Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Sci Transl Med ; 13(575)2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33408188

RESUMO

Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. These data demonstrate that atoxic BoNT derivatives can be harnessed to deliver therapeutic protein moieties to the neuronal cytoplasm where they bind and neutralize intracellular targets in experimental models. The generalizability of this platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets.


Assuntos
Toxinas Botulínicas Tipo A , Botulismo , Anticorpos de Domínio Único , Animais , Botulismo/tratamento farmacológico , Cobaias , Camundongos , Modelos Animais , Neurotoxinas
2.
Sci Rep ; 7: 42923, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28220863

RESUMO

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.


Assuntos
Toxinas Botulínicas/metabolismo , Portadores de Fármacos/química , Sequência de Aminoácidos , Animais , Toxinas Botulínicas/genética , Toxinas Botulínicas/toxicidade , Células Cultivadas , Dimerização , Feminino , Dose Letal Mediana , Camundongos , Microscopia Confocal , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Neurônios/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo
3.
Sci Rep ; 6: 30429, 2016 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-27484492

RESUMO

Cyto-012 is a recombinant derivative of Botulinum neurotoxin Type A (BoNT/A). It primarily differs from wild type (wt) BoNT/A1 in that it incorporates two amino acid substitutions in the catalytic domain of the light chain (LC) metalloprotease (E224 > A and Y366 > A), designed to provide a safer clinical profile. Cyto-012 is specifically internalized into rat cortical and hippocampal neurons, and cleaves Synaptosomal-Associated Protein 25 (SNAP-25), the substrate of wt BoNT/A, but exhibits slower cleavage kinetics and therefore requires a higher absolute dose to exhibit pharmacologic activity. The pharmacodynamics of Cyto-012 and wt BoNT/A have similar onset and duration of action using the Digital Abduction Assay (DAS). Intramuscular LD50 values for Cyto-012 and wt BoNT/A respectively, were 0.63 ug (95% CI = 0.61, 0.66) and 6.22 pg (95% CI = 5.42, 7.02). ED50 values for Cyto-012 and wt BoNT/A were respectively, 0.030 ug (95% CI = 0.026, 0.034) and 0.592 pg (95% CI = 0.488, 0.696). The safety margin (intramuscular LD50/ED50 ratio) for Cyto-012 was found to be improved 2-fold relative to wt BoNT/A (p < 0.001). The DAS response to Cyto-012 was diminished when a second injection was administered 32 days after the first. These data suggest that the safety margin of BoNT/A can be improved by modulating their activity towards SNAP-25.


Assuntos
Toxinas Botulínicas Tipo A/toxicidade , Fármacos Neuromusculares/toxicidade , Animais , Toxinas Botulínicas Tipo A/farmacocinética , Células Cultivadas , Feminino , Dose Letal Mediana , Camundongos , Músculo Esquelético/metabolismo , Fármacos Neuromusculares/farmacocinética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidade
4.
J Neurogenet ; 29(2-3): 135-43, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26100104

RESUMO

Autism spectrum disorder (ASD) is a neurodevelopmental disorder in humans characterized by complex behavioral deficits, including intellectual disability, impaired social interactions, and hyperactivity. ASD exhibits a strong genetic component with underlying multigene interactions. Candidate gene studies have shown that the neurobeachin (NBEA) gene is disrupted in human patients with idiopathic autism ( Castermans et al., 2003 ). The NBEA gene spans the common fragile site FRA 13A and encodes a signal scaffold protein ( Savelyeva et al., 2006 ). In mice, NBEA has been shown to be involved in the trafficking and function of a specific subset of synaptic vesicles. ( Medrihan et al., 2009 ; Savelyeva et al., 2006 ). Rugose (rg) is the Drosophila homolog of the mammalian and human NBEA. Our previous genetic and molecular analyses have shown that rg encodes an A kinase anchor protein (DAKAP 550), which interacts with components of the epidermal growth factor receptor or EGFR and Notch-mediated signaling pathways, facilitating cross talk between these and other pathways ( Shamloula et al., 2002 ). We now present functional data from studies on the larval neuromuscular junction that reveal abnormal synaptic architecture and physiology. In addition, adult rg loss-of-function mutants exhibit defective social interactions, impaired habituation, aberrant locomotion, and hyperactivity. These results demonstrate that Drosophila NBEA (rg) mutants exhibit phenotypic characteristics reminiscent of human ASD and thus could serve as a genetic model for studying ASDs.


Assuntos
Proteínas de Ancoragem à Quinase A/genética , Comportamento Animal/fisiologia , Proteínas de Drosophila/genética , Locomoção/genética , Atividade Motora/genética , Comportamento Social , Sinapses/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Animais Geneticamente Modificados , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Mutação , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Transdução de Sinais/genética , Sinapses/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...