The ATP-binding cassette (ABC) transporters such as P-glycoprotein (Pgp, ABCB1), the multidrug resistance-associated protein (MRP1, ABCC1), and the mitoxantrone-resistance protein (MXR also known as breast cancer resistance protein, BCRP, ABCP or ABCG2), which function as ATP-dependent efflux pumps, play an important role in the development of multidrug resistance in most cancers. There are 48 known ABC transport proteins in the human and at least 14 of these transporters are involved in the movement of a variety of amphipathic agents including anticancer agents, nucleotide analogs and cyclic nucleotides. Thus, some of these transporters also may contribute to the development of multidrug resistance in malignant cells. Our studies are directed toward understanding the mechanism of action of the multidrug resistance-linked ABC transporters. By using a baculovirus-insect cell expression system, a large amount (6-7 mg/ml) of biologically active Pgp has been prepared for biophysical and structural studies, as further understanding of the mechanism of these transporters would be accelerated by resolution of the structure of Pgp. In last couple of years we have directed our efforts towards understanding the catalytic cycle of ATP hydrolysis by Pgp, identification of rate-limiting step(s) and modulation of the ATPase activity by substrates and modulators. Similar studies have been initiated with MRP1, MRP4 and MRP8 to gain insight into the role of the two ATP sites in ATP hydrolysis by these transporters. Such studies will provide an insight into the role of these transporters in the development of multidrug resistance in cancers and aid in the development of new therapeutic strategies.1.Characterization of substrate interaction sites and identification of regions and residues involved in interaction of substrates with Pgp: Our previous studies demonstrated the presence of at least two non-identical substrate interaction sites on Pgp. We have continued our efforts to characterize the interactions of substrates and inhibitors to elucidate the biochemical basis for the broad substrate specificity of Pgp. We have screened a large variety of derivatives of stipiamide, a synthetic polyene antibiotic that reverses Pgp-mediated drug resistance, to gain insight into the substrate interaction sites. We have also studied the mechanism by which disulfiram, a drug used to treat alcoholism, modulates Pgp activity. From a clinical perspective it is interesting that disulfiram also modulates activities of human MRP1, MRP4 as well as fungal ABC drug transporter Cdr1p. Disulfiram, or tetraethylthiuram disulfide, is a relatively nontoxic (oral LD50 8.6 g/kg) dithiocarbamate, which has been used for over half a century for alcohol aversion therapy. Dithiocarbamates react with critical thiols and also complex metal ions. We have demonstrated that in intact cells disulfiram reverses either human MDR1- or MRP1-mediated efflux of fluorescent drug-substrates. Disulfiram inhibits ATP hydrolysis and the binding of a-32P8-azidoATP to P-glycoprotein and MRP1, with inhibition curves comparable to 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, though not in the active site per se. Similarly, we have shown that disulfiram is an effective modulator of the multidrug transporter Cdr1p from Candida albicans, which plays an important role in the development of resistance to a variety of antifungal agents. The biochemical mechanisms for the inhibition of Cdr1p and human P-glycoprotein appear to be similar. Disulfiram inhibits the binding of photoaffinity analogs of both ATP (a-32P8-azidoATP) and drug substrates (125I-Iodoarylazidoprazosin and 3H-Azidopine) to Cdr1p in a concentration-dependent manner. Consistent with these findings, a non-toxic low concentration (1 mM) of disulfiram makes the Cdr1p expressing S. cerevisiae cells more susceptible to antifungal agents such as cycloheximide, fluconazole, miconazole and nystatin. Collectively, our results demonstrate that disulfiram modulates function of drug transporters by interaction with both ATP and substrate-binding sites and thus dithiocarbamates may provide a useful molecular scaffold for developing novel interventions against resistance-mediated by both fungal and mammalian ABC drug transporters.2.Characterization of human MRP4 (ABCC4) and MRP8 (ABCC11): Multidrug Resistance Protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents and prostaglandins. We have characterized ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mM), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. a-32P8azidoATP binds to MRP4 (concentration for half-maximal binding 3 mM) and is displaced by ATP or by its non-hydrolysable analog AMPPNP (concentrations for half-maximal inhibition 13.3 mM and 308 mM). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2 to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonyl-methoxyethyl) adenine and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4a-32P8azidoADPVi and MRP4a-32P8azidoADPBeFx can be generated, nucleotide trapping is 4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis and trapping. However, Co2+ increases a-32P8azidoATP binding and beryllium fluoride-induced a-32P8azidoADP trapping but does not support steady state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mM, respectively) and beryllium fluoride-induced a-32P8azidoADP trapping while Pi has no effect up to 20 mM. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle. Recently, two new members of the MRP subfamily have been identified (MRP8 and MRP9). We have expressed human MRP8 in baculovirus-infected High Five insect cells. The initial results indicate that MRP8, similar to Pgp and MRP1, exhibits substrate-stimulated ATPase activity. Such studies will help us to understand the function of these MRP subfamily members and their role in the development of multidrug resistance in cancer cells.3.Resolution of two- and three-dimensional structure of human Pgp: The high-resolution structure of Pgp at various stages during the catalytic cycle will be essential to understand the transport mechanism. This is one of our major interests and we have invested considerable effort in the past to develop methods for obtaining pure and active Pgp in large amounts.
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