Our studies are directed toward understanding the mechanism of action of the multidrug transporters that function as ATP-dependent efflux pumps for a variety of lipophilic cytotoxic natural product anticancer agents. These transporters play an important role in the development of multidrug resistance in most cancers. Our major goals include elucidation of the mechanism of substrate interaction with the multidrug transporters and how the energy from ATP hydrolysis is coupled to drug transport. We have biochemically characterized the human P-glycoprotein (P-gp) purified from MDR1-baculovirus infected insect cells and have optimized the conditions for obtaining >95% pure P-gp by using metal affinity chromatography followed by gel filtration. The properties such as drug-stimulated ATP hydrolysis, photoaffinity labeling with substrate analogs of the pure protein are similar to those observed in native membranes. We have also optimized conditions for the large-scale purification of P-gp from a baculovirus expression system. We can generate about 15 to 20 mg of pure protein from 1 g of crude membrane protein, which will be used for the resolution of three- dimensional structure. To eliminate the possible interference by glycosylation for the generation of two- and three-dimensional crystals, we have characterized the properties of glycosylation- deficient P-gp in baculovirus and vaccinia virus expression systems. The analysis of substrate binding based on photoaffinity labeling suggests that there are at least two non-identical sites on P-gp formed by both halves of the molecule. For the identification of the residue(s) involved in the drug binding, a 6-kDa peptide retaining most of label has been generated. We have shown that this peptide is generated from the proteolysis of the C-terminal region of P-gp. The analysis of peptides by MALDI-MS following extensive trypsin or cyanogen bromide treatment of the C-terminal 60 kDa fragment of P-gp indicates the presence of two unique prazosin analog labeled peptides which appear to originate from the region in and around TM 11 (residues 935 to 969). By using pure protein and enzyme-linked ATPase assay we have begun to dissect various steps in the catalytic cycle of P-gp. The multidrug resistance protein (MRP1) similar to P-gp plays in important role in the development of multidrug resistance in cancer cells. MRP1 exhibits functional similarities to P-gp and for this reason, understanding of the structure-function relationship of various domains of MRP1 will provide information about the mechanism of action of these ABC transporters. We have expressed functional MRP1 in a vaccinia virus based transient expression system and plan to assess the structure- function relationships of various domains of MRP1 by constructing chimeras containing MRP1 and MDR1 regions. - ABC Transport proteins, ATP hydrolysis, Cancer, Drug binding site , Drug transport, Multidrug resistance, MRP1, P-glycoprotein, - Neither Human Subjects nor Human Tissues

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010030-05
Application #
6433192
Study Section
(LCB)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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Gillet, Jean-Pierre; Calcagno, Anna Maria; Varma, Sudhir et al. (2012) Multidrug resistance-linked gene signature predicts overall survival of patients with primary ovarian serous carcinoma. Clin Cancer Res 18:3197-206
Gillet, Jean-Pierre; Wang, Junbai; Calcagno, Anna Maria et al. (2011) Clinical relevance of multidrug resistance gene expression in ovarian serous carcinoma effusions. Mol Pharm 8:2080-8
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
Chen, Shih-Yin; Pan, Chi-Jiunn; Nandigama, Krishnamachary et al. (2008) The glucose-6-phosphate transporter is a phosphate-linked antiporter deficient in glycogen storage disease type Ib and Ic. FASEB J 22:2206-13
Shukla, S; Sauna, Z E; Ambudkar, S V (2008) Evidence for the interaction of imatinib at the transport-substrate site(s) of the multidrug-resistance-linked ABC drug transporters ABCB1 (P-glycoprotein) and ABCG2. Leukemia 22:445-7
Wu, Chung-Pu; Calcagno, Anna Maria; Ambudkar, Suresh V (2008) Reversal of ABC drug transporter-mediated multidrug resistance in cancer cells: evaluation of current strategies. Curr Mol Pharmacol 1:93-105
Calcagno, A M; Fostel, J M; To, K K W et al. (2008) Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes. Br J Cancer 98:1515-24

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