Targeting Cellular Processes to Counter the Effects of BoNT Intoxication

靶向细胞过程来对抗 BoNT 中毒的影响

• 批准号: 8841460
• 负责人: Sina Bavari
• 金额: $ 37.77万
• 依托单位: GENEVA FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8841460
• 关键词: Address; Advanced Development; Aerosols; Animal Model; Antibodies; Biological; Biological Assay; Biological Testing; Bontoxilysin; Botulinum Toxin Type A; Botulism; Cell physiology; Cells; Chemicals; Cleaved cell; Combined Modality Therapy; Complex; Cosmetics; Data; Disease; Evaluation; Food; Goals; Human; Image; In Vitro; Intoxication; Lead; Life; Light; Limb structure; Measures; Mechanical ventilation; Mediating; Metalloproteases; Methods; Modeling; Molecular Models; Molecular Target; Motor Neurons; Mus; Muscle; Neurons; Organic Synthesis; Paralysed; Pathway interactions; Pharmaceutical Chemistry; Poisoning; Process; Proteolysis; Public Health; Recovery; Research; Respiratory Diaphragm; Route; SNAP receptor; Scheme; Serotyping; Signal Pathway; Site; System; Testing; Therapeutic; Time; Toxin; base; cellular imaging; chemical synthesis; design; drug development; embryonic stem cell; in vivo; inhibitor/antagonist; knowledge base; molecular modeling; neurotransmitter release; novel; pharmacophore; screening; small molecule; synaptosomal-associated protein 25; therapeutic development

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.

项目概要/摘要 神经元功能的恢复对于克服肉毒神经毒素（BoNT）介导的麻痹至关重要。 事实证明，促进这种复苏的策略几乎是棘手的。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，无论 通过抑制毒素酶活性以外的方式来确定血清型。 BoNT 是最有效的生物毒素，可通过食物“掺加”和/或气雾剂传递 路线。目前，一旦 BoNT 介导的呼吸困难，长期机械通气是唯一维持生命的选择。 出现膈肌麻痹。因此，存在一个需要填补的重大空白： 发现和开发能够对抗暴露后的 BoNT 和/或加速神经元损伤的疗法 恢复。几乎所有药物开发工作都集中于抑制锌金属蛋白酶轻链 (LC) 这些毒素的成分； BoNT/LC 选择性裂解参与的不同 SNARE 复合体亚基 神经递质释放。七种生化上不同的 BoNT 血清型 (A-G) 会导致肉毒杆菌中毒，从而使 开发一种针对所有血清型的单一、非混杂的抑制剂的前景不太可能。我们相信瞄准 BoNT/LC 必须与其他增强神经元恢复的努力同时进行。因此，扩大 可行目标的范围，包括参与中毒或恢复的细胞过程，可能会提供新的 对抗多种血清型的方法。然而，这一战略因缺乏高水平的人才而受到阻碍。 测量 BoNT 活性的基于细胞的通量测定。我们的团队开发了一套全面的系统 由源自胚胎干细胞的人类或小鼠运动神经元组成，并使用成像分析 BoNT 裂解敏感抗体。因此，第一次有一个系统可以识别小分子， 除了毒素本身之外，还可以影响神经元通路。我们建议利用我们独特的优势 能够识别具有独特作用机制的药理活性小分子以对抗 BoNT/A、/B 和/E 中毒。鉴定后，先导化合物将通过以下方式进行表征和优化 多轮化学合成和生物学评价。优化的化合物将在动物模型中进行评估 中毒的识别以确定可行的候选者进行高级开发作为治疗对策。指导 在我们的研究中，我们提出以下假设： 通过小量的肉毒杆菌毒素治疗，可以抵消多种 BoNT 的影响。 通过阻断毒素酶位点以外的方式作用于神经细胞功能的分子。 该提案的主要目标是确定对 BoNT A、B 和 E 后有效的化合物。 中毒作为高级开发研究的化学先导物。第二个目标是使用 确定了先导化合物作为化学探针，用于剖析所需的神经元信号传导途径 被这些毒素。为了实现这些目标，我们将进行首次大规模基于细胞的筛选 使用基于药效团的方法识别 BoNT/A、/B 和 /E 抑制剂并优化先导分子 结合药物化学、有机合成、体外和体内测试以及分子建模。