The human plasma membrane protein, P-glycoprotein (P-gp) is an ATP driven drug exporting pump that counteracts chemotherapy in cancer cells and limits the bioavailability of other therapeutic drugs in the body. The mechanism by which P-glycoprotein transports drugs remains enigmatic, thus the long range goal of this proposal is to elucidate this mechanism. Mutagenesis of human P-gp and quantitative kinetic, thermodynamic and spectroscopic methods of enzyme analysis will be employed to investigate the coupling of ATP hydrolysis and drug transport.
The first aim will be to generate mutant forms of P-glycoprotein and screen for transport/ATP hydrolysis coupling mutations. Mutational analysis will also be used to test proposed transport mechanisms and to assign functions to particular amino acid residues. For further assignments of function to structure, site-directed cysteine residues will be placed in the drug and nucleotide binding sites. This will allow for the placement of spectroscopic probes at defined locations that will provide direct observation of binding of ligands and changes in conformation during transport.
The second aim i s to quantitatively measure the functional and coupling interactions between the drug binding sites and nucleotide binding sites. Drug binding and transport will be examined in parallel with the ATP hydrolytic mechanism, and thermodynamic measurements of the interactions between them will be established. Next, the ATP hydrolytic reaction will be investigated to reveal the partial reaction steps involved in catalysis and how they relate to drug binding.
The third aim i s to study the structure and dynamics of the drug transport sites through the application of site-directed spin labeling. Together the results will lead to a detailed understanding of how P-glycoprotein transports drugs and provide a structural model of the drug binding sites. Such knowledge will aid in the rational design of drugs and methodologies to overcome or modulate this transporter, thus enhancing chemotherapy and improving cancer treatment as well as improving AIDS treatment.
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