Elucidation of critical pH-dependent structural changes in Botulinum Neurotoxin E.

Botulinum Neurotoxins (BoNT) are the most potent toxins currently known. However, they also have therapeutic applications for an increasing number of motor related conditions due to their specificity, and low diffusion into the system. Although the start- and end- points for the BoNT mechanism of action are well-studied, a critical step remains poorly understood. It is theorised that BoNTs undergo a pH-triggered conformational shift, activating the neurotoxin by priming it to form a transmembrane (TM) channel. To test this hypothesis, we combined molecular dynamics (MD) simulations and small-angle x-ray scattering (SAXS), revealing a new conformation of serotype E (BoNT/E). This conformation was exclusively observed in simulations below pH 5.5, as determined by principal component analysis (PCA), and its theoretical SAXS profile matched an experimental SAXS profile obtained at pH 4. Additionally, a localised secondary structural change was observed in MD simulations below pH 5.5, in a region previously identified as instrumental for membrane insertion for serotype A (BoNT/A). These changes were found at a critical pH value for BoNTs in vivo, and may be relevant for their therapeutic use.

Correction: Field, M. et al. AbobotulinumtoxinA (Dysport®), OnabotulinumtoxinA (Botox®), and IncobotulinumtoxinA (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients.

The authors wish to make the following corrections to their paper [...].

AbobotulinumtoxinA (Dysport®), OnabotulinumtoxinA (Botox®), and IncobotulinumtoxinA (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients.

Botulinum neurotoxin type-A (BoNT-A) blocks the release of acetylcholine from peripheral cholinergic nerve terminals and is an important option for the treatment of disorders characterised by excessive cholinergic neuronal activity. Several BoNT-A products are currently marketed, each with unique manufacturing processes, excipients, formulation, and non-interchangeable potency units. Nevertheless, the effects of all the products are mediated by the 150 kDa BoNT-A neurotoxin. We assessed the quantity and light chain (LC) activity of BoNT-A in three commercial BoNT-A products (Dysport®; Botox®; Xeomin®). We quantified 150 kDa BoNT-A by sandwich ELISA and assessed LC activity by EndoPep assay. In both assays, we assessed the results for the commercial products against recombinant 150 kDa BoNT-A. The mean 150 kDa BoNT-A content per vial measured by ELISA was 2.69 ng/500 U vial Dysport®, 0.90 ng/100 U vial Botox®, and 0.40 ng/100 U vial Xeomin®. To present clinically relevant results, we calculated the 150 kDa BoNT-A/US Food and Drug Administration (FDA)-approved dose in adult upper limb spasticity: 5.38 ng Dysport® (1000 U; 2 × 500 U vials), 3.60 ng Botox® (400 U; 4 × 100 U vials), and 1.61 ng Xeomin® (400 U; 4 × 100 U vials). EndoPep assay showed similar LC activity among BoNT-A products. Thus, greater amounts of active neurotoxin are injected with Dysport®, at FDA-approved doses, than with other products. This fact might explain the long duration of action reported across multiple indications, which benefits patients, caregivers, clinicians, and healthcare systems.

The Expanding Therapeutic Utility of Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is a major therapeutic agent that is licensed in neurological indications, such as dystonia and spasticity. The BoNT family, which is produced in nature by clostridial bacteria, comprises several pharmacologically distinct proteins with distinct properties. In this review, we present an overview of the current therapeutic landscape and explore the diversity of BoNT proteins as future therapeutics. In recent years, novel indications have emerged in the fields of pain, migraine, overactive bladder, osteoarthritis, and wound healing. The study of biological effects distal to the injection site could provide future opportunities for disease-tailored BoNT therapies. However, there are some challenges in the pharmaceutical development of BoNTs, such as liquid and slow-release BoNT formulations; and, transdermal, transurothelial, and transepithelial delivery. Innovative approaches in the areas of formulation and delivery, together with highly sensitive analytical tools, will be key for the success of next generation BoNT clinical products.

The denatured state under native conditions: a non-native-like collapsed state of N-PGK.

The guanidinium-denatured state of the N-domain of phosphoglycerate kinase (PGK) has been characterized using solution NMR. Rather than behaving as a homogenous ensemble of random coils, chemical shift changes for the majority of backbone amide resonances indicate that the denatured ensemble undergoes two definable equilibrium transitions upon titration with guanidinium, in addition to the major refolding event. (13)C and (15)N chemical shift changes indicate that both intermediary states have distinct helical character. At denaturant concentrations immediately above the mid-point of unfolding, size-exclusion chromatography shows N-PGK to have a compact, denatured form, suggesting that it forms a helical molten globule. Within this globule, the helices extend into some regions that become beta strands in the native state. This predisposition of the denatured state to extensive non-native-like conformation, illustrates that, rather than directing folding, conformational pre-organization in the denatured state can compete with the normal folding direction. The corresponding reduction in control of the direction of folding as proteins become larger, could thus constitute a restriction on the size of protein domains.