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Principal Investigator: WILLIAM M ATKINS
Organization: UNIVERSITY OF WASHINGTON
Fiscal Year: 2019
Award: $299,338
Funding agency: National Institute of General Medical Sciences
Project Summary
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.
Terms: <ABC Transport Protein><ABC Transporter Protein><ABC Transporters><ABC15><ABCG2><ABCG2 gene><ABCP><ATP Hydrolysis><ATP phosphohydrolase><ATP-Binding Cassette Transporters><ATP-Binding Cassette, Sub-Family B, Member 1><ATP-Binding Cassette, Sub-Family G (WHITE), Member 2 Gene><ATP-Binding Cassette, Sub-Family G, Member 2><ATP-Binding Cassette, Subfamily G, Member 2><ATPase><Address><Adenosine Triphosphatase><Adenosinetriphosphatase><Anti-Cancer Agents><Antineoplastic Agents><Antineoplastic Drugs><Antineoplastics><Assay><BCRP><BCRP1><Behavior><Binding><Bioassay><Biochemical><Biologic Assays><Biologic Models><Biological Assay><Biological Models><Breast Cancer Resistance Protein><Cancer Drug><Cell Body><Cells><Communication><Complex><Computer Models><Computer Simulation><Computer based Simulation><Computerized Models><Coupling><Data><Detergents><Dissociation><Drug Design><Drug Interactions><Drug Targeting><Drug resistance><Drugs><EST157481><Environment><Fluorescence><Food Interactions><Goals><Individual><Infrastructure><Kinetics><Knowledge><Ligand Binding><Ligands><Lipid Bilayers><Lipids><Liposomal><Liposomes><MDR1 Protein><MRX><MXR1><Maps><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mathematical Model Simulation><Mathematical Models and Simulations><Measurement><Measures><Mediating><Medication><Membrane><Methodology><Methods><Mitoxantrone Resistance Protein><Model System><Modeling><Molecular><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Interaction><Molecular Modeling Nucleic Acid Biochemistry><Molecular Modeling Protein/Amino Acid Biochemistry><Molecular Models><Molecular Stereochemistry><Monitor><Multidrug Resistance 1><Multidrug Resistance Protein 1><Neoplastic Disease Chemotherapeutic Agents><Nucleotides><Nutrient><P-Glycoprotein><P-Glycoprotein 1><P-Glycoprotein Transporter><PGY-1 Protein><Peptides><Pharmaceutic Preparations><Pharmaceutical Preparations><Physiologic><Physiological><Placenta-Specific ATP-Binding Cassette Transporter><Play><Preparation><Property><Protein Dynamics><Proteins><Research><Resolution><Role><Safety><Solvents><Spectroscopy><Spectrum Analyses><Spectrum Analysis><Surface Plasmon Resonance><Therapeutic><Time><Toxic effect><Toxicities><Tumor-Specific Treatment Agents><Uncertainty><Vanadates><Xenobiotics><absorption><anti-cancer drug><anticancer agent><anticancer drug><base><cancer drug resistance><computational modeling><computational models><computational simulation><computer based models><computerized modeling><computerized simulation><conformation><conformational state><design><designing><doubt><drug disposition><drug resistant><drug/agent><experiment><experimental research><experimental study><improved><in silico><inhibitor><inhibitor/antagonist><lipid bilayer membrane><lipid nanoparticle><membrane structure><molecular dynamics><molecular modeling><nano disk><nanodisc technology><nanodisk><nutrient interaction><pharmacokinetic model><reconstitute><reconstitution><residence><residential site><resistance to Drug><resistance to cancer drugs><resistant to Drug><resistant to cancer drugs><social role><virtual simulation>