Vol. 62, Issue 6, 1288-1298, December 2002

Characterization of Two Pharmacophores on the Multidrug Transporter P-Glycoprotein

Alexia Garrigues, Nicolas Loiseau, Marcel Delaforge, Jacques Ferté, Manuel Garrigos, François André, and Stéphane Orlowski

Service de Biophysique des Fonctions Membranaires, Département de Biologie Joliot Curie (A.G., J.F., M.G., S.O.); Service de Pharmacologie et d'Immunologie, Département de Recherche Médicale (N.L., M.D.), Service de Bioénergétique, Département de Biologie Joliot Curie (F.A.), Commissariat à l'Energie Atomique, and Unité de Recherche Associée 2096 Centre National de la Recherche Scientifique, Laboratoire de Recherche Associé 17V Université Paris-Sud, Paris, France

The multidrug transporter P-glycoprotein is a plasma membrane protein involved in cell and tissue detoxification and the multidrug resistance (MDR) phenotype. It actively expels from cells a number of cytotoxic molecules, all amphiphilic but chemically unrelated. We investigated the molecular characteristics involved in the binding selectivity of P-glycoprotein by means of a molecular modeling approach using various substrates combined with an enzymological study using these substrates and native membrane vesicles prepared from MDR cells. We determined affinities and mutual relationships from the changes in P-glycoprotein ATPase activity induced by a series of cyclic peptides and peptide-like compounds, used alone or in combination. Modeling of the intramolecular distribution of the hydrophobic and polar surfaces of this series of molecules made it possible to superimpose some of these surface elements. These molecular alignments were correlated with the observed mutual exclusions for binding on P-glycoprotein. This led to the characterization of two different, but partially overlapping, pharmacophores. On each of these pharmacophores, the ligands compete with each other. The typical MDR-associated molecules, verapamil, cyclosporin A, and actinomycin D, bound to pharmacophore 1, whereas vinblastine bound to pharmacophore 2. Thus, the multispecific binding pocket of P-glycoprotein can be seen as sites, located near one another, that bind ligands according to the distribution of their hydrophobic and polar elements rather than their chemical motifs. The existence of two pharmacophores increases the possibilities for multiple chemical structure recognition. The size of the ligands affects their ability to compete with other ligands for binding to P-glycoprotein.