Deng Gejing, Gu Rong-Fang, Marmor Stephen, Fisher Stewart L, Jahic Haris, Sanyal Gautam
Infection Discovery, Department of Biochemistry, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
J Pharm Biomed Anal. 2004 Jun 29;35(4):817-28. doi: 10.1016/j.jpba.2004.02.029.
An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated.
基于高效液相色谱(HPLC)分离后特定反应产物的质谱(MS)检测,已开发出一种酶活性测定方法,用于评估从初级高通量筛选(HTS)中鉴定出的针对目标酶大肠杆菌UDP-N-乙酰胞壁酰-L-丙氨酸连接酶(MurC)的药物筛选命中物,MurC是细菌肽聚糖生物合成途径中的一种必需酶,并用于研究该酶的动力学。对这种基于液相色谱-质谱联用(LC-MS)的新测定方法与传统的分光光度法孔雀石绿(MG)测定方法进行了比较分析,MG测定法检测反应中产生的磷酸盐。结果表明,测定MurC特异性连接酶活性的LC-MS测定法具有几个优点,包括背景更低(0.2%对26%)、灵敏度更高(≥10倍)、定量下限(LOQ)更低(0.02 microM对1 microM)以及线性动态范围更宽(≥4倍)。LC-MS测定法的日内和日间变异系数(CV)值较低(分别为3%和6%),证明了其良好的精密度。LC-MS测定法没有孔雀石绿测定法中常见的假象,为筛选命中物评估提供了一种有价值的二级测定方法,可消除初级高通量筛选中的假阳性。此外,该测定法在酶动力学研究中的适用性也得到了证明。