Eubanks Lisa M, Hixon Mark S, Jin Wei, Hong Sukwon, Clancy Colin M, Tepp William H, Baldwin Michael R, Malizio Carl J, Goodnough Michael C, Barbieri Joseph T, Johnson Eric A, Boger Dale L, Dickerson Tobin J, Janda Kim D
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2602-7. doi: 10.1073/pnas.0611213104. Epub 2007 Feb 9.
Among the agents classified as "Category A" by the U.S. Centers for Disease Control and Prevention, botulinum neurotoxin (BoNT) is the most toxic protein known, with microgram quantities of the protein causing severe morbidity and mortality by oral or i.v. routes. Given that this toxin easily could be used in a potential bioterrorist attack, countermeasures urgently are needed to counteract the pathophysiology of BoNT. At a molecular level, BoNT exerts its paralytic effects through intracellular cleavage of vesicle docking proteins and subsequent organism-wide autonomic dysfunction. In an effort to identify small molecules that would disrupt the interaction between the light-chain metalloprotease of BoNT serotype A and its cognate substrate, a multifaceted screening effort was undertaken. Through the combination of in vitro screening against an optimized variant of the light chain involving kinetic analysis, cellular protection assays, and in vivo mouse toxicity assays, molecules that prevent BoNT/A-induced intracellular substrate cleavage and extend the time to death of animals challenged with lethal toxin doses were identified. Significantly, the two most efficacious compounds in vivo showed less effective activity in cellular assays intended to mimic BoNT exposure; indeed, one of these compounds was cytotoxic at concentrations three orders of magnitude below its effective dose in animals. These two lead compounds have surprisingly simple molecular structures and are readily amenable to optimization efforts for improvements in their biological activity. The findings validate the use of high-throughput screening protocols to define previously unrecognized chemical scaffolds for the development of therapeutic agents to treat BoNT exposure.
在美国疾病控制与预防中心归类为“A类”的病原体中,肉毒杆菌神经毒素(BoNT)是已知毒性最强的蛋白质,微克量的该蛋白质通过口服或静脉途径即可导致严重发病和死亡。鉴于这种毒素很容易被用于潜在的生物恐怖袭击,迫切需要采取对策来对抗BoNT的病理生理过程。在分子水平上,BoNT通过细胞内切割囊泡对接蛋白并随后引发全身自主神经功能障碍来发挥其麻痹作用。为了鉴定能够破坏A型肉毒杆菌神经毒素轻链金属蛋白酶与其同源底物之间相互作用的小分子,开展了多方面的筛选工作。通过针对轻链的优化变体进行体外筛选,结合动力学分析、细胞保护试验和体内小鼠毒性试验,鉴定出了能够防止BoNT/A诱导的细胞内底物切割并延长接受致死毒素剂量攻击的动物死亡时间的分子。值得注意的是,体内最有效的两种化合物在旨在模拟BoNT暴露的细胞试验中显示出较低的活性;事实上,其中一种化合物在浓度比其在动物体内有效剂量低三个数量级时就具有细胞毒性。这两种先导化合物具有惊人的简单分子结构,很容易进行优化以提高其生物活性。这些发现验证了使用高通量筛选方案来确定以前未被认识的化学支架,以开发治疗BoNT暴露的治疗药物。