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 | $791,464 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2016 | 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 Disease (5.0%) Leukemia (5.0%) Liver Cancer (5.0%) Lung (5.0%) 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%) Kidney Cancer (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) with P-gp and ABCG2; iv) determination of the binding site of nilotinib, a second generation TKI, on P-gp; v) pharmacophore features required for binding of nilotinib 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 for P-gp and ABCG2. We found that several tyrosine kinase inhibitors, which are used in the clinic for treatment of various types of cancers, are either transport substrates or inhibitors of P-gp and/or ABCG2. 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 have begun to use tmFRET, which is a novel biophysical method developed to determine short range (5 - 25 angstrom) distances within different locations of the protein at very low concentrations. Using this sensitive fluorescence-based method, we have begun to determine the changes in distance associated with the apo and the closed (ATP/Vi trapped) conformations of P-gp. With tmFRET, preliminary results show that there is a significant change in the distance of the two NBDs between the apo and closed conformations (~ 20 angstrom). Similarly, results of disulfide crosslinking studies with the oxidant copper phenanthroline and bi-functional sulfhydryl group reagents indicate that human P-gp is a very flexible molecule and its NBDs are much closer to each other in the apo form. The distance between the C431 and C1074 residues in the Walker A motif of NBDs ranges from ~5 to 25 angstroms in the apo conformation (in the absence of ATP and drug-substrate). In collaboration with Dr. Di Pietro's group at the CNRS, University of Lyon, France, we found that the linker region of human ABCG2 harbors another C- signature motif and point mutations of the first two residues (L and S) of this motif completely abolished the transport-coupled ATPase activity, and resulted in complete reversal of cell resistance to mitoxantrone. These results suggest that the C2-signature motif in the linker region of ABCG2 plays an important mechanistic role in ATP binding and/or hydrolysis of this transporter. 2. Mechanism of 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. Zosuquidar, tariquidar and elacridar, high affinity inhibitors of transport function, also inhibit Pgp ATPase activity, while a variety of substrates including verapamil, paclitaxel and vinblastine stimulate ATP hydrolysis. The molecular mechanisms that are in play, in either case (stimulation or inhibition), remain elusive. The development of an effective P-gp inhibitor certainly would benefit from the understanding of drug-mediated inhibition of ATP hydrolysis. Using directed mutagenesis, we identified a pair of phenylalanine-tyrosine structural motifs of P-gp that are critical for the inhibition of ATP hydrolysis by high-affinity modulators. These structural motifs are located i |