Wang Q M, Johnson R B, Cox G A, Villarreal E C, Loncharich R J
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, 46285, USA.
Anal Biochem. 1997 Oct 15;252(2):238-45. doi: 10.1006/abio.1997.2315.
Human rhinovirus encoded 3C protease is an attractive target for antiviral drug development. However, lack of a convenient and selective assay for 3C protease has been a hindrance in characterization of this enzyme and evaluation of a large number of potential inhibitors. In the present study we describe development of a simple, continuous colorimetric assay for this enzyme using peptide p-nitroanilides (pNA) as substrates. Several peptides mimicking the native 3C cleavage site of HRV-14 polyprotein have been synthesized with an N-acylated p-nitroaniline at position P1' and examined as substrates for the purified 3C protease. In these peptides, amino acids downstream from the original cleavage site have all been replaced with a chromophoric p-nitroaniline moiety which is directly linked to the bond undergoing enzymatic cleavage, thereby generating a new cleavage site Gln-pNA for the enzyme. Hydrolysis of these pNA peptides by 3C at the newly formed scissile bond releases free p-nitroaniline which is yellow-colored and can be continuously monitored at a visible wavelength. Kinetic parameters of 3C protease toward these peptides have been measured and analyzed. In addition, the pNA peptides have been modeled within the active site of the 3C protease to investigate the ability of the pNA group to act as a replacement for Gly-Pro in the prime side. The selectivity and applicability of this assay and its advantages over the previously described methods have been demonstrated and discussed. Since multiple tests can be performed simultaneously in one microtiter plate, the assay is ideal for evaluation of a large number of samples.
人鼻病毒编码的3C蛋白酶是抗病毒药物开发的一个有吸引力的靶点。然而，缺乏一种方便且具有选择性的3C蛋白酶检测方法一直阻碍着对该酶的特性表征以及对大量潜在抑制剂的评估。在本研究中，我们描述了一种使用肽对硝基苯胺（pNA）作为底物针对该酶的简单、连续比色检测方法的开发。已合成了几种模拟HRV - 14多蛋白天然3C切割位点的肽，在P1'位置带有N - 酰化对硝基苯胺，并作为纯化的3C蛋白酶的底物进行检测。在这些肽中，原始切割位点下游的氨基酸均被发色对硝基苯胺部分取代，该部分直接与进行酶促切割的键相连，从而为该酶产生一个新的切割位点Gln - pNA。3C在新形成的可裂解键处对这些pNA肽的水解会释放出黄色的游离对硝基苯胺，其可在可见波长下进行连续监测。已测量并分析了3C蛋白酶对这些肽的动力学参数。此外，已在3C蛋白酶的活性位点内对pNA肽进行建模，以研究pNA基团在原侧替代Gly - Pro的能力。已证明并讨论了该检测方法的选择性、适用性及其相对于先前描述方法的优势。由于可以在一个微量滴定板中同时进行多次测试，该检测方法非常适合评估大量样品。
