. rBoNT/A1‐injected gastrocnemius lateralis (GL) muscle was assessed in the compound muscle action potential (CMAP) test in rats. The rBoNT/A1‐injected GL muscle was evaluated for muscle weight, volume, myofiber composition and immunohistochemical detection of cleaved SNAP25 (c‐SNAP25). Results showed that rBoNT/A1 and nBoNT/A1 were equipotent and had similar onset and duration of action in both mouse and rat DAS assays. rBoNT/A1 caused a dose‐dependent inhibition of muscle force and a rapid long‐lasting reduction in CMAP amplitude that lasted for at least 30 days. Dose‐dependent reductions in GL weight and volume and increases in myofiber atrophy were accompanied by immunohistochemical detection of c‐SNAP25. Overall, rBoNT/A1 and nBoNT/A1 exhibited similar properties following intramuscular administration. rBoNT/A1 inhibited motoneurons neurotransmitter release, which was robust, long‐lasting, and accompanied by cleavage of SNAP25. rBoNT/A1 is a useful tool molecule for comparison with current natural and future modified recombinant neurotoxins products.

Botulinum neurotoxin (BoNT) serotype A inhibits neurotransmitter release by cleaving SNAP-25 and represents an established pharmaceutical for treating medical conditions caused by hyperactivity of cholinergic nerves. Oversecretion from non-neuronal cells is often also the cause of diseases. Notably, excessive release of inflammatory messengers is thought to contribute to diseases such as chronic obstructive pulmonary disease, asthma, diabetes etc. The expansion of its application to these medical conditions is prevented because the major non-neuronal SNAP-25 isoform responsible for exocytosis, SNAP-23, is, in humans, virtually resistant to BoNT/A. Based on previous structural data and mutagenesis studies of SNAP-23 we optimized substrate binding pockets of the enzymatic domain for interaction with SNAP-23. Systematic mutagenesis and rational design yielded the mutations E148Y, K166F, S254A, and G305D, each of which individually increased the activity of LC/A against SNAP-23 between 3- to 23-fold. The assembled quadruple mutant showed approximately 2000-fold increased catalytic activity against human SNAP-23 in in vitro cleavage assays. A comparable increase in activity was recorded for the full-length BoNT/A quadruple mutant tested in cultivated primary neurons transduced with a fluorescently tagged-SNAP-23 encoding gene. Equipped with a suitable targeting domain this quadruple mutant promises to complete successfully tests in cells of the immune system.

Botulinum neurotoxin (BoNT/A) is an FDA and NICE approved second-line treatment for overactive bladder (OAB) in patients either not responsive or intolerant to anti-cholinergic drugs. BoNT/A acts to weaken muscle contraction by blocking release of the neurotransmitter acetyl choline (ACh) at neuromuscular junctions. However, this biological activity does not easily explain all the observed effects in clinical and non-clinical studies. There are also conflicting reports of expression of the BoNT/A protein receptor, SV2, and intracellular target protein, SNAP-25, in the urothelium and bladder. This review presents the current evidence of BoNT/A’s effect on bladder sensation, potential mechanisms by which it might exert these effects and discusses recent advances in understanding the action of BoNT in bladder tissue.