Pfizer Worldwide Research and Development, San Diego, CA 92121, USA.
Antiviral Res. 2011 Aug;91(2):102-11. doi: 10.1016/j.antiviral.2011.05.007. Epub 2011 May 19.
Several potent inhibitors of hepatitis C virus (HCV) NS3/4A protease have been identified that show great clinical potential against genotype 1. Due to the tremendous genetic diversity that exists among HCV isolates, development of broad spectrum inhibitors is challenging. With a limited number of lab strains available for preclinical testing, new tools are required for assessing protease inhibitor activity. We developed a chimeric replicon system for evaluating NS3 protease inhibitor activity against naturally occurring isolates. NS3/4A genes were cloned from the plasma of HCV-infected individuals and inserted into lab strain replicons, replacing the native sequences. The chimeric reporter replicons were transfected into Huh 7.5 cells, their replication monitored by luciferase assays, and their susceptibilities to inhibitors determined. Viable chimeras expressing heterologous genotypes 1, 2, 3, and 4 protease domains were identified that exhibited varying susceptibilities to inhibitors. Protease inhibitor spectrums observed against the chimeric replicon panel strongly correlated with published enzymatic and clinical results. This cell-based chimeric replicon system can be used to characterize the activities of protease inhibitors against diverse natural isolates and may improve the ability to predict dose and clinical efficacy.
已鉴定出几种有效的丙型肝炎病毒 (HCV) NS3/4A 蛋白酶抑制剂，它们对基因型 1 具有很大的临床潜力。由于 HCV 分离株之间存在巨大的遗传多样性，因此开发广谱抑制剂具有挑战性。由于临床前测试可用的实验室菌株数量有限，因此需要新的工具来评估蛋白酶抑制剂的活性。我们开发了一种用于评估 NS3 蛋白酶抑制剂对天然分离株活性的嵌合复制子系统。NS3/4A 基因从 HCV 感染个体的血浆中克隆，并插入实验室株复制子中，取代天然序列。将嵌合报告复制子转染入 Huh 7.5 细胞，通过荧光素酶测定法监测其复制，并确定其对抑制剂的敏感性。鉴定出表达异源基因型 1、2、3 和 4 蛋白酶结构域的可行嵌合体，它们对抑制剂的敏感性不同。针对嵌合复制子组观察到的蛋白酶抑制剂谱与已发表的酶学和临床结果强烈相关。这种基于细胞的嵌合复制子系统可用于表征针对多种天然分离株的蛋白酶抑制剂的活性，并可能提高预测剂量和临床疗效的能力。
