Michael C. Goodnough

Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

Among the agents classified as “Category A” by the U.S. Centers for Disease Control and Prevention, botulinum neurotoxin (BoNT) is the most toxic protein known, with microgram quantities of the protein causing severe morbidity and mortality by oral or i.v. routes. Given that this toxin easily could be used in a potential bioterrorist attack, countermeasures urgently are needed to counteract the pathophysiology of BoNT. At a molecular level, BoNT exerts its paralytic effects through intracellular cleavage of vesicle docking proteins and subsequent organism-wide autonomic dysfunction. In an effort to identify small molecules that would disrupt the interaction between the light-chain metalloprotease of BoNT serotype A and its cognate substrate, a multifaceted screening effort was undertaken. Through the combination of in vitro screening against an optimized variant of the light chain involving kinetic analysis, cellular protection assays, and in vivo mouse toxicity assays, molecules that prevent BoNT/A-induced intracellular substrate cleavage and extend the time to death of animals challenged with lethal toxin doses were identified. Significantly, the two most efficacious compounds in vivo showed less effective activity in cellular assays intended to mimic BoNT exposure; indeed, one of these compounds was cytotoxic at concentrations three orders of magnitude below its effective dose in animals. These two lead compounds have surprisingly simple molecular structures and are readily amenable to optimization efforts for improvements in their biological activity. The findings validate the use of high-throughput screening protocols to define previously unrecognized chemical scaffolds for the development of therapeutic agents to treat BoNT exposure.

Purification of Clostridium botulinum Type A Neurotoxin

1. Introduction Botulinum neurotoxins produced by strains of the spore-bearing bacterium Clostridium botulinum have long been known to cause a distinctive paralytic disease in humans and animals (1). In recent years, injection of crystalline botulinum toxin type A has been demonstrated to provide relief from certain involuntary muscle disorders, dystonic conditions, pain syndromes, and headaches (2,3,4). The use of botulinum toxin for treatment of human disease and its usefulness in cell biology (5) has stimulated interest in the study of the toxin complexes and the neurotoxin component within the complexes. The use of botulinum toxin complex in medicine will benefit from the development of more potent, less antigenic, and longer-lasting toxin preparations. In this chapter we describe methods for production of high-quality botulinum type A toxin complex and neurotoxin. The procedures described in this chapter are not those used for preparation of botulinum toxin for medical use (3). C. botulinum produces seven serotypes of neurotoxin designated A–G. These neurotoxins exist in nature as toxin complexes, in which the neurotoxin is noncovalently bound to various nontoxic protein components and to ribo-nucleic acid (1,3). The neurotoxins are considered the most potent poisons known for humans and certain animals (3,6). The toxicity of type A botulinum neurotoxin has been estimated to be 0.2 ng/kg of body weight (3,6), and as little as 0.1–1 µg may be lethal to humans (3). Consequently, considerable care and safety precautions are necessary in working with botulinum neurotoxins. Because the consequences of an accidental intoxication with botulinum neurotoxins are severe, safety must be a primary concern of scientists interested in the study of these toxins. The Centers for Disease Control and Prevention

Botulinum toxin for aberrant facial nerve regeneration : Double-blind, placebo-controlled trial using subjective endpoints

Background: Aberrant facial nerve regeneration syndrome is systematically studied by constructing a subjective questionnaire organized into pertinent domains (impairment of quality of life, social interactions, perception of self-appearance, visual function, and perception of problem severity) and by physical assessment of degree of involuntary palpebral fissure closure during routine facial movements using videotaping and a physicians’ grading scale. Methods: The videotape measurements of corneal light reflex to upper lid margin distance and vertical palpebral distance were made during adynamic and active facial expression. Thirty-six patients were studied (six in an open-label pilot study design and 30 in a multicenter, double-blind, placebo-controlled trial). Botulinum toxin type A injections consistently suppressed the degree of involuntary eyelid movement associated with smiling, chewing, and puckering by each measurement and in both study designs. Results: In all subjective morbidity domains, improvement was demonstrated in the double-blind study design (quality of life, p < 0.05; social interaction, p < 0.001; personal appearance, p < 0.001; peripheral visual impairment, p < 0.01; and perception of severity, p < 0.05). Correlation between degrees ofphysical impairment was significant but low comparing degree of subjective impairment to direct videotaped measurements. Correlation was more significant comparing subjective scores to the physicians’ grading scale assessment of degree of physical impairment. Conclusions: Impairment in quality of life, social interactions, and self-assessment of appearance closely reflected the nature of the morbidity associated with palpebral fissure asymmetry associated with aberrant facial nerve regeneration. The physicians’ grading scale best reflected subjective morbidity over direct videotaped measurements.

A capillary electrophoresis technique for evaluating botulinum neurotoxin B light chain activity

Botulinum neurotoxin B (BoNT/B) produces muscle paralysis by cleaving synaptobrevin/vesicleassociated membrane protein (VAMP), an 18-kDa membrane-associated protein located on the surface of small synaptic vesicles. A capillary electrophoresis (CE) assay was developed to evaluate inhibitors of the proteolytic activity of BoNT/B with the objective of identifying suitable candidates for treatment of botulism. The assay was based on monitoring the cleavage of a peptide that corresponds to residues 44–94 of human VAMP-2 (V51) following reaction with the catalytic light chain (LC) of BoNT/B. Cleavage of V51 generated peptide fragments of 18 and 33 amino acids by scission of the bond between Q76 and F77. The fragments and parent peptide were clearly resolved by CE, allowing accurate quantification of the BoNT/B LC-mediated reaction rates. The results indicate that CE is suitable for assessing the enzymatic activity of BoNT/B LC.

Small molecule metalloprotease inhibitor with in vitro, ex vivo and in vivo efficacy against botulinum neurotoxin serotype A

ABSTRACT Botulinum neurotoxins (BoNTs) are the most toxic substances known to mankind and are the causative agents of the neuroparalytic disease botulism. Their ease of production and extreme toxicity have caused these neurotoxins to be classified as Tier 1 bioterrorist threat agents and have led to a sustained effort to develop countermeasures to treat intoxication in case of a bioterrorist attack. While timely administration of an approved antitoxin is effective in reducing the severity of botulism, reversing intoxication requires different strategies. In the present study, we evaluated ABS 252 and other mercaptoacetamide small molecule active‐site inhibitors of BoNT/A light chain using an integrated multi‐assay approach. ABS 252 showed inhibitory activity in enzymatic, cell‐based and muscle activity assays, and importantly, produced a marked delay in time‐to‐death in mice. The results suggest that a multi‐assay approach is an effective strategy for discovery of potential BoNT therapeutic candidates. HIGHLIGHTSABS 143 and ABS 252 inhibited the proteolytic activity of BoNT/A light chain.Inhibition involved specific interactions with active site groups in the light chain.ABS 252 antagonized BoNT/A‐mediated cleavage of SNAP‐25 in primary neurons.ABS 252 slowed onset of paralysis in BoNT/A‐intoxicated nerve‐muscle preparations.ABS 252 delayed time‐to‐death in mice intoxicated by BoNT/A.