Sudo K, Inoue H, Shimizu Y, Yamaji K, Konno K, Shigeta S, Kaneko T, Yokota T, Shimotohno K
Rational Drug Design Laboratories, Fukushima 960-12, Japan.
Antiviral Res. 1996 Aug;32(1):9-18. doi: 10.1016/0166-3542(95)00969-8.
The hepatitis C virus (HCV) genome contains the code for a conserved, serine-type protease, called NS3, for the processing of the non-structural protein region of the viral polyproteins. Furthermore, a related protein, NS4A, is an effector or cofactor of NS3 protease activity in the cleavage of NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. To establish an in vitro assay system for the screening of those enzyme inhibitors that inhibit the protease NS3-4A, we prepared a maltose-binding protein-NS3-NS4A fusion protein and a synthetic peptide substrate that mimics the NS5A-5B junction. Cleavage of the synthetic peptide was analyzed by reversed-phase high performance liquid chromatography (HPLC). We showed that the enzymatic activity of the NS3-NS4A fusion protein was enhanced in comparison to the NS3 protein alone. The assay conditions for optimum NS3-4A protease activity were determined to be pH 7.6 and 37 degrees C. In addition, we evaluated several protease inhibitors using the same HPLC assay system. The activity of HCV protease NS3-4A was inhibited by 2714.4 microM diisopropyl fluorophosphate, 270.8 microM N-tosyl-L-lysyl chloromethyl ketone, and 825.5 microM chymostatin. The results of the present study indicated that the synthetic peptide substrate and HPLC assay system are suitable for studying HCV protease activity and may facilitate the development of anti-HCV therapeutic reagents.
丙型肝炎病毒（HCV）基因组包含一种保守的丝氨酸型蛋白酶的编码，该蛋白酶称为NS3，用于处理病毒多聚蛋白的非结构蛋白区域。此外，一种相关蛋白NS4A是NS3蛋白酶活性在切割NS3 - 4A、NS4A - 4B、NS4B - 5A和NS5A - 5B连接点时的效应器或辅助因子。为了建立一种体外检测系统，用于筛选抑制蛋白酶NS3 - 4A的酶抑制剂，我们制备了一种麦芽糖结合蛋白 - NS3 - NS4A融合蛋白和一种模拟NS5A - 5B连接点的合成肽底物。通过反相高效液相色谱（HPLC）分析合成肽的切割情况。我们发现，与单独的NS3蛋白相比，NS3 - NS4A融合蛋白的酶活性有所增强。确定NS3 - 4A蛋白酶最佳活性的检测条件为pH 7.6和37摄氏度。此外，我们使用相同的HPLC检测系统评估了几种蛋白酶抑制剂。HCV蛋白酶NS3 - 4A的活性被2714.4微摩尔的二异丙基氟磷酸酯、270.8微摩尔的N - 甲苯磺酰 - L - 赖氨酰氯甲基酮和825.5微摩尔的抑肽酶所抑制。本研究结果表明，合成肽底物和HPLC检测系统适用于研究HCV蛋白酶活性，并可能有助于抗HCV治疗试剂的开发。
