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：
10624958
负责人：
Kim Janda
金额：
$ 63.34万
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/10624958
关键词：
Accounting Acetylcholine Active Sites Adult Affect Affinity Anaerobic Bacteria Animal Model Antibodies Antibody Therapy Asphyxia Binding Binding Proteins Biological Assay Bioterrorism Botulinum Toxin Type A Botulinum Toxins Botulism Caspase Categories Cells Cellular Assay Centers for Disease Control and Prevention (U.S.)Cessation of life Clinical Clinical Research Clostridium botulinum Complex Contracts Crystallography Data Development Disease Disease Progression Docking Drug Kinetics Drug user Ensure Enzymes Etiology Evaluation Event Food Geometry Goals Half-Life Heroin Users Hospitalization Hour Human Iatrogenesis Infection Inhalation Injectable Intervention Intoxication Intravenous Light Longevity Longitudinal Studies Mediating Membrane 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 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 X-Ray Crystallography Zinc antagonist antitoxin biological systems botulinum data modeling design efficacy evaluation environmental change food preparation improved in vivo inhibitor 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.

项目摘要/摘要引起疾病肉毒杆菌的肉毒神经毒素血清型A（BONT/A）是已知的最有效的毒素给男人。 BONT最常见的是botoxtm，越来越多的使用使医源性肉毒杆菌是一个主要问题。 BONT是CDC指定为A类A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而引起的。此外，肉毒杆菌在海洛因之间的传播用户越来越关注。尽管有潜在的威胁和疾病的严重性，但仍未使用治疗性引起肉毒杆菌的神经元中毒。充其量，通过治疗减轻疾病的进展使用甲毒素的抗毒素，仍需要几个月的住院治疗。我们的长期目标是开发除了阻止进步。由于BONT肠毒性是一个牢固的事件，因此我们肯定的是，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械，从而提供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众多报道，对不可逆抑制作用金属蛋白酶很少见，这是催化机制差异的结果。因此，我们制定了一种策略其中，靶向变构反应驻留的共价弹头被束缚在潜在的活性部位抑制剂上从而产生“双功能”抑制剂。从本质上讲，这本质上是酶的机械问题，现在允许共价靶向BONT/LC。根据承诺的初步数据，我们提出了四个特定目标，这些目标将导致识别电势，反应性和选择性分子。 1）使用对接和结构活动关系（SAR）数据我们将适应先前确定的BONT/A轻链可逆抑制剂，以使双功能共价策略。 2）我们将在存在可逆抑制剂的情况下筛选共价碎片以选择耐受的弹头存在可逆的抑制剂支架，并考虑了由活性位点构象变化引起的可逆抑制剂。 3）我们将获得并分析晶体学，细胞和药代动力学数据改善我们的抑制剂，优先考虑效力，选择性和安全性，以最大程度地提高成功的机会在体内研究中。 4）最后，通过在FDA黄金标准鼠标杀伤力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效率及其对临床前和临床研究的适用性。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Transformation of a Metal Chelate into a "Catch and Anchor" Inhibitor of Botulinum A Protease.

DOI：
10.3390/ijms24054303
发表时间：
2023-02-21
期刊：
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
影响因子：
5.6
作者：
Lin, Lucy;Patel, Ealin N.;Nielsen, Alexander L.;Turner, Lewis D.;Tepp, William H.;Nguyen, Kong;Pellett, Sabine;Janda, Kim
通讯作者：
Janda, Kim

COVALENT INHIBITION OF BOTULINUM NEUROTOXIN A - EXPLORATION OF WARHEAD REACTIVITY AND FUNCTION USING A BIFUNCTIONAL APPROACH.

肉毒杆菌神经毒素 A 的共价抑制 - 使用双功能方法探索弹头反应性和功能。

DOI：
发表时间：
2021
期刊：
Toxicon : official journal of the International Society on Toxinology
影响因子：
0
作者：
Turner,LewisD;Nielsen,AlexanderL;Lin,Lucy;Pellett,Sabine;Sugane,Takashi;Olson,MargaretE;Janda,KimD
通讯作者：
Janda,KimD

Investigation of Salicylanilides as Botulinum Toxin Antagonists.

DOI：
10.1021/acsinfecdis.2c00230
发表时间：
2022-08-12
期刊：
ACS INFECTIOUS DISEASES
影响因子：
5.3
作者：
Patel, Ealin N.;Lin, Lucy;Sneller, Molly M.;Eubanks, Lisa M.;Tepp, William H.;Pellett, Sabine;Janda, Kim D.
通讯作者：
Janda, Kim D.