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Using Allosteric Inhibition as a Means to Ablate Botulinum Neurotoxin Protease

使用变构抑制作为消除肉毒杆菌神经毒素蛋白酶的方法

基本信息

批准号：
9197258
负责人：
Kim Janda
金额：
$ 24.06万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-17 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9197258
关键词：
Acids Active Sites Adverse effects Anaerobic Bacteria Angiotensin-Converting Enzyme Inhibitors Antitoxins Bacteria Binding Biological Assay Bioterrorism Bontoxilysin Botox Botulinum Toxin Type A Botulism Caring Catalytic Domain Cell surface Cells Centers for Disease Control and Prevention (U.S.)Chelating Agents Chemistry Cichorium intybus Cleaved cell Clinical Clostridium botulinum Complex Controlled Environment Cosmetics Data Development Dimensions Disease Endopeptidases Enzymes Event Exocytosis Exposure to Food Contamination GTP-Binding Protein alpha Subunits, Gs Gangliosides Glycoproteins Gram-Positive Bacteria Grant HIV Protease Hand Heart Human Immune response Induction of neuromuscular blockade Intervention Intoxication Kinetics Knowledge Life Ligands Light Liquid substance Matrix Metalloproteinase Inhibitor Matrix Metalloproteinases Mechanical ventilation Medical Membrane Metalloproteases Muscle Mutation Natural Products Nature Neurons Neurotransmitters Overdose Paralysed Pathogenicity Patients Peptide Hydrolases Peripheral Pharmaceutical Preparations Poison Poisoning Procedures Property Protease Inhibitor Proteins Regulation Reporting Research Respiratory Diaphragm Route SNAP receptor Savings Series Serotyping Site Solid Specificity Structure Structure-Activity Relationship Substrate Specificity Synaptic Vesicles Testing Therapeutic Toxic effect Toxin Vesicle Virulence Factors Zinc analog base biothreat botulinum chemical synthesis combat cost design disorder prevention experimental study fighting healing high risk improved in vivo induced pluripotent stem cell inhibitor/antagonist man member presynaptic prevent public health relevance receptor scaffold small molecule inhibitor standard care success theories weapons

项目摘要

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs), from which there are seven serotypes are Gram-positive bacteria. The clinical signature of BoNTs is peripheral neuromuscular blockade and flaccid paralysis, which depending on the serotype can last for months. BoNTs are the most toxic proteins known to man and have been classified by the Centers for Disease Control and Prevention (CDC) as one of the six highest-risk biothreat agents. Despite their toxicity and high potential as a bioterrorist weapon, BoNTs are widely used in medical and cosmetic procedures (i.e., Botox). However, even under a controlled environment unwanted side effects have been reported causing complications for patients and in some cases severe life- threatening disorders. Currently, a botulinum heptavalent antitoxin (BAT) is the only medical intervention for BoNT poisoning and this has limited value since it can only neutralize circulating toxin, being useless once cellular poisoning takes place. At a mechanistic level botulinum intoxication proceeds through a series of three steps to produce its neuroparalytic effects: neuronal membrane binding, internalization, and intracellular poisoning. Our objective is to target the intracellular poisoning stage, which result when the toxins' zinc metalloprotease cleaves neuronal cell SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Proteolytic damage to the SNARE proteins prevents fusion of synaptic vesicles with the presynaptic membrane blocking the exocytosis of essential neurotransmitters. Botulinum neurotoxin proteases are unique among endopeptidases in that they require long substrates (SNAREs) for catalytic efficiency due to their recognition strategy. Further complicating their inhibition are allosteric patches, termed exosites that both determine specificity and also remodel the catalytic cleft upon substrate binding. The challenges in designing small molecule inhibitors to the BoNT proteases are recognized through the historic efforts against matrix metalloproteinases (MMPs) where the potential pitfalls of targeting the active site of Zn+2 proteases are well documented. We plan to develop selective molecules against BoNT serotype A (BoNT/A), the most toxic of the BoNT proteases, by targeting the enzyme's exosite. By using this approach "metalloactive" site redundancy as seen amongst this enzyme class will be avoided as will the use of compounds that chelate zinc. Our proposed research will be centered upon our discovery of the first non-peptide/protein exosite inhibitor, chicoric acid. Our chemistry efforts will be directed toward improving potency while building in drug-like properties. This effort will be assisted by NMR structure guided studies to delineate the BoNT/A exosite structural interactions essential for chicoric acid analogue binding. We anticipate that such studies will identify the key interactions required for exosite BoNT/A light chain protease (BoNT/A LC) inhibition, which in turn will allow additional structures to be prepared and tested in a more rational approach.

描述(申请人提供)：肉毒杆菌神经毒素(BoNTs)，其中有7个血清型是革兰氏阳性菌。BNTs的临床特征是周围神经肌肉阻滞和软性瘫痪，根据血清型的不同，这种症状可以持续几个月。BoNTs是人类已知的毒性最强的蛋白质，已被疾病控制和预防中心(CDC)列为六种风险最高的生物制剂之一。尽管BoNTs具有毒性和作为生物恐怖分子武器的高潜力，但它被广泛用于医疗和美容手术(即肉毒杆菌毒素)。然而，据报道，即使在受控的环境下，不受欢迎的副作用也会给患者造成并发症，在某些情况下还会导致严重的危及生命的疾病。目前，肉毒杆菌七价抗毒素(BAT)是治疗BONT中毒的唯一药物干预措施，其价值有限，因为它只能中和循环毒素，一旦发生细胞中毒就毫无用处。在机械学水平上，肉毒杆菌中毒通过一系列三个步骤产生其神经麻痹效应：神经细胞膜结合、内化和细胞内中毒。我们的目标是针对细胞内中毒阶段，当毒素的锌金属蛋白酶裂解神经细胞SNARE(可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体)蛋白时。SNARE蛋白的蛋白水解性损伤阻止了突触小泡与突触前膜的融合，阻止了必要神经递质的胞吐。肉毒杆菌神经毒素蛋白水解酶在内肽酶中是独一无二的，因为它们的识别策略需要很长的底物(陷阱)来实现催化效率。使它们的抑制进一步复杂化的是变构斑块，称为外位体，它既决定了特异性，也根据底物结合重塑了催化裂解。通过针对基质金属蛋白酶(MMPs)的历史性努力，人们认识到了设计针对BONT蛋白酶的小分子抑制剂的挑战，其中针对锌+2蛋白酶活性部位的潜在陷阱得到了很好的证明。我们计划开发针对BoNT血清型A(BONT/A)的选择性分子，BONT是毒性最大的BONT蛋白酶，通过靶向该酶的外切点。通过使用这种方法，将避免在这类酶中看到的“金属活性”位点冗余，也将避免使用螯合锌的化合物。我们计划的研究将集中在我们发现的第一种非肽/蛋白质外排抑制物--乳糜酸。我们的化学努力将针对提高效力，同时建立类似药物的性质。这项工作将得到核磁共振结构指导研究的帮助，以描绘出 BoNT/A外排体结构相互作用对于菊苣酸类似物结合是必不可少的。我们预计，这些研究将确定抑制外源BONT/A轻链蛋白酶(BONT/A LC)所需的关键相互作用，这反过来将允许以更合理的方法制备和测试更多的结构。