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

基本信息

批准号：
10177867
负责人：
Kim Janda
金额：
$ 63.79万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
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/10177867
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 antitoxin base biological systems botulinum data modeling design environmental change improved in vivo inhibitor/antagonist insight man pathogen pre-clinical preclinical study prevent receptor mediated endocytosis scaffold small molecule small molecule inhibitor success

项目摘要

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于人类来说，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 CDC 指定的 A 类仅有的六种病原体之一。由于其毒性和易于生产，因此成为生物恐怖制剂。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多可以通过治疗减轻疾病的进展。七价抗毒素，仍需要数月的住院治疗我们的长期目标是开发一种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相比之下，神经元中小分子的寿命与神经毒素的持久性之间存在差异。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，金属蛋白酶很罕见，因此我们设计了一种策略。因此，针对变构反应残基的共价弹头被束缚在有效的活性位点抑制剂上，从而创造了一种“双功能”抑制剂，这本质上避免了酶机制问题，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 1) 使用对接和结构活动关系（SAR）数据，我们将将先前确定的 BoNT/A 轻链可逆抑制剂调整为双功能共价策略 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 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 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。