High Throughput Screens for Botulinum Toxin Therapeutics

描述（由申请人提供）：肉毒神经毒素（BoNT）是已知的最有效的毒素之一，并且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，迫切需要这种药剂的治疗方法。我们建议开发并行的高通量筛选（HTS）检测BoNT药物发现，基于这种蛋白酶的天然靶标的切割，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，沿着使用报告毒素活性的培养细胞进行基于伴随细胞的筛选。重要的是，基于细胞的系统的两（2）个版本可以鉴定中断BoNT中毒周期的不同阶段的治疗剂，同时消除通常具有细胞毒性或细胞不可渗透的化合物。体外HTS基于不同BoNT轻链（LC）蛋白酶血清型（如SNAP 25和VAMP）的生物靶标的分子工程。修饰这些底物以产生具有报告结构域的融合蛋白（例如，酶或荧光蛋白）和固定结构域（例如，6 x他的）另一方面。BoNT的切割释放了报道活性， 然后将其与通过固定结构域结合的未切割靶分离。对于一（1）种类型的基于细胞的HTS，表达上述底物-报告蛋白的稳定BoNT敏感性细胞系用LC中毒。使用几种方法（LC的蛋白转导、病毒载体递送、用诱导型载体转染）中的一种（1），之后将细胞 裂解并测量报告子。为了筛选体内活性，我们提出了两（2）种策略， 这两种方法都允许将全毒素添加到经工程改造以报告BoNT活性的稳定细胞系中。在 一种（1）与转录因子结构域融合的底物蛋白在被BoNT切割时激活合适的报道酶的转录。另一方面，在将细胞暴露于BoNT LC后，测量双标记底物-报告物的生物发光共振能量转移（BRET）的实时变化。

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.