Kati W M, Sham H L, McCall J O, Montgomery D A, Wang G T, Rosenbrook W, Miesbauer L, Buko A, Norbeck D W
Pharmaceutical Products Division, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, Illinois, 60064-3500, USA.
Arch Biochem Biophys. 1999 Feb 15;362(2):363-75. doi: 10.1006/abbi.1998.1038.
The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in an Escherichia coli strain which also overexpressed the rare argU tRNA. The protease was isolated from inclusion bodies, refolded, and exhibited a kcat/Km = 3280 M-1 s-1 using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease with kinact/Kinact values as high as 23,400 M-1 s-1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to the kinact/Kinact rate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.
编码人鼻病毒1B株3C蛋白酶的基因在一种同时过表达稀有argU tRNA的大肠杆菌菌株中高效表达。该蛋白酶从包涵体中分离出来，进行复性，使用内部淬灭的肽基荧光底物时，其kcat/Km = 3280 M-1 s-1。这种连续荧光测定法用于测量几种传统肽基氯甲基酮以及一系列新型化合物（溴甲基酮酰肼）对3C蛋白酶的抑制动力学。含有溴甲基酮酰肼主链且在P1位置具有谷氨酰胺样侧链的化合物是鼻病毒3C蛋白酶的强效、时间依赖性抑制剂，其kinact/Kinact值高达23,400 M-1 s-1。含有修饰P1侧链的化合物的抑制活性表明，P1羧酰胺基团与3C蛋白酶之间的相互作用对溴甲基酮酰肼抑制3C蛋白酶的kinact/Kinact速率常数贡献至少30倍。对天然和受抑制的3C蛋白酶分子量的电喷雾电离质谱测量确定了一种抑制机制，该机制涉及酶与抑制剂之间形成共价加合物，同时溴甲基酮酰肼失去一个溴离子。对溴甲基酮酰肼抑制的3C蛋白酶进行胰蛋白酶消化，确定加合物形成于对应于残基145 - 154的肽段，该区域包含活性位点半胱氨酸 - 148残基。溴甲基酮酰肼是胰凝乳蛋白酶、人弹性蛋白酶和组织蛋白酶B的相当弱的抑制剂，其中几种化合物在细胞培养中也显示出抑制人鼻病毒1B复制的证据。
