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  • 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.

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.

Analytical chemistry (2010-04-17)
Vanisree Mulabagal, Angela I Calderón
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.

MATERIALS
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Product Description

Supelco
3-Phosphoshikimic acid lithium salt, ≥95.0% (HPLC)