Chemotherapy often fails as a cure for many cancer patients because tumor cells become resistant to a wide range of anti-cancer chemotherapeutic agents. The human plasma membrane protein, P-glycoprotein, has been found to be overexpressed in many tumors exhibiting this phenomenon of multidrug resistance. P-glycoprotein is thought to function as an ATP-driven drug- exporting pump in cancer cells, thus protecting tumors from chemotherapy. Little is known about the molecular mechanism by which P-glycoprotein transports a large and diverse number of cytotoxic compounds out of cells. The long range goal of this proposal is to understand how the structure of P-glycoprotein accomplishes its active transport mechanism. With this information, rational design of drugs and methodologies to overcome this clinically important transport will be much closer at hand. A detailed characterization of the fundamental properties of P- glycoprotein will be undertaken to understand how energy derived from the hydrolysis of ATP is used to transport drugs of a wide range of diverse chemical structures. We will employ genetic, kinetic and thermodynamic methods of enzyme analysis, and site specific probe placements.
Aim 1 is to further develop yeast expression systems for normal and mutated forms of human P-glycoprotein. This will facilitate genetic manipulations of the protein and provide a cost effective system for producing large quantities of purified P-glycoprotein for biochemical and structural studies. We have already found that this system can be used for selection of deleterious mutations and return-of-function secondary mutations to map important residues and protein sequences. In addition, we will continue to develop assays to measure fundamental transport properties such as turnover rates, drug specificities, cooperativity between drug and nucleotide sites and extent of coupling and slippage between transport and hydrolysis. These assays will allow quantitative analyses and an understanding of the effects of mutations.
Aim 2 is to test structural and chemical models proposed for P-glycoprotein. Mutational analysis will be used to assign functions to particular amino acid residues and domain structures. Random mutagenesis and reversion analysis will be used to find new important residues and domains, and to map the interactions within the protein between its functional domains. Site directed mutagenesis will be used to test for mechanistic functions that have been attributed to particular residues.
Aim 3 is to elicit structural information by site directed mutagenesis. Specific cysteine and tryptophan replacements will be made for assignments of function to structure by placing spectroscopic probes at defined locations in the nucleotide and drug binding sites. This will allow distance mapping and direct monitoring of binding of ligands and changes in conformation during transport. Taken together, the results of this research should lead to a detailed understanding of the molecular mechanisms underlying drug transport by P-glycoprotein.
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