title: Development of an ultrafiltration-liquid chromatography/mass spectrometry (UF-LC/MS) based ligand-binding assay and an LC/MS based functional assay for Mycobacterium tuberculosis shikimate kinase.
abstract: Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets in the discovery of antimicrobial agents. In the current study, an ultrafiltration-liquid chromatography/mass spectrometry (UF-LC/MS) ligand based binding assay and an LC/MS based functional assay for Mycobacterium tuberculosis shikimate kinase (MtSK) were developed. Compounds 1, 2, 3, and 4 were tested for MtSK (1 microM) at a concentration of 1 microM. In order to evaluate the MtSK inhibitory activity, compounds 1-4 were tested at concentrations ranging from 0.05 to 1 microM, and the enzymatic activity was assessed by quantifying shikimate-3-phosphate (S3P) by LC/MS after 60 min incubation with 2 mM shikimic acid as a substrate. The EC(50) values of compounds 1, 2, 3, and 4 were 0.30, 0.24, 0.07, and 0.18 microM, respectively. The ligands and the S3P were analyzed using positive and negative electrospray LC/MS, respectively. The calibration curve for S3P was prepared with concentrations ranging from 4 to 125 microg/mL, and the lower detection limit (LOD) of S3P was identified as 1.95 microg/mL (9.75 ng on-column). This is the first application of UF-LC/MS and LC/MS in the development of ligand-binding and functional assays, respectively as a useful approach to screen MtSK inhibitors.
authors.affiliation: Department of Pharmacal Sciences, Harrison School of Pharmacy, 4306B Walker Building, Auburn University, Auburn, Alabama 36849, USA.
authors.forename: Vanisree
authors.initials: V
authors.lastname: Mulabagal
authors.forename: Angela I
authors.initials: AI
authors.lastname: Calderón
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D008024:Ligands; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D012765:Shikimic Acid / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D012765:Shikimic Acid; D010770:Phosphotransferases