High Throughput Screens for Botulinum Toxin Therapeutics

描述（由申请人提供）：肉毒杆菌神经毒素（BoNT）是已知最有效的毒素之一，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，迫切需要这种药物的治疗方法。我们建议开发并行高通量筛选（HTS）方法来发现BoNT药物，基于这种蛋白酶对天然靶点的切割，这种策略可以推广到其他a类生物威胁，如炭疽致死因子。我们的方法利用体外HTS来快速选择候选药物，以及使用报告毒素活性的培养细胞进行基于细胞的伴随筛选。重要的是，两种基于细胞的系统可以识别出阻断BoNT中毒周期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性的化合物。体外HTS是基于针对不同BoNT轻链（LC）蛋白酶血清型（如SNAP25和VAMP）的生物靶点的分子工程。这些底物经过修饰后产生具有报告结构域的融合蛋白。（一种酶或荧光蛋白）在一端和固定结构域(例如；(6)他)在另一边。BoNT的裂解释放报告蛋白活性，

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.