Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University , 4306 Walker Building, Auburn, Alabama 36849, United States.
Anal Chem. 2015 Feb 17;87(4):2129-36. doi: 10.1021/ac503210n. Epub 2015 Jan 28.
A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.
已经开发并验证了一种简单可靠的液相色谱-质谱（LC-MS）分析方法，用于对结核分枝杆菌（Mycobacterium tuberculosis， MtSK）的莽草酸激酶的抑制剂进行动力学特征描述和评估，该酶是开发新型抗结核药物的潜在靶标。该测定法基于使用电喷雾电离（ESI）和四极杆飞行时间（Q-TOF）检测器直接测定反应产物莽草酸-3-磷酸（S3P）。对通过 LC-MS 分析获得的 MtSK 的动力学参数与通过常规 UV 分析获得的动力学参数进行了比较分析。通过 LC-MS 确定的动力学参数与通过 UV 分析获得的动力学参数非常吻合，证明了该方法的准确性和可靠性。经过验证的分析方法成功应用于已知的莽草酸激酶抑制剂的动力学特征描述，通过 LC-MS 准确描绘了抑制常数和抑制模式。
