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1.
Toxins (Basel) ; 3(3): 207-17, 2011 03.
Artigo em Inglês | MEDLINE | ID: mdl-22069707

RESUMO

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.


Assuntos
Aconitina/análogos & derivados , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Imidazóis/farmacologia , Neurônios Motores/efeitos dos fármacos , Ftalimidas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Aconitina/administração & dosagem , Aconitina/química , Aconitina/farmacologia , Animais , Western Blotting , Técnicas de Cultura de Células , Células Cultivadas , Embrião de Galinha , Citosol/efeitos dos fármacos , Citosol/metabolismo , Imidazóis/administração & dosagem , Imidazóis/química , Macrolídeos/administração & dosagem , Macrolídeos/farmacologia , Estrutura Molecular , Neurônios Motores/metabolismo , Ftalimidas/administração & dosagem , Ftalimidas/química , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
2.
Stem Cell Res ; 6(3): 195-205, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21353660

RESUMO

Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins, there are elevated concerns regarding their possible use as bioterrorism agents. Moreover, their widespread use for cosmetic purposes, and as medical treatments, has increased the potential risk of accidental overdosing and environmental exposure. Hence, there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs; however, due to the difficulty and poor efficiency of the procedures required to isolate the cells, they are not suitable for high-throughput drug screening assays. Here, we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable, reproducible, and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally, we demonstrated that several BoNT/A inhibitors protected SNAP-25, the BoNT/A substrate, in the ES-derived motoneuron system. Furthermore, this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics.


Assuntos
Antitoxina Botulínica/farmacologia , Toxinas Botulínicas/antagonistas & inibidores , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Embrionárias/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Neurônios Motores/efeitos dos fármacos , Animais , Toxinas Botulínicas/toxicidade , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/instrumentação , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo
3.
J Biomol Screen ; 15(1): 42-51, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19965805

RESUMO

Botulinum neurotoxins (BoNTs) are zinc-metalloproteases that cleave components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complex, inhibiting acetylcholine release into neuromuscular junctions, resulting in flaccid paralysis and eventual death. The potential for the malicious misuse of these toxins as bioweapons has created an urgent need to develop effective therapeutic countermeasures. Robust cell-based assays will be essential for lead identification and the optimization of therapeutic candidates. In this study, the authors developed novel BoNT serotype A (BoNT/A) cleavage-sensitive (BACS) antibodies that only interact with full-length SNAP-25 (synaptosomal-associated protein of 25 kDa), the molecular target of the BoNT/A serotype. These antibodies exhibit high specificity for full-length SNAP-25, allowing the BoNT/A-mediated proteolysis of this protein to be measured in diverse assay formats, including several variations of enzyme-linked immunosorbent assay and multiple immunofluorescence methods. Assays built around the BACS antibodies displayed excellent sensitivity, had excellent reproducibility, and were amenable to multiwell formats. Importantly, these assays provided novel methods for evaluating BoNT/A activity in cellular models of intoxication and allowed for the high-throughput evaluation of experimental compounds.


Assuntos
Anticorpos/imunologia , Toxinas Botulínicas Tipo A/análise , Toxinas Botulínicas Tipo A/imunologia , Ensaios de Triagem em Larga Escala/métodos , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Western Blotting , Galinhas , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Peptídeos/química , Sorotipagem , Proteína 25 Associada a Sinaptossoma/metabolismo
4.
J Biomol Screen ; 12(3): 370-7, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17332092

RESUMO

Botulinum toxin is an exceedingly potent inhibitor of neurotransmission across the neuromuscular junction, causing flaccid paralysis and death. The potential for misuse of this deadly poison as a bioweapon has added a greater urgency to the search for effective therapeutics. The development of sensitive and efficient cell-based assays for the evaluation of toxin antagonists is crucial to the rapid and successful identification of therapeutic compounds. The authors evaluated the sensitivity of primary cultures from 4 distinct regions of the embryonic chick nervous system to botulinum neurotoxin A (BoNT/A) cleavage of synaptosomal-associated protein of 25 kD (SNAP-25). Although differences in sensitivity were apparent, SNAP-25 cleavage was detectable in neuronal cells from each of the 4 regions within 3 h at BoNT/A concentrations of 1 nM or lower. Co-incubation of chick neurons with BoNT/A and toxin-neutralizing antibodies inhibited SNAP-25 cleavage, demonstrating the utility of these cultures for the assay of BoNT/A antagonists.


Assuntos
Bioensaio/métodos , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Toxinas Botulínicas Tipo A/farmacologia , Avaliação Pré-Clínica de Medicamentos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Células Cultivadas , Embrião de Galinha , Sensibilidade e Especificidade , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de Tempo
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