Targeting Cellular Processes to Counter the Effects of BoNT Intoxication

项目摘要/摘要 神经功能的恢复是克服肉毒杆菌神经毒素(BoNT)介导的瘫痪的关键。 事实证明，促进这种复苏的战略几乎难以解决。这项拟议的研究试图确定 直接作用于神经元过程/功能以减轻BONT中毒的新型小分子，无论如何 通过抑制毒素的酶活性以外的方法，对血清型的影响。 BoNTs是最有效的生物毒素，可通过食物“尖峰”和/或气雾剂传递 路线。目前，长期机械通气是唯一的维持生命的选择一旦BONT中介 表现为横隔肌麻痹。因此，有一个重要的空白需要填补： 发现和开发将对抗暴露后的BoNTs和/或加速神经元的治疗方法 恢复。几乎所有的药物开发工作都集中在抑制锌金属蛋白酶轻链(LC)上。 这些毒素的成分；BONT/LCS选择性地切割参与其中的不同的SNAR复合亚单位 神经递质释放。七种生化上截然不同的BONT血清型(A-G)会导致肉毒杆菌中毒，使 开发一种单一的、非混杂的所有血清型抑制物的前景不太可能。我们相信目标是 BONT/LCS必须伴随其他促进神经元恢复的努力。因此，扩大了 包括参与中毒或恢复的细胞过程的可行靶点的范围可能提供新的 对抗多种血清型的方法。然而，这一战略因缺乏高质量的 测量BONT活性的基于细胞的吞吐量分析。我们小组开发了一套全面的系统 由来自胚胎干细胞的人类或小鼠运动神经元组成，并使用 BONT裂解敏感抗体。因此，第一次有了一个系统来识别小分子 除了毒素本身外，还可以影响神经元通路。我们建议利用我们独特的 识别具有独特作用机制的药理活性小分子的能力 BOT/A、/B和/E中毒。在鉴定之后，将通过以下方式来表征和优化先导化合物 进行了几轮化学合成和生物评价。优化的化合物将在动物模型中进行评估 以确定可作为治疗对策的晚期发展的可行候选者。去指导 在我们的研究中，我们提出了以下假设：多个BoNTs的影响是可能的 通过阻断毒素的酶部位以外的方式作用于神经细胞功能的分子。 这项建议的主要目标是确定对BNTs A、B和E有效的化合物。 醉酒作为高级发展研究的化学先导。第二个目标是使用 将先导化合物确定为解剖所需神经元信号通路的化学探针 由这些毒素所致。为了实现这些目标，我们将进行有史以来第一次大规模的基于细胞的筛查 使用基于药效团的方法确定BONT/A、/B和/E抑制剂并优化铅分子 集药物化学、有机合成、体外和体内试验、分子建模于一体。

PROJECT SUMMARY/ABSTRACT Recovery of neuronal function is critical for overcoming botulinum neurotoxin (BoNT)-mediated paralysis. Strategies for promoting such recovery have proven nearly intractable. The proposed research seeks to identify novel small molecules that act directly on neuronal processes/functions to mitigate BoNT intoxication, regardless of the serotype, by means other than inhibition of the toxins' enzymatic activities. BoNTs are the most potent of the biological toxins and may be delivered via food "spiking" and/or aerosol route. Currently, long-term mechanical ventilation is the only life-sustaining option once the BoNT-mediated paralysis of diaphragm muscles manifests. Consequently, there is a significant void that needs to be filled: the discovery and development of therapeutics that will counter BoNTs post-exposure and/or expedite neuronal recovery. Nearly all drug development efforts focus solely on inhibiting the Zn metalloprotease light chain (LC) components of these toxins; BoNT/LCs selectively cleave different SNARE complex subunits involved in neurotransmitter release. Seven biochemically distinct BoNT serotypes (A-G) cause botulism, making the prospect of developing a single, non-promiscuous inhibitor of all serotypes unlikely. We believe targeting BoNT/LCs must be accompanied by other endeavors to enhance neuronal recovery. Hence, broadening the scope of viable targets to include cellular processes involved in either intoxication or recovery may provide novel methods for countering multiple serotypes. However, this strategy has been hampered by a lack of high- throughput cell-based assays that measure BoNT activity. Our group developed a comprehensive system consisting of human or mouse motor neurons derived from embryonic stem cells, and imaging assays using BoNT cleavage-sensitive antibodies. Thus, for the first time, a system is available to identify small molecules that can impinge on neuronal pathways in addition to the toxin itself. We propose taking advantage of our unique capabilities to identify pharmacologically active small molecules with unique mechanisms of action for countering BoNT/A, /B and /E poisoning. Following identification, lead compounds will be characterized and optimized via rounds of chemical synthesis and biological evaluation. Optimized compounds will be evaluated in animal models of intoxication to identify viable candidates for advanced development as therapeutic countermeasures. To guide our studies, we offer the following hypothesis: It is possible to counter the effects of multiple BoNTs by small molecules which act on neuronal cell functions by a means other than blockade of the enzymatic site of the toxin. The main goal of this proposal is to identify compounds that are effective against BoNTs A, B and E post- intoxication to serve as chemical leads for advanced development studies. A second goal is to use the identified lead compounds as chemical probes for dissecting the neuronal signaling pathways that are required by these toxins. To accomplish these goals, we will conduct the first ever large-scale cell-based screening to identify BoNT/A, /B, and /E inhibitors and optimize lead molecules using pharmacophore-based approaches incorporating medicinal chemistry, organic synthesis, in vitro and in vivo testing, and molecular modeling.