title: Identification of ligand-binding regions of P-glycoprotein by activated-pharmacophore photoaffinity labeling and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.
abstract: 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.
authors.affiliation: Institute of Pharmaceutical Chemistry, University of Vienna, Austria.
authors.forename: Gerhard F
authors.initials: GF
authors.lastname: Ecker
authors.forename: Edina
authors.initials: E
authors.lastname: Csaszar
authors.forename: Stephan
authors.initials: S
authors.lastname: Kopp
authors.forename: Brigitte
authors.initials: B
authors.lastname: Plagens
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Holzer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Ernst
authors.forename: Peter
authors.initials: P
authors.lastname: Chiba
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D011869:Radioligand Assay; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels