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Characteristics of Botulinum Neurotoxins that determine potency

决定效力的肉毒杆菌神经毒素的特征

基本信息

批准号：
10539300
负责人：
Joseph T Barbieri
金额：
$ 63.6万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-18 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10539300
关键词：
Affect Amaze Amino Acid Substitution Amino Acids Animals Area Binding Biological Biological Assay Biological Products Botulinum Toxins Botulism C-terminal CCRL2 gene Caring Categories Cell model Cells Cessation of life Characteristics Chimera organism Classification Data Development Disease Dose Elements Engineering Esthetics Gangliosides Human Immunologics In Vitro Individual Intoxication Investigation Kinetics Knowledge Laboratories Light Maps Medical Medicine Membrane Modeling Molecular Mus Mutagenesis Mutate Mutation N-terminal Neuromuscular Diseases Neurons Paralysed Pathogenicity Pathologic Pathology Patients Pharmacologic Substance Property Protein Family Proteins Receptor Cell Recombinants Research Rodent Role Serotyping Severity of illness Structural Models Supportive care System Testing Therapeutic Time Toxic effect Toxin Variant antitoxin base beta-Lactamase bioweapon cost digital experimental study holotoxins improved in vivo insight mouse model nervous system disorder novel novel therapeutics pathogen pharmacologic receptor receptor binding respiratory trafficking uptake weapons

项目摘要

Abstract Botulinum Neurotoxins (BoNTs) are a large family of protein toxins and are of great significance due to their extreme potency and the severity of the disease they cause in humans and animals. Botulism is a neuroparalytic disease of long duration, lasting up to several months. Without proper medical care, naturally occurring botulism is lethal in up to 50% of cases, and even with respiratory and other supportive care and antitoxin administration, up to 5 % of patients die. While naturally occurring botulism is rare, BoNTs are classified as a Tier 1 Category A Select Agent due to their threat as potential bioterrorist weapons. Amazingly, BoNTs are also widely used in medicine to treat more than 100 neuromuscular disorders and for aesthetic purposes. BoNTs are immunologically divided into 7 serotypes, which are further subdivided into subtypes. Today, 100s of BoNT variants have been identified by sequencing efforts, but only few have been investigated at the protein level. It is noteworthy that out of all the BoNT variants only two (BoNT/A1 and /B1) are currently used as therapeutics, and studies examining properties of other variants are infrequent. Our research groups have determined for the first time that subtypes within the BoNT/A serotype have distinct biologic properties, including cell entry kinetics, duration of action, cell and mouse toxicity, and immunological variations. This RO1 project proposes to determine on a molecular and structural level the mechanisms underlying these unique properties of A subtypes and the chimeric BoNT/FA. Specifically, this project will investigate the mechanisms underlying the shorter duration of action of BoNT/A3, the faster and more efficient cell entry by BoNT/A2, /A6, and /FA, the 1,000 fold lower toxicity of BoNT/A4. Our collaborative efforts are unique in this area, as we are able to combine detailed mechanistic studies on subdomains with cell-based and in vivo studies on the holotoxin level. The Barbieri lab will use structural modeling to guide mutagenesis studies on functional domains of BoNTs and investigate mechanisms of receptor binding and cell trafficking using neuronal cell models. The Johnson/Pellett laboratory will utilize these data to create targeted holotoxin constructs in their native hosts and conduct detailed investigations in various rodent and human cell models. Finally, based on the data from these studies, select holotoxin constructs will be investigated in mice to determine pathologic and pharmacologic consequences of structural alterations. Using this approach, we will investigate a large number of amino acid substitutions in functional domain studies and select specific alterations for the more effort- and cost-intensive construction of recombinant holotoxins. By utilizing several cell models before conducting in vivo studies, we are able to reduce the number of required animals and also use human specific models. Finally, the combination of in vitro, cell-based, and in vivo studies will provide novel insight into the mechanisms underlying the observed pathologic and pharmacologic properties of these toxins.

摘要肉毒杆菌神经毒素(BoNTs)是一大类蛋白质毒素，具有重要的意义。它们在人类和动物中引起的疾病的极端效力和严重程度。肉毒杆菌中毒是一种一种持续时间较长的神经性瘫痪疾病，可持续数月。如果没有适当的医疗护理，自然会发生肉毒杆菌中毒在高达50%的病例中是致命的，即使在呼吸和其他支持性护理和服用抗毒素药物，高达5%的患者死亡。虽然自然发生的肉毒杆菌中毒很少见，但BoNTs是由于其潜在的生物恐怖武器的威胁，被归类为一级A类精选特工。令人惊讶的是， BoNTs还被广泛用于医学，治疗100多种神经肌肉疾病和美容目的。BNTs在免疫学上分为7个血清型，并进一步细分为亚型。今天，通过测序工作已经确定了100个BONT变体，但只有几个被调查过在蛋白质水平上。值得注意的是，在所有的BONT变体中，目前只有两个(BONT/A1和/B1 用于治疗，而检验其他变种的特性的研究并不频繁。我们的研究小组首次确定了BONT/A血清型中的亚型具有不同的生物学特性，包括细胞进入动力学、作用持续时间、细胞和小鼠毒性以及免疫学变化。这 RO1项目建议在分子和结构水平上确定这些机制 A亚型和嵌合的BONT/FA的独特性质。具体地说，这个项目将调查 BONT/A3作用持续时间较短的机制，通过以下方式更快、更有效地输入细胞 BONT/A2、/A6和/FA，毒性比BONT/A4低1000倍。我们的合作努力在这方面是独一无二的区域，因为我们能够将亚域的详细机制研究与基于细胞和体内的研究相结合全毒素水平的研究。Barbieri实验室将使用结构建模来指导对 BoNTs的功能区及其受体结合和细胞转运机制的研究神经细胞模型。约翰逊/佩莱特实验室将利用这些数据来制造有针对性的全息毒素并在各种啮齿动物和人类细胞模型中进行详细的研究。最后，根据这些研究的数据，将在小鼠身上研究选定的全毒素结构，以确定结构改变的病理和药理学后果。使用这种方法，我们将在功能域研究中调查大量的氨基酸替换，并选择特定的对于更费力和成本更密集的重组全毒素构建的改造。通过利用几个细胞模型在进行体内研究之前，我们能够减少所需的动物数量，还使用特定于人类的模型。最后，体外、基于细胞和体内研究的结合将提供对这些观察到的病理和药理学特性背后的机制的新见解毒素。