Molecular mechanisms of botulinum neurotoxin neutralization

肉毒杆菌神经毒素中和的分子机制

• 批准号: 9160875
• 负责人: Rongsheng Jin
• 金额: $ 63.62万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-10 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9160875
• 关键词: Affinity; Animals; Antidotes; Antitoxins; Bacterial Toxins; Binding; Binding Sites; Bontoxilysin; Botulinum Toxin Type A; Botulism; Cell surface; Centers for Disease Control and Prevention (U.S.); Cleaved cell; Clostridium botulinum; Communities; Complement; Complex; Data; Development; Diagnostic; Disease; Endopeptidases; Enteral; Epitopes; Exposure to; Gene Therapy Agent; Goals; Health; Human; Immune Sera; Infection; Infectious Agent; Intoxication; Invaded; Knowledge; Lead; Length; Link; Lung; Maps; Mediating; Membrane; Methods; Microbe; Modeling; Molecular; Mutation; Neurons; Paralysed; Pathology; Peptide Hydrolases; Peptides; Prevention approach; Prevention strategy; Process; Property; Proteins; Reagent; Research; Risk; Role; SNAP receptor; Serotyping; Serum; Severities; Site; Specificity; Structural Models; Structure; Symptoms; Syndrome; Technology; Testing; Therapeutic; Time; Toxic effect; Toxin; Virus; base; biodefense; biothreat; design; flexibility; gene therapy; improved; inhibitor/antagonist; innovation; interest; mouse model; neurotoxin receptor; neurotransmission; novel; novel strategies; pathogen; prevent; public health relevance; receptor; receptor binding; small molecule; treatment strategy

Project summary Botulism is caused by exposure to protein toxins called botulinum neurotoxins (BoNTs) that are produced by Clostridium botulinum. BoNTs are CDC Tier 1 select agent for which no antidote currently exists. Seven different BoNT serotypes have been discovered to date (BoNT/A-G), many having numerous additional BoNT subtypes. However the only currently available treatments are serum based antitoxin products derived from large animals that are only effective if administered soon after BoNT intoxication. The challenge of developing BoNT therapeutics is exacerbated by the fact that the seven known BoNT serotypes are each distinct toxins with distinct receptor specificities and proteases that cleave at distinct sites on SNARE proteins to disrupt nerve transmission. Due to the severity of the risk, the paucity of treatment options, and the complexity of the challenge, novel approaches to the prevention and treatment of BoNT intoxication are clearly needed. We now have extensive evidence in multiple toxin models demonstrating that bispecific VHH-based neutralizing agents (VNAs), consisting of two covalently linked, toxin-neutralizing VHHs, are antitoxins with potencies that often exceed that of current monoclonal and polyclonal antitoxin agents. Furthermore, VNAs offer substantial advantages over serum and mAb antitoxin products as they are economical to produce and highly versatile; offering innovative new prevention and treatment strategies for toxin exposures and infections with toxin-producing pathogens such as gene therapies and direct delivery to enteric and pulmonary sites of challenge. In this proposal, we test the hypothesis that integrating structural and mechanistic information into VNA design will lead to even greater antitoxin efficacy and versatility. The Specific Aims are to (1) determine the crystal structures of selected BoNT-binding VHHs in complex with their target BoNTs; (2) define the mechanisms by which VHHs selected in Aim 1 block BoNT toxicity, and; (3) design and test bispecific VNAs with enhanced antitoxin properties by exploiting structure/function data from Aims 1 and 2. This will be the first comprehensive structural mapping of BoNT neutralizing epitopes, which will be complemented with mechanistic studies of BoNT function and BoNT-host interactions. Furthermore, this study will improve general understanding of how structural and mechanistic information can inform the design of even more effective VNA antitoxin agents and should permit rapid development of commercial antitoxin therapeutics to treat exposures to all BoNT serotypes and other toxin biothreat agents.

项目概要 肉毒杆菌中毒是由于接触由梭状芽胞杆菌产生的称为肉毒杆菌神经毒素 (BoNT) 的蛋白质毒素引起的 肉毒杆菌。 BoNT 是 CDC Tier 1 选择药物，目前尚无解毒剂。七种不同的 BoNT 血清型 迄今为止已发现 (BoNT/A-G)，其中许多具有许多其他 BoNT 亚型。然而唯一的 目前可用的治疗方法是源自大型动物的基于血清的抗毒素产品，只有在以下情况下才有效 BoNT 中毒后立即给药。开发 BoNT 疗法的挑战因以下事实而加剧： 七种已知的 BoNT 血清型各自是不同的毒素，具有不同的受体特异性和在 SNARE 蛋白上的不同位点可破坏神经传递。由于风险严重，治疗手段匮乏 选项，以及挑战的复杂性，预防和治疗 BoNT 中毒的新方法是 显然需要。我们现在在多种毒素模型中拥有大量证据，证明基于双特异性 VHH 的 中和剂 (VNA) 由两个共价连接的毒素中和 VHH 组成，是具有效力的抗毒素 这通常超过了目前的单克隆和多克隆抗毒素剂。此外，VNA 还提供大量 与血清和 mAb 抗毒素产品相比，它们具有生产经济且用途广泛的优势；奉献 针对毒素暴露和产毒病原体感染的创新预防和治疗策略 例如基因疗法和直接递送至肠道和肺部的攻击部位。在这个提案中，我们测试了 假设将结构和机械信息整合到 VNA 设计中将产生更强的抗毒素 功效和多功能性。具体目标是 (1) 确定所选 BoNT 结合 VHH 的晶体结构 与目标 BoNT 复合； (2) 定义目标 1 中选择的 VHH 阻断 BoNT 毒性的机制， 和; (3) 通过利用结构/功能数据设计和测试具有增强抗毒素特性的双特异性 VNA 目标 1 和 2。这将是 BoNT 中和表位的第一个全面结构图谱，这将是 辅以 BoNT 功能和 BoNT-宿主相互作用的机制研究。此外，这项研究将 提高对结构和机械信息如何为更多设计提供信息的一般理解 有效的 VNA 抗毒素剂，应该允许快速开发商业抗毒素疗法来治疗 暴露于所有 BoNT 血清型和其他毒素生物威胁剂。