The primary research focus in our laboratory is to further characterize ATP binding/hydrolysis and the relationship of ATP hydrolysis and anticancer drug transport by multidrug resistance-associated protein (MRP1). The over-expression of MRP1 and/or P-glycoprotein (P-gp) in cancer cells confers multidrug resistance, which is one of the major obstacles to the successful treatment of many types of cancers. In order to combat the multidrug resistance caused by the over-expression of MRP1 protein we?d like to investigate the mechanism of coupling ATP hydrolysis and anticancer drug transport by the protein, to determine the drug binding site(s) and to screen compounds inhibiting the protein function.

MRP1 protein is a member of ATP-binding cassette (ABC) transporters containing 17 transmembrane segments and two nucleotide binding domains (NBD). It is widely accepted that the protein couples ATP hydrolysis and drug transport. The consequence of the transport is that the drug concentration inside of the cell is too low to kill the cancer cell during chemotherapy. Although the multidrug resistance profile caused by the over-expression of MRP1 is similar to the one caused by over-expression of P-gp, the ways they pump drugs out of the cells are somewhat different, i.e, P-gp pumps hydrophobic compounds out of cells directly, whereas MRP1 transports anionic conjugates, such as glutathione-, glucuronide-, or sulfate-conjugated aliphatic, prostanoid and heterocyclic compounds. In addition, the two NBDs in P-gp have been shown to be functionally equivalent with identical ATP hydrolysis steps occurring alternatively at each NBD, whereas the two NBDs in MRP1 do not seem to be functionally equivalent. We have found that ATPase activity at NBD2 is much higher than at NBD1 and ATP binding occurs exclusively at NBD1 and ADP trapping predominantly at NBD2. Interestingly, the trapping of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1 and the binding of ATP at NBD1 allosterically enhances the trapping of ADP at NBD2. Although the mechanism of how the protein couple ATP hydrolysis and drug transport is not clear yet, these findings indicate that the separations of functions in the two NBDs and the mutual cooperative enhancements between the two NBDs may play an important role in the ATP-dependent drug transport.

As I mentioned above that P-gp pumps hydrophobic compounds out of cells directly, the drug binding sites have been thought to be located in the hydrophobic transmembrane segments. Whereas MRP1 transports anionic conjugates out of the cell, there must be a binding site for the glutathione or glucuronide in the hydrophilic cytoplasmic domains. The picture for the conjugates binding sites is not clear yet. We are going to use cysteine scanning method to define the conjugates binding sites. We are also going to screen some compounds to inhibit either the conjugates binding or the ATPase activity of the MRP1 protein to combat multidrug resistance.