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首页> 外文期刊>Journal of mass spectrometry: JMS >Screening and rank ordering of reversible mechanism-based inhibitors of hepatitis C virus NS3 protease using electrospray ionization mass spectrometry
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Screening and rank ordering of reversible mechanism-based inhibitors of hepatitis C virus NS3 protease using electrospray ionization mass spectrometry

机译：基于电喷雾电离质谱的丙型肝炎病毒NS3蛋白酶可逆机制抑制剂的筛选和排序

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摘要

An affinity-selection study using size exclusion chromatography (SEC) combined with off-line electrospray ionization mass spectrometry (ESI-MS) was performed on libraries of peptidic α-ketoamide inhibitors directed against the hepatitis C virus (HCV) NS3 protease. A limiting amount of HCV NS3 protease (25 μM) was incubated with equimolar amounts (100 μM) of 49 reversible mechanism-based ketoamide inhibitors, previously grouped into seven sets to ensure clearly distinguishable mass differences of the enzyme-inhibitor complexes (>10 Da). The unbound compounds were separated rapidly from the protease and the protease-inhibitor complexes by SEC spin columns. The eluate of the SEC was immediately analyzed by direct-infusion ESI-MS. An enzyme-inhibitor complex, with a molecular mass corresponding to the NS3 protease binding to the preferred inhibitor, SCH212986, was the only molecular species detected. By increasing the molar ratio of HCV NS3 protease to inhibitors to 1:2 while keeping the inhibitors' concentration constant, the complex of the second most tightly bound inhibitor, SCH215426, was also identified. Although the potencies of these inhibitors were virtually un-measurable by kinetic assays, a rank order of CVS4441 > SCH212986 > SCH215426 was deduced for their inhibition potencies by direct competition experiment with CVS4441 (K_i~*> 80 μ M). As discussed in the article, through judicious application of this strategy, even large libraries of fairly weak, reversible and slow-binding inhibitors could be rapidly screened and rank ordered to provide critical initial structure-activity insights.
机译：在针对丙型肝炎病毒（HCV）NS3蛋白酶的肽α-酮酰胺抑制剂库中进行了使用尺寸排阻色谱（SEC）结合离线电喷雾电离质谱（ESI-MS）的亲和力选择研究。将有限量的HCV NS3蛋白酶（25μM）与等摩尔量（100μM）的49种可逆的基于机理的酮酰胺抑制剂一起孵育，先前分为7组以确保酶抑制剂复合物（> 10 Da ）。通过SEC旋转柱将未结合的化合物与蛋白酶和蛋白酶抑制剂复合物快速分离。立即通过直接输注ESI-MS分析SEC洗脱液。一种酶抑制剂复合物是唯一检测到的分子种类，其分子质量对应于与优选抑制剂SCH212986结合的NS3蛋白酶。通过将HCV NS3蛋白酶与抑制剂的摩尔比提高至1：2，同时保持抑制剂的浓度恒定，还鉴定出第二紧密结合的抑制剂SCH215426的复合物。尽管这些抑制剂的效力实际上无法通过动力学测定法测量，但通过与CVS4441的直接竞争实验（K_i〜*> 80μM）推导了CVS4441> SCH212986> SCH215426的排名。如本文所述，通过明智地应用该策略，甚至可以迅速筛选出相当弱，可逆和缓慢结合的抑制剂的大型文库，并对其进行排序，以提供关键的初始结构活性见解。

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