Competitive and allosteric interactions in ligand binding to P-glycoprotein as observed on an immobilized P-glycoprotein liquid chromatographic stationary phase

A liquid chromatographic stationary phase containing immobilized P-glycopro tein (Pgp) was synthesized using cell membranes obtained from Pgp-expressin g cells. The resulting Pgp-stationary phase was used in frontal and zonal c hromatographic studies to investigate the binding of vinblastine (VBL), dox orubicin (DOX), verapamil (VER), and cyclosporin A (CsA) to the immobilized Pgp. The compounds were added individually to the chromatographic system w ith or without ATP in the running buffer. Using this approach, dissociation constants were calculated for VBL (23.5 +/- 7.8 nM), DOX (15.0 +/- 3.2 muM ), VER (54.2 +/- 4.7 muM), and CsA [97.9 +/- 19.4 nM (without ATP) and 62.5 +/- 4.6 nM (with ATP)]. The compounds were also added in pairs using stand ard competitive chromatography procedures. The results of the study demonst rate that competitive interactions occurred between VBL and DOX, cooperativ e allosteric interactions occurred between VBL and CsA and ATP and CsA, and anticooperative allosteric interactions occurred between ATP and VBL and V ER. The chromatographic studies indicate that the immobilized Pgp was modif ied by ligand and cofactor binding and that the stationary phase can be use d to study drug-drug binding interactions on the Pgp molecule.