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 | $768,848 | Project Dates | 01/01/2001 - 00/00/0000 |
Fiscal Year | 2015 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Digestive Diseases (25.0%) |
Brain (10.0%) Breast (25.0%) Colon/Rectum (10.0%) Kidney Cancer (5.0%) Kidney Disease (10.0%) Liver Cancer (10.0%) Nervous System (10.0%) Non Hodgkins Lymphoma (10.0%) Ovarian Cancer (10.0%) Pancreas (5.0%) Prostate (5.0%) Urinary System (10.0%) |
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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 Pgp; ii) the molecular basis of the polyspecificity of Pgp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) with Pgp and ABCG2; iv) determination of the binding site of nilotinib, a second generation TKI, on Pgp; v) pharmacophore features required for binding of nilotinib to Pgp and ABCG2; and vi) the fate of cell surface Pgp, its folding and stability. 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 efforts to the screening and development of TKIs and small molecule modulators for Pgp 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 Pgp and/or ABCG2. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of Pgp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of Pgp. 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 Pgp. 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 Pgp 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 angstrom in the apo (in the absence of ATP and drug-substrate) conformation. 2. Mechanism of the drug-mediated inhibition of Pgp ATPase activity. Most of the substrates or modulators of Pgp 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 Pgp 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 Pgp that are critical for the inhibition of ATP hydrolysis by high-affinity modulators. These structural motifs are located at the drug-binding pocket of Pgp. We found that drugs that inhibit the ATPase activity switch to stimulating the ATPase activity when any of these residues are mutated. For instance, zosuquidar inhibits the basal ATP hydrolysis of cysless WT Pgp with high affinity (IC50 = 10 nM). The inhibition is completely lost upon mutation of Y953 to alanine and is switched to stimulation when three polar residues are mutated (Y307A/Q725A/Y953A). Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with the tyrosines Y953 and Y310, respectively, in an edge-to-face conformation, helping the tyrosine |