Expression of Recombinant Clostridial Neurotoxin by .

Tetanus neurotoxins (TeNT) and botulinum neurotoxins (BoNTs) are closely related ~150 kDa protein toxins that together comprise the group of clostridial neurotoxins (CNTs) expressed by various species of . While TeNT is expressed as a single polypeptide, BoNTs are always produced alongside multiple non-toxic proteins that form a stabilizing complex with BoNT and are encoded in a conserved toxin gene cluster. It is unknown how evolved without a similar gene cluster and why complex-free TeNT is secreted as a stable and soluble protein by , whereas complexing proteins appear to be essential for BoNT stability in culture supernatants of .

Investigations of Thiosemicarbazides as Botulinum Toxin Active-Site Inhibitors: Enzyme, Cellular, and Rodent Intoxication Studies.

Botulinum neurotoxin type A (BoNT/A) is an exceptionally potent neurotoxin of great therapeutic value; however, it is also considered a weapon of mass destruction, as it is one of the most poisonous biological substances known to man. The etiology behind BoNT/A is its action as a zinc-dependent protease, which can cause extended paralysis through the cleavage of SNARE proteins. Thiosemicarbazones, known zinc chelators, provide a privileged scaffold that can be leveraged for the development of BoNT/A LC inhibitors.

Potency Evaluations of Recombinant Botulinum Neurotoxin A1 Mutants Designed to Reduce Toxicity.

Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2-3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold-12.

Botulinum neurotoxin X lacks potency in mice and in human neurons.

Unlabelled: Botulinum neurotoxins (BoNTs) are a class of toxins produced by () and other species of . BoNT/X is a putative novel botulinum neurotoxin identified through genome sequencing and capable of SNARE cleavage, but its neurotoxic potential in humans and vertebrates remained unclear. The strain producing BoNT/X, Strain 111, encodes both a plasmid-borne as well as the chromosomal putative .

Transformation of a Metal Chelate into a "Catch and Anchor" Inhibitor of Botulinum A Protease.

Targeting the botulinum neurotoxin light chain (LC) metalloprotease using small-molecule metal chelate inhibitors is a promising approach to counter the effects of the lethal toxin. However, to overcome the pitfalls associated with simple reversible metal chelate inhibitors, it is crucial to investigate alternative scaffolds/strategies. In conjunction with Atomwise Inc.

Investigation of Salicylanilides as Botulinum Toxin Antagonists.

Botulinum neurotoxin serotype A (BoNT/A) is recognized by the Centers for Disease Control and Prevention (CDC) as the most potent toxin and as a Tier 1 biowarfare agent. The severity and longevity of botulism stemming from BoNT/A is of significant therapeutic concern, and early administration of antitoxin-antibody therapy is the only approved pharmaceutical treatment for botulism. Small molecule therapeutic strategies have targeted both the heavy chain (HC) and the light chain (LC) catalytic active site and α-/β-exosites.

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1-A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol.

Posttranslational Regulation of Botulinum Neurotoxin Production in Clostridium botulinum Hall A-.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind and are the causative agents of the neuroparalytic disease botulism. Despite the overall importance of BoNTs in public health and safety, as a bioterrorism concern, and in pharmaceutical development, little is known about the molecular mechanisms mediating BoNT stability and degradation in various environments. Previous studies using Clostridium botulinum strain ATCC 3502 revealed that high levels of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold.

A Novel High-Potency Tetanus Vaccine.

Chemically inactivated tetanus toxoid (CITT) is clinically effective and widely used. However, CITT is a crude nonmalleable vaccine that contains hundreds of proteins, and the active component is present in variable and sometimes minor percentages of vaccine mass. Recombinant production of a genetically inactivated tetanus vaccine offers an opportunity to replace and improve the current tetanus vaccine.

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins.

Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis of botulism, basic research, drug development, potency determination, and detection in clinical, environmental, and food samples.

Botulinum neurotoxins A, B, C, E, and F preferentially enter cultured human motor neurons compared to other cultured human neuronal populations.

Human-induced pluripotent stem cell (hiPSC)-derived neurons can be exquisitely sensitive to botulinum neurotoxins (BoNTs), exceeding sensitivity of the traditionally used mouse bioassay. In this report, four defined hiPSC-derived neuronal populations including primarily GABAergic, glutamatergic, dopaminergic, and motor neurons were examined for BoNT/A, B, C, D, E, and F sensitivity. The data indicate that sensitivity varies markedly for the BoNTs tested.

Comparative functional analysis of mice after local injection with botulinum neurotoxin A1, A2, A6, and B1 by catwalk analysis.

Botulinum neurotoxins (BoNTs) are potent neurotoxins and are the causative agent of botulism, as well as valuable pharmaceuticals. BoNTs are divided into seven serotypes that comprise over 40 reported subtypes. BoNT/A1 and BoNT/B1 are currently the only subtypes approved for pharmaceutical use in the USA.

Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6.

Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals used to treat more than 200 neuronal disorders. BoNTs comprise 7 serotypes and more than 40 isoforms (subtypes).

The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes.

Botulinum neurotoxin (BoNT) is the causative agent of botulism and a widely used pharmaceutical to treat a variety of neurological diseases. BoNTs are 150-kDa protein toxins organized into heavy chain (HC) and light chain (LC) domains linked by a disulfide bond. The HC selectively binds to neurons and aids cell entry of the enzymatically active LC.

Enhancing toxin-based vaccines against botulism.

Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HC) and a receptor binding domain (HC).

Substrate cleavage and duration of action of botulinum neurotoxin type FA ("H, HA").

Botulinum neurotoxin (BoNT) type FA is the only known naturally occurring chimeric BoNT of domains of BoNT/A and BoNT/F. BoNT/FA consists of an F5-like light chain (LC), a unique heavy chain (HC) translocation domain, and a HC receptor binding domain similar to BoNT/A1. Previous analyses of purified BoNT/FA have indicated a 5-10-fold greater potency in cultured human or rat neurons as compared to BoNT/A1 and a 400-500-fold greater potency compared to BoNT/B1.

Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC.

Botulinum neurotoxins (BoNTs), the most potent toxins known, are potential bioterrorism agents. It is well established that all seven serotypes of BoNTs (BoNT/A-G) require complex gangliosides as co-receptors. Here, we report that BoNT/DC, a presumed mosaic toxin between BoNT/D and BoNT/C1, binds and enters efficiently into neurons lacking complex gangliosides and shows no reduction in toxicity in mice deficient in complex gangliosides.

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

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.

Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A.

Botulinum neurotoxin serotype A (BoNT/A) causes a debilitating and potentially fatal illness known as botulism. The toxin is also a bioterrorism threat, yet no pharmacological antagonist to counteract its effects has reached clinical approval. Existing strategies to negate BoNT/A intoxication have looked to antibodies, peptides, or organic small molecules as potential therapeutics.

Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons.

Introduction: Botulinum neurotoxins (BoNTs), the causative agents of botulism, are widely used as powerful bio-pharmaceuticals to treat neuro-muscular disorders. Due to the high potency and potential lethality of BoNTs, careful monitoring of the biologic activity of BoNT-based pharmaceuticals is required to ensure safe usage. For decades, the only approved method for potency determination of pharmaceutical BoNTs was the mouse bioassay (MBA), but in recent years improvements in cell-assay technologies have enabled MBA replacement by cell-based assays for specific product evaluations.

Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons.

Recent reports suggest that botulinum neurotoxin (BoNT) A, which is widely used clinically to inhibit neurotransmission, can spread within networks of neurons to have distal effects, but this remains controversial. Moreover, it is not known whether other members of this toxin family are transferred between neurons. Here, we investigate the potential distal effects of BoNT/A, BoNT/D, and tetanus toxin (TeNT), using central neurons grown in microfluidic devices.

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.

Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated "type H," has been controversial.

Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin.

Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by "thiol-ene" photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces.