Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin A inhibitors
Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin A inhibitors
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DOI：
10.1021/cc060010h
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发表时间：
2006-07-10
影响因子：
--
通讯作者：
Janda, Kim D.
中科院分区：
文献类型：
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作者：
Boldt, Grant E.;Kennedy, Jack P.;Janda, Kim D.
肉毒神经毒素是目前已知的毒性最强的蛋白质，是引起食物中毒的病原体。虽然在美国每年只有几百例肉毒杆菌中毒的报道，但由于其作为生物战剂的潜在用途，人们对肉毒杆菌毒素的兴趣越来越大。神经毒性由BoNT轻链亚基（一种Zn内肽酶）对突触前囊泡的可溶性NSF-附着蛋白受体复合物蛋白的裂解引起。已知BoNT/A LC（轻链）活性的有效抑制剂很少，并且发现过程由于缺乏用于筛选化合物文库的有效高通量测定而受到阻碍。为了缓解这一瓶颈，我们合成了肽SNAPtide，并开发了一种用于BoNT/A LC抑制剂高通量评价的稳健测定法。开发这种优化测定的关键方面包括添加一系列去污剂、共溶剂和盐，包括0.01% w/v吐温20，以增加BoNT/A LC催化、稳定性和小分子筛选的容易性。为了评价测定的有效性，合成了一系列基于异羟肟酸盐的小分子，并用BoNT/A LC进行检查。所描述的方法是上级到目前为止报道的BoNT/A抑制剂的高通量鉴定的其他测定。
Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. Although only a few hundred cases of botulism are reported in the United States annually, there is growing interest in BoNTs attributable to their potential use as biological warfare agents. Neurotoxicity results from cleavage of the soluble NSF-attachment protein receptor complex proteins of the presynaptic vesicles by the BoNT light chain subunit, a Zn endopeptidase. Few effective inhibitors of BoNT/A LC (light chain) activity are known, and the discovery process is hampered by the lack of an efficient high-throughput assay for screening compound libraries. To alleviate this bottleneck, we have synthesized the peptide SNAPtide and have developed a robust assay for the high-throughput evaluation of BoNT/A LC inhibitors. Key aspects for the development of this optimized assay include the addition of a series of detergents, cosolvents, and salts, including 0.01% w/v Tween 20 to increase BoNT/A LC catalysis, stability, and ease of small molecule screening. To evaluate the effectiveness of the assay, a series of hydroxamate-based small molecules were synthesized and examined with BoNT/A LC. The methodology described is superior to other assays reported to date for the high-throughput identification of BoNT/A inhibitors.