Overexpression of P-glycoprotein (P-gp) and/or multidrug resistance-associated protein (MRP1) is associated with resistance of tumors to a wide range of chemotherapeutic drugs. Many cancers initially respond well to chemotherapeutic treatment, but these tumors eventually become resistant to those cytotoxic drugs. Some other cancers are resistant to anticancer drugs, even at the beginning of treatment. Therefore, no matter whether the resistance is """"""""acquired"""""""" or """"""""intrinsic,"""""""" multidrug-resistance is one of the major obstacles to the successful treatment of many types of cancers. The mechanism of multidrug resistance conferred by either P-gp or MRP1 involves extrusion of the drugs out of the tumor cells. Both P-gp and MRP1 are members of the ABC superfamily of transport proteins, typically containing two membrane-spanning domains and two nucleotide binding domains. However, MRP1 differs from P-gp in that it contains an extra membrane-spanning domain at the N-terminus. ATP (binding and hydrolysis) is required in both cases. However, the transport steps differ in that P-gp extrudes the unmodified drugs directly, while the drugs transported by MRP1 protein require the presence of a hydrophilic compound, e.g., glutathione- or glucuronide-conjugates. Since the mechanism of multidrug resistance conferred by P-gp or MRP1 is to reduce the level of drug accumulation inside of the cell, inhibiting of the function of P-gp or MRP1 may reverse the drug resistance. Therefore, much effort is being devoted to discover specific inhibitors of these pumps. Present candidates as modulators of the process include verapamil, cyclosporin A, Cremophor, ardeemins, PSC833, rifamycins, RU486, MS-209, non-steroidal inflammatory drugs, acrolein, pyridine analogues, ONO-1078, chloroacetaldehyde, imidazothiazole derivatives, and even some protein kinase inhibitors. However, in order to develop well-designed specific modulators, it is important to understand the mechanisms of drug transport carried out by each individual protein. The first specific aim is to further characterize the ATP finding/hydrolysis and substrate transport by MRP1 protein.
The second aim i s to determine whether MRP1 protein phosphorylates itself or is a substrate for other protein kinases. This is based on our hypotheses that the phosphorylated MRP1 protein is an intermediate (i.e., there is an autophosphorylation) during ATP hydrolysis and substrate transport.
The third aim i s to define the substrate binding site(s). New insights gained from these aims should provide the basis for novel means of combating multidrug resistance.
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