Identification of ligand-binding regions of P-glycoprotein by activated-pharmacophore photoaffinity labeling and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry
- Author(s)
- Gerhard Ecker, Edina Csaszar, Brigitte Plagens, Wolfgang Holzer, Wolfgang Ernst, Peter Chiba
- 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.
- Organisation(s)
- Department of Biochemistry and Cell Biology, Department of Analytical Chemistry
- External organisation(s)
- Universität Wien
- Journal
- Molecular Pharmacology
- Volume
- 61
- Pages
- 637-648
- No. of pages
- 12
- ISSN
- 0026-895X
- DOI
- https://doi.org/10.1124/mol.61.3.637
- Publication date
- 2002
- Peer reviewed
- Yes
- Austrian Fields of Science 2012
- 3012 Pharmacy, Pharmacology, Toxicology
- Portal url
- https://ucris.univie.ac.at/portal/en/publications/identification-of-ligandbinding-regions-of-pglycoprotein-by-activatedpharmacophore-photoaffinity-labeling-and-matrixassisted-laser-desorptionionizationtimeofflight-mass-spectrometry(42790fda-a880-4a0d-b557-da759a17f5b0).html