Targeting Cellular Processes to Counter the Effects of BoNT Intoxication

项目总结/摘要 神经元功能的恢复对于克服肉毒杆菌神经毒素（BoNT）介导的麻痹至关重要。 事实证明，促进这种复苏的战略几乎难以实施。拟议的研究旨在确定 直接作用于神经元过程/功能以减轻BoNT中毒的新型小分子， 通过抑制毒素的酶活性以外的方式对血清型进行抑制。 BoNTs是最有效的生物毒素，可通过食物"掺入"和/或气溶胶递送 路线目前，长期机械通气是一旦BoNT介导的 出现横隔膜肌麻痹。因此，需要填补一个重大空白： 发现和开发对抗暴露后BoNT和/或促进神经元凋亡的治疗剂 复苏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。 BoNT/LC选择性切割参与这些毒素的不同SNARE复合物亚基， 神经递质释放七种生物化学上不同的BoNT血清型（A-G）引起肉毒中毒，使得 开发一种单一的、非混杂的所有血清型抑制剂的前景不太可能。我们认为目标 BoNT/LC必须伴随着其他努力，以提高神经元的恢复。因此，扩大 包括参与中毒或恢复的细胞过程的可行靶点范围可以提供新的 对抗多种血清型的方法。然而，这一战略受到缺乏高... 通过基于细胞的测定来测量BoNT活性。我们的团队开发了一个全面的系统 由源自胚胎干细胞的人或小鼠运动神经元组成，以及使用 BoNT切割敏感性抗体。因此，第一次，一个系统可用于识别小分子， 除了毒素本身还能影响神经通路我们建议利用我们独特的 识别具有独特作用机制的生物活性小分子的能力， BoNT/A、/B和/E中毒。鉴定后，将通过以下方法表征和优化先导化合物： 化学合成和生物学评估的回合。将在动物模型中评价优化的化合物 中毒，以确定可行的候选人先进的发展作为治疗对策。引导 在我们的研究中，我们提出了以下假设： 通过阻断毒素的酶位点以外的方式作用于神经元细胞功能的分子。 该提案的主要目标是鉴定有效对抗BoNT A、B和E后的化合物。 中毒，作为化学线索，为先进的发展研究。第二个目标是使用 确定了先导化合物作为化学探针，用于解剖神经元信号通路， 这些毒素。为了实现这些目标，我们将进行有史以来第一次大规模的细胞筛选， 使用基于药效团方法鉴定BoNT/A、/B和/E抑制剂并优化先导分子 包括药物化学、有机合成、体外和体内测试以及分子建模。

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.