ZIA BC 010030 (ZIA) | |||
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Title | Biochemical Analysis of Multidrug Resistance-linked Transport Proteins | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Ambudkar, Suresh | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $755,842 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Chemoprevention (30.0%) Childhood Cancers (5.0%) Digestive Diseases (20.0%) Inflammatory Bowel Disease (10.0%) |
Brain (5.0%) Breast (15.0%) Childhood Leukemia (5.0%) Colon/Rectum (5.0%) Kidney Cancer (5.0%) Kidney Disease (5.0%) Leukemia (5.0%) Liver Cancer (5.0%) Lung (15.0%) Melanoma (10.0%) Nervous System (5.0%) Non Hodgkins Lymphoma (5.0%) Ovarian Cancer (5.0%) Pancreas (5.0%) Prostate (5.0%) Stomach (5.0%) Urinary System (5.0%) |
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Research Type | |||
Systemic Therapies - Discovery and Development Complementary and Alternative Treatment Approaches |
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Abstract | |||
We have focused our efforts to elucidate: i) the catalytic cycle and transport pathway of human P-gp; ii) the molecular basis of the polyspecificity of P-gp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) and other natural product modulators with P-gp and ABCG2; iv) pharmacophore features required for binding of third generation cyclic peptide inhibitor derivatives (BTT compounds) to P-gp and ABCG2; and vi) the use of single particle cryo-EM for the analysis of the conformational landscape of human P-gp during its catalytic cycle. We have been employing cell-based, biochemical, biophysical, pharmacological, and physiological techniques along with molecular biology and molecular modeling approaches to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we have devoted considerable effort to the screening and development of TKIs and small molecule modulators of P-gp and ABCG2 that are used in the clinic for treatment of various types of cancers. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of P-gp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of P-gp. We are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. We recently identified a phenylalanine-tyrosine structural motif in the drug-binding pocket of P-gp that is critical for the inhibition of ATP hydrolysis by zosuquidar and other high-affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, we made a global alteration of the drug-binding pocket using gene synthesis to introduce fifteen tyrosine residues in the drug-binding pocket to study its effect on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine, two leucine, one isoleucine, and one methionine. Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size fluorescent substrates, its ability to transport large ( above 1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased, thus revealing a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. To our knowledge, this is the first report demonstrating that the global alteration of the drug-binding pocket with substitution of fifteen residues is well tolerated. The 15Y mutant has six mutated residues located in TMD1 and nine in TMD2. The generation of mutants with either six substitutions in TMD1 or nine in TMD2 will be useful to assess the role of each TMD of the transporter and to narrow the number of residues contributing to substrate size determination. 2. The mechanism of the molecular basis of polyspecificity, which is an important property of multidrug transporters, by using molecular modeling and mutagenesis approaches: Towards the goal of understanding the molecular basis of broad substrate specificity of P-gp, we characterized the effect of a detergent micelle environment on the drug-mediated inhibition of P-gp ATPase activity. Most of the substrates or modulators of P-gp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Several structural studies on purified P-gp have bee |