Characteristics of Botulinum Neurotoxins that determine potency

决定效力的肉毒杆菌神经毒素的特征

• 批准号: 10326384
• 负责人: Joseph T Barbieri
• 金额: $ 64.02万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-18 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326384
• 关键词: Affect; Amino Acid Substitution; Amino Acids; Animals; Area; Binding; Biological; Biological Assay; Bontoxilysin; Botulism; C-terminal; CCRL2 gene; Caring; Categories; Cell model; Cells; Cessation of life; Characteristics; Chimera organism; Data; Development; Disease; Dose; Elements; Engineering; Esthetics; GTP-Binding Protein alpha Subunits, Gs; 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; Pharmacology; Property; Protein Family; Proteins; Receptor Cell; Recombinants; Research; Rodent; Role; Serotyping; Severity of illness; Structural Models; Supportive care; System; Testing; Therapeutic Studies; Time; Toxic effect; Toxin; Variant; antitoxin; base; beta-Lactamase; cost; digital; experimental study; holotoxins; improved; in vivo; insight; mouse model; nervous system disorder; novel; novel therapeutics; pathogen; receptor; receptor binding; respiratory; trafficking; uptake; weapons

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 肉毒神经毒素（BotanicNeurotoxins，BoNT）是蛋白质毒素的一个大家族，并且由于它们的生物活性而具有重要意义。 极端的效力和它们在人类和动物中引起的疾病的严重性。肉毒杆菌中毒是一种 持续时间长的神经麻痹性疾病，可持续数月。如果没有适当的医疗护理， 肉毒杆菌中毒在高达50%的病例中是致命的，即使有呼吸和其他支持性护理， 抗毒素给药后，高达5%的患者死亡。虽然自然发生的肉毒杆菌中毒是罕见的， 由于其潜在的生物恐怖主义武器的威胁，被归类为第1级A类选择代理。令人惊讶的是， BoNT还广泛用于医学中以治疗100多种神经肌肉疾病和用于美学目的。 目的BoNT在免疫学上分为7种血清型，其进一步细分为亚型。 今天，测序工作已经鉴定了100多个BoNT变体，但只有少数被调查 在蛋白质水平上。值得注意的是，在所有BoNT变体中，目前只有两种（BoNT/A1和/B1）是已知的。 用作治疗药物，并且检查其他变体特性的研究很少。我们的研究小组 首次确定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 RO 1项目建议在分子和结构水平上确定这些潜在的机制， A亚型和嵌合BoNT/FA的独特性质。具体而言，本项目将调查 BoNT/A3的作用持续时间越短，其进入细胞的速度越快， BoNT/A2、/A6和/FA，毒性比BoNT/A4低1,000倍。我们的合作努力在这方面是独一无二的 区域，因为我们能够联合收割机结合详细的机制研究的亚结构域与细胞为基础的和在体内 全毒素水平的研究Barbieri实验室将使用结构建模来指导诱变研究， BoNTs的功能结构域，并研究受体结合和细胞运输的机制， 神经元细胞模型。约翰逊/佩利特实验室将利用这些数据来创造有针对性的全毒素 构建在其天然宿主中，并在各种啮齿动物和人类细胞模型中进行详细研究。 最后，基于来自这些研究的数据，将在小鼠中研究选择的全毒素构建体， 确定结构改变的病理学和药理学后果。通过这种方法，我们将 在功能结构域研究中调查大量的氨基酸取代，并选择特异性的 用于更费力和成本密集型的重组全毒素构建的改变。通过利用几个 在进行体内研究之前，我们能够减少所需动物的数量， 使用人类特定的模型。最后，体外、基于细胞和体内研究的结合将提供 新的洞察机制的基础上观察到的病理和药理学特性，这些 毒素