Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) is a significant problem in the treatment of many cancers, HIV, and psychiatric illnesses. Pgp is an ATP-binding cassette transporter that pumps many structurally unrelated drugs out of the cell through an ATP-dependent mechanism. Our recent X- ray structure of Pgp identified hydrophobic and aromatic amino acids that contribute to binding of two different inhibitors to the drug-binding site. In this proposal, we will test the hypothesis tht anticancer drugs bind to different subsets of residues within defined subpockets in the transmembrane regions of the protein. Using tryptophan (Trp) fluorescence quenching, we will map out sites of interaction of the purified protein with three prototypical substrates that occupy biochemically defined and distinct binding sites, as well as those of common anticancer drugs and newly identified inhibitors. The novelty of this proposal is our development of a functional Trp-free Pgp, and the introduction into this Trp-free background of one or more Trps at strategic positions to monitor drug binding. With this new approach, we will address the molecular mechanism and kinetics of drug/inhibitor binding and determine the mechanisms of action of the recently-identified blockers. We plan to obtain direct information on how different surfaces of the protein subpockets interact with anticancer drugs, and how different blockers work. The latter will be invaluable to develop mechanistically- and structurally-based panels of potential blockers for high-throughput screening.

Public Health Relevance

P-glycoprotein (Pgp) is the cell's cleaning machine, pumping harmful substances to the outside of the cell. In cancer chemotherapy, Pgp can cause problems by removing chemotherapy drugs from the tumor cells that they were intended to kill. By learning more about how Pgp recognizes the chemicals that it carries out of the cell, scientists may devise new drugs to prevent Pgp from interfering with the valuable effects of anticancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM102928-01A1
Application #
8365444
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2012-09-21
Project End
2014-09-20
Budget Start
2012-09-21
Budget End
2014-09-20
Support Year
1
Fiscal Year
2012
Total Cost
$347,454
Indirect Cost
$112,454
Name
Texas Tech University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430