A Multidisciplinary Approach for the Treatment of Botulinum Intoxication

项目概要/摘要。肉毒杆菌神经毒素（BoNT）的临床特征是外周 神经肌肉阻滞和弛缓性麻痹，这取决于血清型（A-G），可持续数月。 BoNTs是人类已知的毒性最大的蛋白质，已被疾病控制中心分类 和预防作为六个最高风险的生物威胁剂之一。尽管作为生物恐怖武器的潜力很大， BoNT广泛用于医疗和美容程序（即，Botox）。然而，即使在一个受控制的 环境有害的副作用已经被报道，并且在某些情况下严重危及生命的疾病。 BoNT中毒的临床干预不仅因其极端毒性而且因其长半衰期而变得复杂 （BoNT/A的月数）。目前，肉毒杆菌七价抗毒素（BAT）是唯一被批准的药物。 然而，干预的价值有限，因为抗体只能中和循环毒素，而循环毒素一旦被中和， 就会发生细胞中毒没有药理学拮抗剂，一旦细胞 中毒发生，甚至没有进展到I期临床试验。禁止开发一个 我们已经表明，BoNT/A中毒可以使用协同治疗来减弱， 在一些实施方案中，本发明涉及抗体和药理学拮抗剂的组合。尽管这一发现对 BoNT领域，进一步掌握将是使用独特的药理学试剂，以减弱 BoNT/A的毒性。我们目前拥有几类小分子抑制剂，可以调解在三个 连接关键BoNT的病理：陷阱蛋白裂解，神经毒素内吞作用，并封锁 乙酰胆碱释放从这个武器库中，我们发现了分子产生，以阻止多个 与BoNT/A神经毒性相关的过程。令人印象深刻的是，这种双重通道机制策略提供了 第一个可以延长暴露后发生BoNT/A死亡时间的小分子。我们有 建立了钾通道阻滞剂如何在术后瘫痪的逆转中提供充分的支持， BoNT/A中毒。对于这项研究，我们提供分子3，4-二氨基吡啶（3，4-DAP）和3，4，5-二氨基吡啶（3，4-DAP）。 三氨基吡啶（3，4，5-TAP）;两者都对BoNT/A拯救非常有效。然而，氨基吡啶 其短的半衰期严重损害了有效性。我们将在我们的 建议的研究。最后，作为加强我们小分子药理学拮抗剂研究的一种手段， 我们将利用药物化学、X射线晶体学和药代动力学， 针对肉毒杆菌神经毒素的选择性和更有效的抑制剂。成功整合我们的 研究目标将把治疗肉毒杆菌毒性这一复杂且具有挑战性的问题推向临床 可行的治疗

Project Summary/Abstract. The clinical signature of botulinum neurotoxin (BoNT) is peripheral neuromuscular blockade and flaccid paralysis, which depending on the serotype (A-G) 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 as one of the six highest-risk biothreat agents. Despite 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 and in some cases severe life-threatening disorders. Clinical intervention of BoNT poisoning is complicated by not only its extreme toxicity but also its long half-life (months for BoNT/A). Currently a botulinum heptavalent antitoxin (BAT) is the only approved medical intervention, yet, has limited value since antibodies can only neutralize circulating toxin, which is negated once cellular poisoning takes place. There are no pharmacologic antagonists available that act once cellular intoxication takes place and none that even advanced to Phase I Clinical Trials. Prohibitive as developing a therapeutic might seem we have shown that BoNT/A intoxication can be attenuated using a synergistic combination of an antibody and a pharmacological antagonist. As significant as this finding has been to the BoNT field, a further mastery would be the use of singular pharmacological agents fashioned to attenuate BoNT/A's toxicity. We currently possess several classes of small molecule inhibitors that can intercede at three junctions critical to BoNT's pathology: SNARE protein cleavage, neurotoxin endocytosis, and blockade of acetylcholine release. From this arsenal we have discovered molecules engendered to block multiple processes associated with BoNT/A neurotoxicity. Impressively, this dual inhibitor-mechanism strategy provides the first small molecule that can extend time to death from a BoNT/A post exposure occurrence. We have established how potassium channel blockade can provide full sustenance in the reversal of paralysis for post- intoxication of BoNT/A. For this research we offer the molecules 3,4-diaminopyridine (3,4-DAP) and 3,4,5- triaminopyridine (3,4,5-TAP); both are highly effective for BoNT/A rescue. However, aminopyridine's effectiveness is severely compromised by their short half-life. We will address this limitation within our proposed studies. Finally, as a means to augment our small molecule pharmacological antagonist research initiative we will use medicinal chemistry, X-ray crystallography and pharmacokinetics to develop both greater selectivity and more potent inhibitors against the botulinum neurotoxins. The successful integration of our research goals will bring the complex and challenging problem of treating botulinum toxicity toward a clinically viable treatment.