Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having α,β-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme’s catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

人鼻病毒（Human rhinoviruses）是引发普通感冒最重要的病原，属于具有信使活性的单链单顺反子RNA病毒，演化出高度复杂的蛋白水解级联反应以调控病毒基因表达与复制。前体多蛋白的多数成熟裂解步骤由鼻病毒3C蛋白酶（3C protease）及其直接前体3CD介导，该酶属于具有胰蛋白酶样多肽折叠的半胱氨酸蛋白酶。针对三种病毒血清型的该酶所解析的高分辨率晶体结构，已被用于设计并开发不同结构与化学类别的3C蛋白酶抑制剂。携带α,β-不饱和羰基基团与针对3C蛋白酶的特异性肽基结合元件的抑制剂，可通过酶的催化残基Cys-147的亲核加成发生迈克尔反应，最终形成共价键并使病毒蛋白酶发生不可逆失活。通过对放射性标记蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS/PAGE）分析，可观察到病毒前体多蛋白的积累呈剂量依赖性，由此可推断这类化合物可直接抑制病毒感染细胞内的3C蛋白水解活性。借助共晶体结构辅助优化靶向3C蛋白酶的迈克尔受体类抑制剂，已获得可在体外极快速灭活病毒蛋白酶、对多种鼻病毒血清型具有强效抗病毒活性且细胞毒性较低的化合物。近期，该系列中的化合物AG7088已进入临床试验阶段。