P-glycoprotein (P-gp) is an energy-dependent drug extrusion pump with broad specificity for diverse hydrophobic anticancer agents and compounds known to reverse multidrug resistance (MDR). Among MDR reversing agents, phenothiazines (PTZs) and related compounds may sensitize MDR by interacting with a specific binding site(s) on P-gp and by other mechanisms. In order (1) to identify a binding site for PTZs and related compounds on P-gp, (2) to examine whether these compounds and other MDR modulators bind to the same domains of P-gp, and (3) to identify proteins with high specificity for these neuroleptic agents and other MDR modulators, we used a butyrophenone D2-dopamine receptor photoaffinity probe, N-(p-azido-3-[125I]iodophenethyl)spiperone ([125I]NAPS). [125I]NAPS was actively effluxed from vincristine (VCR)-resistant SH-SY5Y/VCR human neuroblastoma cells, and nonradioactive I-NAPS was a potent chemosensitizing agent. After photolabeling, the probe bound specifically and with high efficiency to P-gp and to another multidrug binding 17-kDa membrane-bound protein, spiperophilin, in these cells. The efficiency of [125I]NAPS binding to P-gp was 5-6-fold more than [3H]azidopine and [125I]arylazidoprazosin ([125I]AAP), known photoaffinity analogs for P-gp. [125I]NAPS photolabeling of P-gp was preferentially competed by MDR-related drugs, with vinblastine > VCR > colchicine > doxorubicin > actinomycin D. Many drugs that are known to reverse MDR were potent inhibitors of [125I]NAPS binding to P-gp. While PTZs and related compounds were potent inhibitors of [125I]NAPS binding to P-gp, most of them enhanced the binding of [125I]AAP significantly. cis-Flupentixol increased the binding of [125I]AAP to P-gp 9-fold more than did trans-flupentixol, but both were potent inhibitors of [125I]NAPS binding, suggesting their stereoselective effect on the [125I]AAP binding site. Proteolysis of [125I]NAPS-bound P-gp with Staphylococcus aureus V8 protease revealed that this probe binds to two major peptides, 6 and 8 kDa, and a number of minor ones, while [125I]AAP binds to only an 8-kDa peptide. These results suggest that modulators of MDR may interact with separate or overlapping domains. Furthermore, most MDR modulators, dopaminergic drugs, and β-adrenergic antagonists used also inhibited binding of [125I]-NAPS to spiperophilin, suggesting that this protein may be a target for these drugs.
ASJC Scopus subject areas