azidopine and 8-azidoadenosine-5--triphosphate

The human ATP-binding cassette transporter, ABCG2, confers resistance to multiple chemotherapeutic agents and also affects the bioavailability of different drugs. [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine were used for photoaffinity labeling of ABCG2 in this study. We show here for the first time that both of these photoaffinity analogues are transport substrates for ABCG2 and that [(3)H]azidopine can also be used to photolabel both wild-type R482-ABCG2 and mutant T482-ABCG2. We further used these assays to screen for potential substrates or modulators of ABCG2 and observed that 1,4-dihydropyridines such as nicardipine and nifedipine, which are clinically used as antihypertensive agents, inhibited the photolabeling of ABCG2 with [(125)I]IAAP and [(3)H]azidopine as well as the transport of these photoaffinity analogues by ABCG2. Furthermore, [(3)H]nitrendipine and bodipy-Fl-dihydropyridine accumulation assays showed that these compounds are transported by ABCG2. These dihydropyridines also inhibited the efflux of the known ABCG2 substrates, mitoxantrone and pheophorbide-a, from ABCG2-overexpressing cells, and nicardipine was more potent in inhibiting this transport. Both nicardipine and nifedipine stimulated the ATPase activity of ABCG2, and the nifedipine-stimulated activity was inhibited by fumitremorgin C, suggesting that these agents might interact at the same site on the transporter. In addition, nontoxic concentrations of dihydropyridines increased the sensitivity of ABCG2-expressing cells to mitoxantrone by 3-5-fold. In aggregate, results from the photoaffinity labeling and efflux assays using [(125)I]IAAP and [(3)H]azidopine demonstrate that 1,4-dihydropyridines are substrates of ABCG2 and that these photolabels can be used to screen new substrates and/or inhibitors of this transporter.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Azides; Calcium Channel Blockers; Cell Line; Chlorophyll; Dihydropyridines; Drug Interactions; Humans; Mitoxantrone; Neoplasm Proteins; Nicardipine; Nifedipine; Photoaffinity Labels; Prazosin

Curcumin (curcumin I), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the major forms of curcuminoids found in the turmeric powder, which exhibit anticancer, antioxidant, and anti-inflammatory activities. In this study, we evaluated the ability of purified curcuminoids to modulate the function of either the wild-type 482R or the mutant 482T ABCG2 transporter stably expressed in HEK293 cells and drug-selected MCF-7 FLV1000 and MCF-7 AdVp3000 cells. Curcuminoids inhibited the transport of mitoxantrone and pheophorbide a from ABCG2-expressing cells. However, both cytotoxicity and [(3)H]curcumin I accumulation assays showed that curcuminoids are not transported by ABCG2. Nontoxic concentration of curcumin I, II, and III sensitized the ABCG2-expressing cells to mitoxantrone, topotecan, SN-38, and doxorubicin. This reversal was not due to reduced expression because ABCG2 protein levels were unaltered by treatment with 10 mumol/L curcuminoids for 72 hours. Curcumin I, II, and III stimulated (2.4- to 3.3-fold) ABCG2-mediated ATP hydrolysis and the IC(50)s were in the range of 7.5 to 18 nmol/L, suggesting a high affinity of curcuminoids for ABCG2. Curcuminoids also inhibited the photolabeling of ABCG2 with [(125)I]iodoarylazidoprazosin and [(3)H]azidopine as well as the transport of these two substrates in ABCG2-expressing cells. Curcuminoids did not inhibit the binding of [alpha-(32)P]8-azidoATP to ABCG2, suggesting that they do not interact with the ATP-binding site of the transporter. Collectively, these data show that, among curcuminoids, curcumin I is the most potent modulator of ABCG2 and thus should be considered as a treatment to increase the efficacy of conventional chemotherapeutic drugs.

Topics: Adenosine Triphosphate; Anticarcinogenic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Azides; Biological Transport; Chlorophyll; Curcumin; Dihydropyridines; Drug Resistance, Neoplasm; Humans; Hydrolysis; Mitoxantrone; Mutation; Neoplasm Proteins; Prazosin; Toxicity Tests; Tumor Cells, Cultured

The overexpression of multidrug resistance protein 1 (MDR1) and multidrug resistance protein 1 (MRP1) gene products is a major cause of multidrug resistance in cancer cells. A recent study suggested that disulfiram, a drug used to treat alcoholism, might act as a modulator of P-glycoprotein. In this study, we investigated the molecular and chemical basis of disulfiram as a multidrug resistance modulator. We demonstrate that in intact cells, disulfiram reverses either MDR1- or MRP1-mediated efflux of fluorescent drug substrates. Disulfiram inhibits ATP hydrolysis and the binding of [alpha-32P]8-azidoATP to P-glycoprotein and MRP1, with inhibition curves comparable with those of N-ethylmaleimide, a cysteine-modifying agent. However, if the ATP sites are protected with excess ATP, disulfiram stimulates ATP hydrolysis by both transporters in a concentration-dependent manner. Thus, in addition to modifying cysteines at the ATP sites, disulfiram may interact with the drug-substrate binding site. We demonstrate that disulfiram, but not N-ethylmaleimide, inhibits in a concentration-dependent manner the photoaffinity labeling of the multidrug transporter with 125I-iodoarylazidoprazosin and [3H]azidopine. This suggests that the interaction of disulfiram with the drug-binding site is independent of its role as a cysteine-modifying agent. Finally, we have exploited MRP4 (ABCC4) to demonstrate that disulfiram can inhibit ATP binding by forming disulfide bonds between cysteines located in the vicinity of, although not in, the active site. Taken together, our results suggest that disulfiram has unique molecular interactions with both the ATP and/or drug-substrate binding sites of multiple ATP binding cassette transporters, which are associated with drug resistance, and it is potentially an attractive agent to combat multidrug resistance.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Binding Sites; Cells, Cultured; Dihydropyridines; Disulfiram; Drug Interactions; Drug Resistance, Multiple; Enzyme Inhibitors; Humans; Hydrolysis; Mice; Multidrug Resistance-Associated Proteins; Phosphorus Radioisotopes; Prazosin

In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [(3)H]cyclosporine and [(3)H]azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [(3)H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [(3)H]daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P-gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe(335) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe(335) also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe(335) results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Amino Acid Sequence; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Binding Sites; Calcium Channel Blockers; Cyclosporine; Dihydropyridines; Drug Interactions; Enzyme Activation; Humans; Molecular Sequence Data; Phenylalanine; Photoaffinity Labels; Tritium; Tumor Cells, Cultured; Vanadates; Verapamil

P-glycoprotein (P-gp) is thought to function as a drug efflux pump in multidrug resistant (MDR) cells. The functional form of P-gp in its native state is not known. Previous results from radiation target size analysis have suggested that P-gp occurs as dimers in MDR cell plasma membranes [Boscoboinik et al. (1990) Biochim. Biophys. Acta 1027, 225-228]. In this study, we used sucrose gradient velocity sedimentation to determine if P-gp oligomers could be retrieved from detergent extracts of hamster and human MDR cell lines. The proportion of P-gp recovered as higher order oligomers was dependent on the detergents used for solubilization of the cells. When a detergent such as CHAPS was used, 50% or more of the P-gp sedimented as higher order oligomers. In contrast, in the presence of SDS, only monomers were retrieved, but naturally occurring oligomers could be preserved if the cells were treated with a cross-linker prior to detergent solubilization. The oligomers and monomers were both able to bind the photoactive analog of ATP (8-azido[alpha-32P]ATP) or the drug [3H]azidopine in membrane preparations. P-gp is a phosphoprotein, and its phosphorylated state is thought to be important for function. When MDR cells were labeled with [32P]orthophosphate in vivo, we observed that the monomer and dimer were more highly phosphorylated than the larger oligomers, suggesting that these different forms of P-gp may be functionally distinct. The assembly of oligomers appears to occur in an early bisynthetic compartment, and asparagine-linked glycosylation is not required for their formation. Our findings indicate that oligomers of P-gp exist in MDR cells and raise the possibility that the dynamics of oligomer formation and dissociation may be important in the mechanism of action of P-gp.

Topics: Adenosine Triphosphate; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Carrier Proteins; Cell Compartmentation; Cell Line; Cell Membrane; Centrifugation; CHO Cells; Cricetinae; Dihydropyridines; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Humans; Membrane Glycoproteins

The role of P-glycoprotein in mediating the drug-resistance phenotype in multidrug resistant cells is now well documented. It is thought to function as an energy-dependent drug-efflux pump of broad specificity. Structurally, P-glycoprotein is an internally duplicated molecule containing two large multi-spanning transmembrane domains and two cytoplasmic ATP binding domains. In this report we demonstrate that monoclonal antibodies C219, C494, and C32 directed against short linear regions of the P-glycoprotein molecule inhibit ATP binding to P-glycoprotein in vitro. We also provide direct evidence that both predicted ATP-binding domains bind ATP and that there is co-operativity between the two sites. In addition, the capacity of P-glycoprotein to bind the calcium channel blocker, azidopine, is inhibited differentially by the antibodies. These observations are the first evidence linking specific perturbations of the P-glycoprotein molecule with ATP and drug binding.

Topics: Adenosine Triphosphate; Affinity Labels; Amino Acid Sequence; Animals; Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Binding Sites; Cell Membrane; CHO Cells; Cricetinae; Dihydropyridines; Drug Resistance; Membrane Glycoproteins; Models, Structural; Molecular Sequence Data; Protein Conformation