Gaining ground: assays for therapeutics against botulinum neurotoxin

Hakami, Ramin M.; Ruthel, Gordon; Stahl, Andrea M.; Bavari, Sina

doi:10.1016/j.tim.2010.02.001

Cited by 31 publications

(32 citation statements)

References 63 publications

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“…BoNT is a potent neurotoxin (Hakami et al, 2011). It inhibits the release of neurotransmitters from the presynaptic buds and thereby impairs the signal transmission between nerves or between nerves and skeletal muscle at the motor end plate.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, the LD50 with respect to the amount of protein fluctuates considerably from batch to batch. Because of the small therapeutic index and potential lethal complication of an overdose, a reliable determination of the biological activity of the BoNT preparation is mandatory (Hakami et al, 2010). The gold standard method to quantify the activity of BoNT is the mouse lethality assay (Pearce et al, 1994(Pearce et al, , 1997Singh et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-targeted luciferase from neuronal cells

Pathe-Neuschäfer-Rube

Neuschäfer-Rube

Genz

et al. 2015

ALTEX

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SummaryBotulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations. Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-targeted luciferase from neuronal cells

Pathe-Neuschäfer-Rube

Neuschäfer-Rube

Genz

et al. 2015

ALTEX

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“…It is well established that BoNT-mediated SNARE protein cleavage is sufficient to inhibit neurotransmission (Apland et al 1999; Bajohrs et al 2004), indicating that the cleavage products of these target proteins provide critical markers to assess the effects of potential BoNT antagonists. Hence, neuron-based assays can provide a simple format for identifying small molecules that protect SNARE proteins during BoNT exposure (Hakami et al 2010; Kota et al 2014). To this end, we and others have shown that mouse ES-derived neurons and more specifically motor neurons can be used for BoNT research (Kiris et al 2014b; Pellett 2013; McNutt et al 2011).…”

Section: Discussionmentioning

confidence: 99%

Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

et al. 2015

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Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins’ proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin’s enzymatic components, to antagonize multiple BoNT serotypes in motor neurons.

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“…The LC of different serotypes targets different SNARE proteins. BoNT serotypes A, C and E target SNAP-25, and serotypes B, D, F, and G target VAMP2, while serotype C targets syntaxin (Hakami et al, 2010). Different SNARE targets of BoNTs have been used to develop in vitro assays for toxin activity.…”

Section: Overview Of Botulinum Neurotoxin Structure and Function As Tmentioning

confidence: 99%

Current Methods for Detecting the Presence of Botulinum Neurotoxins in Food and Other Biological Samples

Cheng¹,

Land²,

Stanker³

2012

Bioterrorism

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Gaining ground: assays for therapeutics against botulinum neurotoxin

Cited by 31 publications

References 63 publications

Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-targeted luciferase from neuronal cells

Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-targeted luciferase from neuronal cells

Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

Current Methods for Detecting the Presence of Botulinum Neurotoxins in Food and Other Biological Samples

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