The long-term objectives of the application are to understand the structure, mechanism and biosynthesis of the human multidrug resistance P-glycoprotein (P-gp). This knowledge will then be used to develop strategies to shut off the transporter during cancer chemotherapy or treatment of AIDS. P-gp interferes in the treatment of cancer and AIDS by preventing anticancer drugs or HIV-1 protease inhibitors from reaching their targets. Efforts to develop effective inhibitory drugs that specifically bind to P-gp have been hampered by the lack of structural information about the drug-binding domain and the conformational changes that take place during transport. These issues will be addressed by testing the hypothesis that transmembrane segments 5, 6, 11 and 12 of P-gp form the drug-binding domain and that they undergo conformational changes during drug transport. These hypotheses will be tested using a Cys-less mutant of P-gp together with cysteine-scanning mutagenesis, thiol-reactive substrates and disulfide crosslinking. A new approach will also be developed to inhibit P-gp function by inhibiting folding and exit of P-gp from the endoplasmic reticulum. Preliminary evidence suggests that some compounds can block the conversion of a biosynthetic intermediate of P-gp into mature enzyme.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA080900-01
Application #
2824929
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Forry, Suzanne L
Project Start
1999-07-01
Project End
2004-04-30
Budget Start
1999-07-01
Budget End
2000-04-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Toronto
Department
Type
DUNS #
259999779
City
Toronto
State
ON
Country
Canada
Zip Code
M5 1-S8
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2004) Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane. J Biol Chem 279:7692-7
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2004) Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein. J Biol Chem 279:18232-8
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2003) Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site. J Biol Chem 278:20449-52
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2003) Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains. J Biol Chem 278:1575-8
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2003) Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites. J Biol Chem 278:50136-41
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2003) Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein. J Biol Chem 278:39706-10
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2003) Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding. J Biol Chem 278:13603-6
Loo, Tip W; Clarke, David M (2003) Application of chemical chaperones to the rescue of folding defects. Methods Mol Biol 232:231-43
Chen, Eva Y; Clarke, David M (2002) The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator. BMC Biochem 3:29
Loo, Tip W; Bartlett, M Claire; Clarke, David M (2002) The ""LSGGQ"" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence. J Biol Chem 277:41303-6

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