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

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....

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2015-02, Vol.87 (4), p.2129-2136
Main Authors: Simithy, Johayra, Gill, Gobind, Wang, Yu, Goodwin, Douglas C, Calderón, Angela I
Format: Article
Language:English
Subjects:
Antitubercular Agents - pharmacology
Assaying
Bacteria
Bioassays
Chromatography
Chromatography, Liquid - methods
Enzyme Assays - methods
Humans
Inhibition
Inhibitors
Kinases
Liquids
Mass spectrometry
Mycobacterium tuberculosis
Mycobacterium tuberculosis - drug effects
Mycobacterium tuberculosis - enzymology
Phosphotransferases (Alcohol Group Acceptor) - antagonists & inhibitors
Phosphotransferases (Alcohol Group Acceptor) - metabolism
Protein Kinase Inhibitors - pharmacology
Reaction kinetics
Shikimic Acid - analogs & derivatives
Shikimic Acid - analysis
Shikimic Acid - metabolism
Spectrometry
Spectrometry, Mass, Electrospray Ionization - methods
Tuberculosis
Tuberculosis - drug therapy
Tuberculosis - microbiology
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac503210n