High Throughput Screens for Botulinum Toxin Therapeutics

肉毒毒素治疗的高通量筛选

• 批准号: 7020809
• 负责人: George A. Oyler
• 金额: $ 46.27万
• 依托单位: VERITAS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2007-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020809
• 关键词: bioluminescence; bioterrorism /chemical warfare; botulinum toxins; cytotoxicity; drug discovery /isolation; fluorescence resonance energy transfer; high throughput technology; immunocytochemistry; neurotoxicology; neurotoxins; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): Botulinum neurotoxin (BoNT) is among the most potent toxins known and no therapy exists to terminate its action after intoxication. Because of the risk for BoNT use by bioterrorists, therapeutics for this agent are urgently needed. We propose developing parallel high-throughput screening (HTS) assays for BoNT drug discovery, based on cleavage of natural targets by this protease, a strategy that can be generalized to other class A biothreats such as anthrax lethal factor. Our approach utilizes in vitro HTS to allow rapid selection of drug candidates, along with a companion cell-based screens using cultured cells that report toxin activity. Importantly, two (2) versions of the cell-based system may identify therapeutics that interrupt different stages of the BoNT intoxication cycle while eliminating compounds that are generally cytotoxic, or cell-impermeable. The in vitro HTS is based on molecular engineering of the biological targets for different BoNT light chain (LC) protease serotypes, such as SNAP25 and VAMP. These substrates are modified to create fusion proteins with a reporter domain (eg., an enzyme or flourescent protein) on one (1) end and an immobilization domain (eg., 6x His) on the other. Cleavage by BoNT releases the reporter activity, which is then separated from non-cleaved target bound via the immobilization domain. For one (1) type of cell-based HTS stable BoNT sensitive cell lines which express the substrate-reporter proteins described above are intoxicated with LC. using one (1) of several methods (protein transduction of LC, viral vector delivery, transfection with inducible vector), after which cells are lysed and reporter is measured. For screening of activity in vivo, we propose two (2) strategies, both of which allow addition of holotoxin to a stable cell line engineered to report BoNT activity. In one (1), substrate protein, fused to a transcription factor domain, activates transcription of a suitable reporter enzyme upon cleavage by BoNT. In the other, real-time changes in bioluminescence resonance energy transfer (BRET) of a dual label substrate - reporter is measured after exposing cells to BoNT LC.

描述(申请人提供)：肉毒杆菌神经毒素(BONT)是已知的最有效的毒素之一，目前还没有治疗方法可以在中毒后终止其作用。由于生物恐怖分子使用BONT的风险，迫切需要针对这种制剂的治疗方法。我们建议开发并行高通量筛选(HTS)方法用于BONT药物发现，基于这种酶对天然靶点的切割，这一策略可以推广到其他A类生物制剂，如炭疽致命因子。我们的方法利用体外HTS来快速选择候选药物，同时使用报告毒素活性的培养细胞进行基于细胞的筛选。重要的是，基于细胞的系统的两(2)个版本可以识别中断BONT中毒循环的不同阶段的治疗方法，同时消除通常具有细胞毒性或细胞不渗透的化合物。体外HTS是基于SNAP25和VAMP等不同BONT轻链(LC)酶血清型的生物靶标的分子工程。这些底物被修饰以产生融合蛋白，在一端具有报告结构域(例如，酶或荧光蛋白)，在另一端具有固定化结构域(例如，6x His)。被BONT割裂释放记者活动， 然后将其与通过固定化结构域结合的未切割靶标分离。对于一(1)种基于细胞的HTS，表达上述底物报告蛋白的稳定的BONT敏感细胞系被LC陶醉。使用几种方法中的一种(LC蛋白转导、病毒载体传递、可诱导载体转染)，之后细胞 解说和记者都是有分寸的。对于体内活性的筛选，我们提出了两(2)种策略， 这两种方法都允许向稳定的细胞系中添加全毒素，该细胞系被设计成报告BONT活性。在……里面 一(1)，底物蛋白，融合到转录因子结构域，在被BONT切割时激活合适的报告酶的转录。另一方面，测量了细胞暴露于BoNT LC后，双标记底物-报告器的生物发光共振能量转移(BRET)的实时变化。