http://www.mdpi.com/journal/toxins/special_issues/botulinum-toxin/ {snippet name="submission_info"} http://www.mdpi.com/2072-6651/3/5/489/ The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K+ had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca2+ influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery. http://www.mdpi.com/2072-6651/3/5/489/ Fri, 13 May 2011 00:00:00 CEST Toxins 2011-05-13 3 5 Article 489 503 2072-6651 Alpha-Latrotoxin Rescues SNAP-25 from BoNT/A-Mediated Proteolysis in Embryonic Stem Cell-Derived Neurons 2011-05-13 doi: 10.3390/toxins3050489 Mariano Mesngon Patrick McNutt http://www.mdpi.com/2072-6651/3/5/469/ Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15–20 kDa single domain antibody (VHH) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/VHH phage display library. The VHH has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the VHH and the toxin but also an insertion of the VHH CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the VHH to a cell penetrating peptide (CPP), the CPP-VHH fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme. http://www.mdpi.com/2072-6651/3/5/469/ Fri, 13 May 2011 00:00:00 CEST Toxins 2011-05-13 3 5 Review 469 488 2072-6651 Botulinum Neurotoxins and Botulism: A Novel Therapeutic Approach 2011-05-13 doi: 10.3390/toxins3050469 Jeeraphong Thanongsaksrikul Wanpen Chaicumpa http://www.mdpi.com/2072-6651/3/4/345/ The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications. http://www.mdpi.com/2072-6651/3/4/345/ Thu, 24 Mar 2011 00:00:00 CET Toxins 2011-03-24 3 4 Article 345 355 2072-6651 Re-Assembled Botulinum Neurotoxin Inhibits CNS Functions without Systemic Toxicity 2011-03-24 doi: 10.3390/toxins3040345 Enrico Ferrari Elizabeth S. Maywood Laura Restani Matteo Caleo Marco Pirazzini Ornella Rossetto Michael H. Hastings Dhevahi Niranjan Giampietro Schiavo Bazbek Davletov http://www.mdpi.com/2072-6651/3/3/207/ Botulinum neurotoxins (BoNTs) comprise seven distinct serotypes that inhibit the release of neurotransmitter across neuromuscular junctions, resulting in potentially fatal flaccid paralysis. BoNT serotype A (BoNT/A), which targets synaptosomal-associated protein of 25kDa (SNAP-25), is particularly long-lived within neurons and requires a longer time for recovery of neuromuscular function. There are currently no treatments available to counteract BoNT/A after it has entered the neuronal cytosol. In this study, we examined the ability of small molecule non-peptidic inhibitors (SMNPIs) to prevent SNAP-25 cleavage post-intoxication of neurons. The progressive cleavage of SNAP-25 observed over 5 h following 1 h BoNT/A intoxication was prevented by addition of SMNPIs. In contrast, anti-BoNT/A neutralizing antibodies that strongly inhibited SNAP-25 cleavage when added during intoxication were completely ineffective when added post-intoxication. Although Bafilomycin A1, which blocks entry of BoNT/A into the cytosol by preventing endosomal acidification, inhibited SNAP-25 cleavage post-intoxication, the degree of inhibition was significantly reduced versus addition both during and after intoxication. Post-intoxication application of SMNPIs, on the other hand, was nearly as effective as application both during and after intoxication. Taken together, the results indicate that competitive SMNPIs of BoNT/A light chain can be effective within neurons post-intoxication. http://www.mdpi.com/2072-6651/3/3/207/ Tue, 15 Mar 2011 00:00:00 CET Toxins 2011-03-15 3 3 Article 207 217 2072-6651 Post-Intoxication Inhibition of Botulinum Neurotoxin Serotype A within Neurons by Small-Molecule, Non-Peptidic Inhibitors 2011-03-15 doi: 10.3390/toxins3030207 Gordon Ruthel James C. Burnett Jonathan E. Nuss Laura M. Wanner Lyal E. Tressler Edna Torres-Melendez Sarah J. Sandwick Cary J. Retterer Sina Bavari