Our efforts are directed toward understanding the mechanism of action of the multidrug resistance linked P-glycoprotein (P-gp) or the multidrug transporter. P-gp, a product of the MDR1 gene in humans, is a member of the ATP-Binding Cassette (ABC) superfamily of transporters. P-gp functions as an ATP-dependent efflux pump for a variety of hydrophobic agents including cytotoxic natural product anticancer drugs, reversing agents and peptides. Our major goals include the determination of the interaction between the substrate-binding domain(s), the ATP binding/utilization sites of the transporter, and also the role of ATP hydrolysis in drug translocation by P-gp through the cell membrane. We are employing biochemical and molecular biological approaches for the development of model systems such as an in vitro artificial membrane system and the heterologous baculovirus-insect cell expression system for the analysis of wild- type and mutant P-gps. We have developed procedures for large scale purification of recombinant P-gp with full retention of biological activity from a baculovirus expression system. To facilitate the structural studies, we have expressed the glycosylation-deficient P-gp in the insect cell system and the mutant P-gp retains drug transport function similar to the wild-type protein. The analysis of effect of various anticancer drugs as well as reversing agents and peptides on the ATPase activity indicates that the ATP hydrolysis of P-gp is modulated by interaction between overlapping catalytic and inhibitory sites. We have used an antibody- depletion method to determine the number of P-gp molecules present at the cell surface of a variety of multidrug resistant cells, and this information was used to determine the stoichiometry of vinblastine transport and vinblastine-stimulated ATP hydrolysis. Our results demonstrate that there is a tight coupling between drug transport and ATP hydrolysis and that hydrolysis of 2-3 molecules of ATP is required to transport one molecule of vinblastine. Using highly efficient photoaffinity labeling of P-gp with a prazosin analog, we have presented evidence for the presence of two non-identical drug binding sites on the multidrug transporter. In addition, drug binding to P-gp appears to be modulated by agents that bind to a modulator site that is separate from the substrate binding site.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010030-03
Application #
6101022
Study Section
Special Emphasis Panel (LCB)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Macalou, S; Robey, R W; Jabor Gozzi, G et al. (2016) The linker region of breast cancer resistance protein ABCG2 is critical for coupling of ATP-dependent drug transport. Cell Mol Life Sci 73:1927-37
Wu, Chung-Pu; Sim, Hong-May; Huang, Yang-Hui et al. (2013) Overexpression of ATP-binding cassette transporter ABCG2 as a potential mechanism of acquired resistance to vemurafenib in BRAF(V600E) mutant cancer cells. Biochem Pharmacol 85:325-34
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Kannan, Pavitra; Telu, Sanjay; Shukla, Suneet et al. (2011) The ""specific"" P-glycoprotein inhibitor Tariquidar is also a substrate and an inhibitor for breast cancer resistance protein (BCRP/ABCG2). ACS Chem Neurosci 2:82-9
Sharma, Monika; Manoharlal, Raman; Shukla, Suneet et al. (2009) Curcumin modulates efflux mediated by yeast ABC multidrug transporters and is synergistic with antifungals. Antimicrob Agents Chemother 53:3256-65
Kim, In-Wha; Booth-Genthe, Catherine; Ambudkar, Suresh V (2008) Relationship between drugs and functional activity of various mammalian P-glycoproteins (ABCB1). Mini Rev Med Chem 8:193-200
Wright, Mollie H; Calcagno, Anna Maria; Salcido, Crystal D et al. (2008) Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res 10:R10
Robey, Robert W; Shukla, Suneet; Finley, Elizabeth M et al. (2008) Inhibition of P-glycoprotein (ABCB1)- and multidrug resistance-associated protein 1 (ABCC1)-mediated transport by the orally administered inhibitor, CBT-1((R)). Biochem Pharmacol 75:1302-12
Shukla, Suneet; Wu, Chung-Pu; Ambudkar, Suresh V (2008) Development of inhibitors of ATP-binding cassette drug transporters: present status and challenges. Expert Opin Drug Metab Toxicol 4:205-23

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