Failure of chemotherapy against cancer and deadly microbial diseases is often correlated to cellular expression of the human multidrug transporter P-glycoprotein (Pgp). ATP-dependent drug transport by Pgp, in vivo, actively contributes to poor bioavailability of many therapeutic drugs as well as cellular resistance against various anticancer and antimicrobial agents. To overcome the clinical impasse created by Pgp, efforts have been directed towards development of pharmacological inhibitors capable of preventing Pgp-mediated drug transport. Despite this initiative, successful clinical circumvention of Pgp-mediated drug resistance is yet to be achieved. This is largely due to the lack of adequate knowledge of the sites and mechanism of inhibitor interaction with Pgp. Our preliminary data leads to the hypothesis that Pgp is an allosteric enzyme and its function as a drug transporter can be modulated by inhibitor interaction at a site distinct from the site of substrate recognition. Since allosteric inhibitors do not directly compete for substrate binding and translocation, it is likely that they inhibit drug transport by interrupting the normal catalytic turnover of Pgp. The goal of our study is to understand the molecular basis of allosteric modulation of Pgp-mediated drug transport. In particular, we will 1) determine the effect of allosteric modulators on the key catalytic events leading to ATP-driven drug transport by Pgp, 2) identify and map modulator interaction sites within Pgp mediating allosteric inhibition of transport, and 3) describe the molecular properties of Pgp-modulator interaction generating allosteric changes. Using transition state analogs of ATP hydrolysis, we will stabilize the major catalytic intermediate conformations of Pgp, and study their modulator-induced changes in the normal transition from one conformation to the next. Alanine-scanning mutagenesis of the transmembrane domains will be performed to identify and map the allosteric site(s) within Pgp. Using radioactive and photoaffinity analogs of the allosteric modulators, the structural determinants for Pgp-modulator interaction will be determined. Using a conformation sensitive monoclonal antibody of Pgp and intrinsic tryptophan fluorescence, we will study the nature and significance of conformational changes induced by allosteric modulators. The outcome of the study will advance our knowledge on the mechanism of functional regulation of Pgp, and at the same time help rational designing of Pgp modulators with improved efficacy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067926-05
Application #
7227051
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Okita, Richard T
Project Start
2003-05-01
Project End
2011-08-31
Budget Start
2008-03-01
Budget End
2011-08-31
Support Year
5
Fiscal Year
2008
Total Cost
$230,237
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
144676566
City
Bethesda
State
MD
Country
United States
Zip Code
20817