N-(p-azido-3-[125I]iodophenethyl)spiperone binds to specific regions of P-glycoprotein and another multidrug binding protein, spiperophilin, in human neuroblastoma cells

Abstract

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 beta-adrenergic antagonists used also inhibited binding of [125I]-NAPS to spiperophilin, suggesting that this protein may be a target for these drugs.

Allosteric Modulation of Human P-glycoprotein

“…A number of these thioxanthene derivatives do not physically compete for the substrate-binding site of Pgp (21,23), but instead they seem to interact at a distinct (allosteric) site within the protein (22), indicating an allosteric mode of action. However, the molecular events leading to inactivation of the pump remained unresolved.…”

“…Characterization of its effect on substrate binding (23) suggested an allosteric mode of action for the modulator (21,22); however, the exact mechanism of action remained unresolved. To understand the mechanism better, we developed a cell-based assay suitable for studying both Pgp-mediated transport and Pgp- (Fig.…”

Identification and Characterization of the Binding Sites of P-Glycoprotein for Multidrug Resistance-Related Drugs and Modulators

“…These photoaffinity analogs are B9209-005, an azido derivative of dexniguldipine [92] [93], N-(p-azido-3-125 I]iodophenethyl) spiperone ([ 125 I]NAPS) [94] (Fig. 3) and radiolabeled photoaffinity analogs of synthetic isoprenoid [95], cyclosporin A [96], forskolin [97] and estramustine [98] (Figs.…”

“…(6), we have demonstrated that P-glycoprotein may interact with some of its substrates stereoselectively. For instance, cis-and trans-flupentixol can reverse MDR, but cisfiupentixol increased the binding of [ 125 I]AAP to P-glycoprotein nine-fold more than transflupentixol [94]. However, both of these MDR reversing agents were potent inhibitors of [113] and Shao et al [119] proposed two separate binding sites, one for P-glycoprotein substrates, such as vinblastine, mefloquine and tamoxifen, and the other one for substrates such as verapamil.…”

“…The results of Shapiro et al [123] strongly support the idea that these two compounds bind to the third stimulatory allosteric site on P-glycoprotein and exert the same effect. The foregoing discussion fayors a multisite model of the P-glycoprotein drug binding sites originally proposed by Tamai and Safa [116] and Safa et al [94], rather than a universal site model with broad substrate specificity as proposed by Borgnia et al [124]. Martin et al [118] employed radioligand-binding techniques to directly characterize drug interaction sites on P-glycoprotein and how these multiple sites interact.…”