title: Development of an ESI-LC-MS-based assay for kinetic evaluation of Mycobacterium tuberculosis shikimate kinase activity and inhibition.
abstract: 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.
authors: Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University , 4306 Walker Building, Auburn, Alabama 36849, United States.
authors: Johayra
authors: J
authors: Simithy
authors: Gobind
authors: G
authors: Gill
authors: Yu
authors: Y
authors: Wang
authors: Douglas C
authors: DC
authors: Goodwin
authors: Angela I
authors: AI
authors: Calderón
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D002853:Chromatography, Liquid / Q000379:methods; D057075:Enzyme Assays / Q000379:methods; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014376:Tuberculosis / Q000188:drug therapy / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D047428:Protein Kinase Inhibitors; D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase