Online Biochemical Detection of Glutathione-S-Transferase P1-Specific Inhibitors in Complex Mixtures

A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC50 values of 8 ligands thus obtained online agreed well with the IC50 values obtained with microplate reader—based assays. For ethacrynic acid, an IC50 value of 1.8 ± 0.4 µM was obtained with the cGSTs EAD system in FIA mode and 0.8 ± 0.6 µM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC50 values of 6.0 ± 2.9 and 3.6 ± 2.8 µM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures. (Journal of Biomolecular Screening 2007:396-405)