Covalent Inhibition as a Method to Counteract Botulinum Intoxication

共价抑制作为对抗肉毒杆菌中毒的方法

• 批准号: 10408004
• 负责人: Kim Janda
• 金额: $ 63.46万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-02 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408004
• 关键词: Accounting; Acetylcholine; Active Sites; Adult; Affect; Affinity; Anaerobic Bacteria; Animal Model; Antibodies; Antibody Therapy; Asphyxia; Binding; Binding Proteins; Biological Assay; Bioterrorism; Bontoxilysin; Botulinum Toxin Type A; Botulism; Caspase; Categories; Cells; Cellular Assay; Centers for Disease Control and Prevention (U.S.); Cessation of life; Clinical; Clinical Research; Clostridium botulinum; Complex; Contracts; Crystallization; Data; Development; Disease; Disease Progression; Docking; Drug Kinetics; Drug user; Ensure; Enzymes; Etiology; Evaluation; Event; Food; Geometry; Goals; Gold; Half-Life; Heroin Users; Hospitalization; Hour; Human; Iatrogenesis; Infection; Inhalation; Injectable; Intervention; Intoxication; Intravenous; Light; Longevity; Longitudinal Studies; Mediating; Membrane Proteins; Metalloproteases; Metals; Methods; Modeling; Modification; Molecular Conformation; Mus; Muscle; Nerve; Neurons; Neurotoxins; Paralysed; Patients; Peptide Hydrolases; Permeability; Poison; Production; Protease Inhibitor; Protein Dynamics; Proteins; Reporting; Roentgen Rays; Role; Safety; Series; Serine; Serotyping; Severity of illness; Soil; Specificity; Structure; Structure-Activity Relationship; Sulfhydryl Compounds; Surface; Symptoms; Synaptosomes; Testing; Therapeutic; Time; Toxic effect; Toxin; Vulnerable Populations; Work; Wound Infection; Zinc; antagonist; antitoxin; base; biological systems; botulinum; data modeling; design; environmental change; improved; in vivo; inhibitor; insight; man; pathogen; pre-clinical; preclinical study; prevent; receptor mediated endocytosis; scaffold; small molecule; small molecule inhibitor; success

PROJECT SUMMARY/ABSTRACT Botulinum neurotoxin serotype A (BoNT/A), which causes the disease botulism, is the most potent toxin known to man. BoNTs are most commonly encountered as BotoxTM, the increasing use of which has made iatrogenic botulism a major concern. BoNTs are one of only six pathogens designated by the CDC as a category A bioterrorism agent due to its toxicity and ease of production. Furthermore, the spread of botulism among heroin users is a growing concern. Despite the potential threat and the severity of the disease, there is no therapeutic available for rescuing the neuronal intoxication that causes botulism. At best, the progression of the disease is mitigated by treatment with a heptavalent antitoxin, which still requires months of hospitalization. Our long-term goal is to develop a clinically viable therapeutic capable of reversing the effects of botulinum neurotoxin, in addition to arresting progress. As BoNT intoxication is a solitary event, we posit that an irreversible covalent inhibitor capable of entering muscle neurons could permanently compromise its catalytic machinery, providing a solution to the discrepancy between the lifetime of a small molecule in neurons and the persistence of the neurotoxin. In contrast to the numerous reports of irreversible inhibitors of serine/cysteine proteases, irreversible inhibition of metalloproteinases is rare, a result of differences in catalytic mechanisms. As such we have devised a strategy wherein a covalent warhead that targets an allosteric reactive residue is tethered to a potent active site inhibitor, thus creating a “bifunctional” inhibitor. This in essence skirts enzyme mechanistic issues and now allows covalent targeting of the BoNT/LC. Based on promising preliminary data, we propose four specific aims that will lead to the identification of potent, reactive, and selective molecules. 1) Using docking and structure activity relationship (SAR) data we will adapt previously identified reversible inhibitors of BoNT/A light chain to the bifunctional covalent strategy. 2) We will screen covalent fragments in the presence of reversible inhibitors to select for warheads that tolerate the presence of the reversible inhibitor scaffold, and to account for active site conformational changes induced by the reversible inhibitor. 3) We will obtain and analyze crystallographic, cell and pharmacokinetic data to iteratively improve our inhibitors, prioritizing potency, selectivity, and safety in order to maximize the chance for success during in vivo studies. 4) Finally, by testing our compounds in the FDA gold standard mouse lethality model, we will assess the efficacy of our developed compounds and their suitability for pre-clinical and clinical studies.

项目概要/摘要 肉毒杆菌神经毒素血清型 A (BoNT/A) 会导致肉毒杆菌中毒，是已知最有效的毒素 对男人。 BoNT 最常见的是 BotoxTM，其使用的增加已导致医源性 肉毒杆菌中毒是一个主要问题。 BoNT 是 CDC 指定为 A 类的仅有的六种病原体之一 生物恐怖剂由于其毒性和易于生产。此外，肉毒杆菌中毒在海洛因中的传播 用户日益关注。 尽管存在潜在的威胁和疾病的严重性，但目前还没有可用于挽救的治疗方法 导致肉毒杆菌中毒的神经元中毒。最好的情况是，通过治疗可以减缓疾病的进展 使用七价抗毒素，仍需要住院数月。我们的长期目标是发展 临床上可行的治疗方法，除了阻止肉毒杆菌神经毒素的作用外，还能够逆转肉毒杆菌神经毒素的作用 进步。由于 BoNT 中毒是一个孤立的事件，我们假设一种不可逆的共价抑制剂能够 进入肌肉神经元可能会永久损害其催化机制，从而提供了解决方案 神经元中小分子的寿命与神经毒素的持久性之间的差异。相比之下 对于丝氨酸/半胱氨酸蛋白酶不可逆抑制剂的大量报道，不可逆抑制 由于催化机制的差异，金属蛋白酶很少见。因此我们制定了一个策略 其中靶向变构反应残基的共价弹头与有效的活性位点抑制剂相连， 从而创造出一种“双功能”抑制剂。这本质上回避了酶机制问题，现在允许共价 BoNT/LC 的靶向。 基于有希望的初步数据，我们提出了四个具体目标，这些目标将导致确定 有效的、反应性的和选择性的分子。 1) 使用对接和结构活动关系（SAR）数据，我们将 使先前确定的 BoNT/A 轻链可逆抑制剂适应双功能共价策略。 2）我们 将在可逆抑制剂存在的情况下筛选共价片段，以选择能够耐受的弹头 可逆抑制剂支架的存在，并解释由以下因素引起的活性位点构象变化 可逆抑制剂。 3）我们将获取并分析晶体学、细胞和药代动力学数据，以迭代 改进我们的抑制剂，优先考虑效力、选择性和安全性，以最大限度地提高成功机会 在体内研究期间。 4) 最后，通过在 FDA 金标准小鼠致死模型中测试我们的化合物，我们 将评估我们开发的化合物的功效及其对临床前和临床研究的适用性。