期刊
BIOCHEMICAL PHARMACOLOGY
卷 92, 期 4, 页码 558-566出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bcp.2014.10.006
关键词
Tariquidar; Inhibitor P-glycoprotein drug pump; Cysteine cross-linking; Rescue of folding mutants
资金
- Cystic Fibrosis Canada
- Canadian Institutes for Health Research
- Canadian Chair in Membrane Biology
P-glycoprotein (P-gp, ABCB1) is a drug pump that confers multidrug resistance. Inhibition of P-gp would improve chemotherapy. Tariquidar is a potent P-gp inhibitor but its mechanism is unknown. Here, we tested our prediction that tariquidar inhibits P-gp cycling between the open and closed states during the catalytic cycle. Transition of P-gp to an open state can be monitored in intact cells using reporter cysteines introduced into extracellular loops 1 (A80C) and 4 (R741C). Residues A80C/R741C come close enough (<7 angstrom) to spontaneously cross-link in the open conformation (<7 A) but are widely separated (>30 angstrom) in the closed conformation. Cross-linking of A80C/R741C can be readily detected because it causes the mutant protein to migrate slower on SOS-PAGE gels. We tested whether drug substrates or inhibitors could inhibit cross-linking of the mutant. It was found that only tariquidar blocked A80C/R741C cross-linking. Tariquidar was also a more potent pharmacological chaperone than other P-gp substrates/modulators such as cyclosporine A. Only tariquidar promoted maturation of misprocessed mutant F804D to yield mature P-gp. Tariquidar interacted with the transmembrane domains because it could rescue a misprocessed truncation mutant lacking the nucleotide-binding domains. These results show that tariquidar is a potent pharmacological chaperone and inhibits P-gp drug efflux by blocking transition to the open state during the catalytic cycle. (C) 2014 Elsevier Inc. All rights reserved.
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