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

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 mu M) at a concentration of 1 mu M. In order to evaluate the MtSK inhibitory activity, compounds 1-4 were tested at concentrations ranging from 0.05 to 1 mu M, 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 EC50 values of compounds 1, 2, 3, and 4 were 0.30, 0.24, 0.07, and 0.18 mu M, 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 mu g/mL, and the lower detection limit (LOD) of S3P was identified as 1.95 mu g/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.