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 drug transporters. By using a baculovirus-insect cell expression system, a large amount (9-10 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. Currently, we have also developed methods for large-scale purification of mutant Pgp variants that are trapped in a transition state during catalytic cycle. At present our collaborator, Dr. Di Xia has tested >25,000 conditions for generation of crystals of wild-type and mutant Pgps. 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. In addition, we have characterized curcumin, a natural product modulator that inhibits the function of Pgp, MRP1 and ABCG2 -the major three ABC drug transporters. Curcumin, which is relatively nontoxic, might have a beneficial effect on cancer chemotherapy with respect to the possibility of long-term use without concerns regarding MDR1, MRP1 or ABCG2 activation. Similarly we also found that major dietary flavonoids such as quercetin and silymarin can modulate function of MRPs--1, 4, and 5. 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. Development of natural product modulators for the reversal of drug resistance-mediated by Pgp, MRP1 and ABCG2: Our previous studies demonstrated that curcumin; an active ingredient of turmeric powder which is consumed in many parts of world modulates the function of Pgp. The curcumin is a mixture of three curcuminoids-curcumin I, II, and III. We purified each curcuminoid from curcumin mixture by using solvent extraction and HPLC chromatography. Among curcuminoids, curcumin I is the most effective modulator of Pgp, MRP1 as well as ABCG2. We characterized the interaction of curcuminoids with these drug transporters and found that these curcuminoids also interact at the drug-substrate-binding sites on these transporters. It is interesting to note that curcumin is one of the few modulators so far known which inhibit the function of the three major ABC drug transporters. It remains to be seen whether these transporters have a common modulator-binding site.2. Characterization of human MRP1 (ABCC1), MRP4 (ABCC4) and MRP5 (ABCC5): Multidrug Resistance Proteins 4 (MRP4/ABCC4) and 5 (MRP5/ABCC5) transport cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents and prostaglandins. We have characterized the effect of mefloquine-a quinoline based antimalarial drug on the function of MRP4 and 5, which are also expressed in human red blood cells. Mefloquine appears to be a transport substrate for MRP1 and MRP4 but not for MRP5.
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