TY - JOUR T1 - Identification of Ligand-Binding Regions of P-Glycoprotein by Activated-Pharmacophore Photoaffinity Labeling and Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry JF - Molecular Pharmacology JO - Mol Pharmacol SP - 637 LP - 648 DO - 10.1124/mol.61.3.637 VL - 61 IS - 3 AU - Gerhard F. Ecker AU - Edina Csaszar AU - Stephan Kopp AU - Brigitte Plagens AU - Wolfgang Holzer AU - Wolfgang Ernst AU - Peter Chiba Y1 - 2002/03/01 UR - http://molpharm.aspetjournals.org/content/61/3/637.abstract N2 - Energy dependent efflux pumps confer resistance to anticancer, antimicrobial, and antiparasitic drugs. P-glycoprotein (Pgp, ABCB1) mediates resistance to a broad spectrum of antitumor drugs. Compounds that themselves are nontoxic to cells have been shown to act as inhibitors of Pgp. The mechanism of binding and transport of low-molecular-mass ligands by Pgp is still incompletely understood. This study introduces a series of propafenone-related photoaffinity ligands, which combine high specificity and selectivity for Pgp with high labeling efficiency. Molecules are intrinsically photoactivatable in the arylcarbonyl group, which represents a pharmacophoric substructure for this group of ligand molecules. A detailed study of the structure-activity relationship for this type of photoligand is presented. In subsequent experiments, these ligands were used to characterize the drug-binding domain of propafenone-type analogs. Matrix-assisted laser desorption/ionization—time-of-flight (MALDI-TOF) mass spectrometry shows that propafenone-type ligands preferentially label fragments assigned to putative transmembrane segments 3, 5, 6, 8, 10, 11, and 12. Labeled fragments are also identified in a highly charged region of 15 amino acids in the second cytoplasmic loop. This region corresponds to the so-called EAA-like motif, which has been proposed to play a role in the interaction between transmembrane domain and nucleotide binding domain of peroxisomal ATP-binding cassette transporters. In addition, a region in cytoplasmic loop 3 and between TM12 and the N terminus of the Walker A sequence of NBD2 are labeled by the ligands. Therefore, a number of confined protein regions contribute to the drug-binding domain of propafenone-type analogs. ER -