Targeting Cellular Processes to Counter the Effects of BoNT Intoxication

描述（由申请人提供）：神经元功能的恢复对于克服肉毒杆菌神经毒素（BoNT）介导的瘫痪至关重要。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/功能的新型小分子，以减轻BoNT中毒，无论血清型如何，通过抑制毒素酶活性以外的方式。 BoNTs是最有效的生物毒素，可以通过食物“掺入”和/或气溶胶途径递送。目前，一旦BoNT介导的膈肌麻痹表现出来，长期机械通气是唯一的维持生命的选择。因此，有一个重要的空白需要填补：发现和开发治疗方法，将对抗暴露后的BoNT和/或加速神经元的恢复。几乎所有的药物开发工作都只集中在抑制这些毒素的锌金属蛋白酶轻链（LC）组分上; BoNT/LC选择性地切割参与神经递质释放的不同SNARE复合物亚基。七种生物化学上不同的BoNT血清型（A-G）引起肉毒中毒，使得开发所有血清型的单一、非混杂抑制剂的前景不太可能。我们认为靶向BoNT/LCs必须伴随着其他努力来增强神经元的恢复。因此，扩大可行靶标的范围以包括涉及中毒或恢复的细胞过程可以提供对抗多种血清型的新方法。然而，这种策略受到缺乏高通量的基于细胞的测定BoNT活性的阻碍。我们的小组开发了一个综合系统，包括来自胚胎干细胞的人类或小鼠运动神经元，以及使用BoNT切割敏感抗体的成像分析。因此，第一次，一个系统可以识别除了毒素本身之外还可以影响神经通路的小分子。我们建议利用我们独特的能力来鉴定具有独特作用机制的抗BoNT/A、/B和/E中毒的抗肿瘤活性小分子。鉴定后，将通过化学合成和生物学评价对先导化合物进行表征和优化。将在中毒动物模型中评价优化的化合物，以确定作为治疗对策的先进开发的可行候选物。为了指导我们的研究，我们提供了以下假设：有可能通过小分子对抗多种BoNT的作用，这些小分子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。该提案的主要目标是鉴定对中毒后BoNT A、B和E有效的化合物，以作为高级开发研究的化学先导。第二个目标是使用已鉴定的先导化合物作为化学探针，用于解剖这些毒素所需的神经元信号通路。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选，以确定BoNT/A，/B和/E抑制剂，并优化使用基于药效团的方法结合药物化学，有机合成，体外和体内测试，分子建模的铅分子。 公共卫生相关性：肉毒神经毒素是肉毒中毒的病原体。这些毒素普遍用于美容和治疗目的，沿着其潜在的破坏性恶意滥用，对公共健康构成了真实的威胁。这项研究计划开发了一种新的方案，用于开发对抗这些毒素的治疗方法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Botulinum neurotoxins are the causative agents of the disease botulism. The ubiquitous use of these toxins for cosmetic and therapeutic purposes, along with their potential devastating malicious misuse presents a real threat to public health. This research plan develops a novel scheme for developing therapeutics to counteract these toxins.