Development of an ESI-LC-MS-Based Assay for Kinetic Evaluation of Mycobacterium tuberculosis Shikimate Kinase Activity and Inhibition

作者：Simithy Johayra; Gill Gobind; Wang Yu; Goodwin Douglas C; Calderon Angela I^*

来源：Analytical Chemistry, 2015, 87(4): 2129-2136.

DOI：10.1021/ac503210n

A simple and reliable liquid chromatographymass 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.

• 出版日期2015-2-17