Using Allosteric Inhibition as a Means to Ablate Botulinum Neurotoxin Protease

 描述（由申请方提供）：肉毒神经毒素（BoNT），其中有7种血清型是革兰氏阳性菌。BoNTs的临床特征是外周神经肌肉阻滞和弛缓性麻痹，根据血清型的不同，这种情况可能持续数月。BoNTs是人类已知的毒性最大的蛋白质，已被疾病控制和预防中心（CDC）列为六种风险最高的生物威胁因子之一。尽管其毒性和作为生物恐怖武器的高潜力，但BoNT广泛用于医疗和美容程序（即，Botox）。然而，即使在受控的环境下，也有报道称不希望的副作用会导致患者并发症，在某些情况下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BoNT中毒的唯一医疗干预措施，这具有有限的价值，因为它只能中和循环毒素，一旦发生细胞中毒就毫无用处。在机械水平上，肉毒杆菌中毒通过一系列的三个步骤产生其神经麻痹作用：神经元膜结合，内化和细胞内中毒。我们的目标是靶向细胞内中毒阶段，这是当毒素的锌金属蛋白酶切割神经元细胞SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）蛋白时产生的。对SNARE蛋白的蛋白水解损伤阻止突触囊泡与突触前膜的融合，从而阻断必需神经递质的胞吐作用。肉毒杆菌神经毒素蛋白酶在内肽酶中是独特的，因为它们由于其识别策略而需要长底物（SNARE）以获得催化效率。使它们的抑制进一步复杂化的是变构斑块，称为外位点，其既决定特异性又重塑底物结合后的催化裂缝。设计BoNT蛋白酶的小分子抑制剂的挑战通过针对基质金属蛋白酶（MMP）的历史性努力而被认识到，其中靶向Zn+2蛋白酶的活性位点的潜在陷阱被充分记录。我们计划开发针对BoNT血清型A（BoNT/A）的选择性分子，这是最具毒性的BoNT蛋白酶，通过靶向酶的外位点。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。我们提出的研究将集中在我们发现的第一个非肽/蛋白质外位点抑制剂，菊苣酸。我们的化学努力将致力于提高效力，同时建立药物样特性。这一努力将得到NMR结构指导研究的协助，以描绘 BoNT/A外位点结构相互作用对菊苣酸类似物结合至关重要。我们预计，这样的研究将确定所需的关键相互作用外切位点BoNT/A轻链蛋白酶（BoNT/A LC）抑制，这反过来将允许额外的结构，以更合理的方法制备和测试。

 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.