A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
复制标题
DOI：
10.3791/50908
复制
发表时间：
2013-12-01
影响因子：
1.2
通讯作者：
Dickerson, Tobin J.
中科院分区：
文献类型：
--
作者：
Caglic, Dejan;Bompiani, Kristin M.;Dickerson, Tobin J.
肉毒杆菌神经毒素(BoNT)是一种强大的、具有潜在致命性的细菌毒素，它与宿主运动神经元结合，内化到细胞内，裂解细胞内对神经递质释放至关重要的蛋白质。BoNT由一个重链(HC)和一个轻链(LC)组成，重链(HC)负责调节宿主细胞的结合和内化，轻链(LC)负责裂解细胞内的乙酰胆碱释放所必需的宿主蛋白。虽然抑制毒素结合/内化的疗法给药时间窗口很小，但针对细胞内LC活性的化合物给药时间窗口要长得多，特别是考虑到毒素的半衰期非常长。近年来，小分子作为潜在的LC抑制剂被大量分析，因为它们相对于大的治疗药物(多肽、适配子等)具有更高的细胞通透性。铅的识别通常涉及高通量筛选(HTS)，即根据小分子调节治疗靶功能的能力来筛选大的小分子文库。在这里，我们描述了一种基于FRET的分析方法，它使用商业BONT/A LC底物和重组LC，可以自动检测潜在BONT抑制剂的HTS。此外，我们描述了一种手动技术，可用于后续二次筛选，或用于比较几个候选化合物的效力。
Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.