P-glycoprotein (P-gp) is an ATP-dependent efflux transporter that plays a critical role in drug and xenobiotic distribution, drug-drug interactions, and drug-nutrient interactions. Efforts to modulate P-gp activity to control cellular drug resistance or to modulate the action of existing drugs have been only modestly successful. A barrier to progress in the design of P-gp inhibitors has been the uncertainty about its catalytic mechanism. Different types of drugs or ligands elicit different behaviors, wherein some stimulate ATP hydrolysis and are transported, while others stimulate ATP hydrolysis but are not transported. Other drugs inhibit P-gp without stimulating ATP hydrolysis. The mechanism by which different drugs elicit different behaviors is unclear. Specifically, the conformational changes that mediate communication between the nucleotide binding domains (NBDs) that hydrolyze ATP and the transmembrane helices (TMHs) that bind and release xenobiotics remain unknown.
One aim of this proposal is to map by H/D exchange mass spectrometry the ligand-dependent conformational changes in the NBDs and the TMHs. This will be performed with Pgp incorporated into lipid bilayer nanodiscs of defined lipid composition. By monitoring the nucleotide-dependent and drug-dependent changes in solvent exposure and dynamics of specific peptides in the sequence of each protein, with inhibitors, substrates, uncouplers and allosteric modulators, the conformational changes that correlate with each behavior will be identified.
A second aim of these studies is to measure the on rates and off rates of drug binding to and dissociating from P-gp in varying conformational states. There are currently no data concerning these rates, which are likely to define ligand behavior, as a substrate vs. inhibitor vs. uncoupler. These measurements will be made via surface plasmon resonance and fluorescence correlation spectroscopy with P-gp nanodiscs. In order to correlate the conformational mapping and off rate information with physiologic behavior, cell based transport activity will be measured for representative drugs with different behaviors. Finally, this proposal aims to explore the methodological advancements offered by nanodiscs with an related drug transporter, BCRP. These studies will add methodological infrastructure to the larger transporter field, increase our fundamental understanding of P-gp, further inform pharmacokinetic models, and facilitate drug design aimed to modulate P-gp.

Public Health Relevance

Mechanisms of drug transporters such as P-glycoprotein (P-gp) play a central role in drug disposition, efficacy, and safety, but are poorly understood because they are difficult to purify in quantities sufficient for biochemical characterization. This proposal aims to study P-gp and the breast cancer resistance protein (BCRP) in lipid nanoparticles that facilitate biochemical studies. The results will add to our understanding of P-gp and BCRP and could enhance efforts to design inhibitors that could improve the therapeutic benefit of other drugs that are cleared by them.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM121603-02
Application #
9398139
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
2017-01-01
Project End
2020-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Washington
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195