Critical analysis in the advancement of cell-based assays for botulinum neurotoxin

Ambrin, Ghuncha; Cai, Shuowei; Singh, Bal Ram

doi:10.1080/1040841x.2022.2035315

Cited by 3 publications

(2 citation statements)

References 147 publications

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“…Recent developments of cell-based assays provide detection methods that are more sensitive than the MBA, and reflect all BoNT steps of activity; however, they have to be adapted for each BoNT type, whereas the MBA is sensitive to all BoNT types [ 107 , 109 ].…”

Section: Discussion: Suitability Of Each Methods For Their Applicationsmentioning

confidence: 99%

Recent Developments in Botulinum Neurotoxins Detection

Rasetti-Escargueil

Popoff

2022

Microorganisms

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Botulinum neurotoxins (BoNTs) are produced as protein complexes by bacteria of the genus Clostridium that are Gram-positive, anaerobic and spore forming (Clostridium botulinum, C. butyricum, C. baratii and C. argentinense spp.). BoNTs show a high immunological and genetic diversity. Therefore, fast, precise, and more reliable detection methods are still required to monitor outbreaks and ensure surveillance of botulism. The botulinum toxin field also comprises therapeutic uses, basic research studies and biodefense issues. This review presents currently available detection methods, and new methods offering the potential of enhanced precision and reproducibility. While the immunological methods offer a range of benefits, such as rapid analysis time, reproducibility and high sensitivity, their implementation is subject to the availability of suitable tools and reagents, such as specific antibodies. Currently, the mass spectrometry approach is the most sensitive in vitro method for a rapid detection of active or inactive forms of BoNTs. However, these methods require inter-laboratory validation before they can be more widely implemented in reference laboratories. In addition, these surrogate in vitro models also require full validation before they can be used as replacement bioassays of potency. Cell-based assays using neuronal cells in culture recapitulate all functional steps of toxin activity, but are still at various stages of development; they are not yet sufficiently robust, due to high batch-to-batch cell variability. Cell-based assays have a strong potential to replace the mouse bioassay (MBA) in terms of BoNT potency determination in pharmaceutical formulations; they can also help to identify suitable inhibitors while reducing the number of animals used. However, the development of safe countermeasures still requires the use of in vivo studies to complement in vitro immunological or cell-based approaches.

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Section: Discussion: Suitability Of Each Methods For Their Applicationsmentioning

confidence: 99%

Recent Developments in Botulinum Neurotoxins Detection

Rasetti-Escargueil

Popoff

2022

Microorganisms

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“…However, the properties of this toxin as such are well-known. Very reliable NAM have been established for quality testing, and they make animal testing redundant in several use domains (Ambrin et al, 2022;Hartung, 2015Hartung, , 2021.…”

Section: Fig 5: Schematic Representation Of Net Benefitmentioning

confidence: 99%

On the usefulness of animals as a model system (part II): Considering benefits within distinct use domains

Pallocca

2022

ALTEX

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To make this less theoretical, we introduce examples from the everyday world (outside science, considering well-known leisure activities). They may help to illustrate a scientific problem, and this way we hope to offer an easy entry into a complex discussion. For instance, one may ask whether skis are a useful tool. It becomes immediately evident that this question is pointless without specification of a purpose (e.g., opening a bottle, descending a mountain slope, crossing the jungle). Even when the purpose (ascending or descending a mountain slope) is defined, the tool's usefulness is determined by the exact situation (snow coverage, etc.). Another easily accessible example is the use of a full-face helmet. It may be argued that helmets are very useful because

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Tirbanibulin (KX2‐391) analog KX2‐361 inhibits botulinum neurotoxin serotype A mediated SNAP‐25 cleavage in pre‐ and post‐intoxication models in cells

Koc,

Ibis,

Besarat

et al. 2024

Drug Development Research

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Botulinum neurotoxins (BoNT) inhibit neuroexocytosis, leading to the potentially lethal disease botulism. BoNT serotype A is responsible for most human botulism cases, and there are no approved therapeutics to treat already intoxicated patients. A growing body of research has demonstrated that BoNT/A can escape into the central nervous system, and therefore, identification of BoNT/A inhibitors that can penetrate BBB and neutralize the toxin within intoxicated neurons would be important. We previously identified an FDA‐approved, orally bioavailable compound, KX2‐391 (Tirbanibulin) that inhibits BoNT/A in motor neuron assays. Recently, a structural analog of KX2‐391, KX2‐361, has been shown to exhibit good oral bioavailability and cross BBB with high efficiency in mouse experiments. Therefore, in this work, we evaluated the inhibitory effects of KX2‐361 against BoNT/A. Toward this goal, we first evaluated the compound for its effects on cell viability in PC12 cells, via MTT assay, and in mouse embryonic stem cell (mESC)‐derived motor neurons, with imaging‐based assays. Following, we tested KX2‐361 in mESC‐derived motor neurons intoxicated with BoNT/A holotoxin, and the compound exhibited activity against the toxin in both pre‐ and post‐intoxication conditions. Excitingly, KX2‐361 also inhibited BoNT/A enzymatic component (light chain; LC) in PC12 cells transfected with BoNT/A LC. Furthermore, our molecular docking analyses suggested that KX2‐361 can directly bind to BoNT/A LC. Medicinal chemistry approaches to develop structural analogs of KX2‐361 to increase its efficacy against BoNT/A may provide a critical lead compound with BBB penetration capacity for drug development efforts against BoNT/A intoxication.

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Critical analysis in the advancement of cell-based assays for botulinum neurotoxin

Cited by 3 publications

References 147 publications

Recent Developments in Botulinum Neurotoxins Detection

Recent Developments in Botulinum Neurotoxins Detection

On the usefulness of animals as a model system (part II): Considering benefits within distinct use domains

Tirbanibulin (KX2‐391) analog KX2‐361 inhibits botulinum neurotoxin serotype A mediated SNAP‐25 cleavage in pre‐ and post‐intoxication models in cells

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