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 | $709,489 | Project Dates | 10/01/2001 - 00/00/0000 |
Fiscal Year | 2014 | 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 | |||
Our work is focused on the elucidation of the role of ATP-binding cassette (ABC) drug transporters in the development of multidrug resistance (MDR) in cancers and on the development of new therapeutic strategies to increase efficiency of chemotherapy for cancer patients. For these studies we are working with human P-glycoprotein (Pgp, ABCB1) and ABCG2 and have employed innovative approaches including biophysical techniques, directed mutagenesis, molecular modeling to elucidate molecular mechanisms of polyspecificity, the ATP hydrolysis catalytic cycle and drug transport, the use of Fab of monoclonal antibodies and various mutant proteins arrested at various steps in the catalytic cycle to enable us to fix the transporter in a particular conformation for resolution of the structure of Pgp by X-ray crystallography and for 3-D image analysis of single molecules by cryo-electron microscopy. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of Pgp and role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of Pgp. To monitor the conformational changes occurring during ATP hydrolysis and drug transport, based on a homology model, we have introduced either a single cys residue or two cys residues at various locations in cys-less Pgp, including regions from extracellular loops, transmembrane domains, intracellular loops, and nucleotide-binding domains (NBDs). We have begun to use the transition metal ion Forster resonance energy transfer (tmFRET) technique, which is a novel biophysical method developed to determine short range (5 - 20 angstroms) 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/vanadate trapped) conformations of Pgp. With tmFRET, preliminary results show that there is a small change in the distance of the two NBDs between the apo and closed conformations (less than 20 angstroms). Similarly, results of chemical crosslinking studies with bi-functional sulfhydryl group reagents indicate that human Pgp is a very flexible molecule and that its NBDs are much closer to each other than those in the published mouse Pgp structure. 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. Our studies with a triple mutant Pgp in a cys-less background demonstrate that when the primary binding site for cyclosporine A, tariquidar or valinomycin is disabled by mutagenesis, these drugs bind to alternate sites, which are capable of transport. Collectively, these data demonstrate that each substrate can bind to more than one site and all sites are capable of transport function, indicating that Pgp exhibits exceptional chemical flexibility for interaction with substrates and modulators. We also observed that certain point mutations in residues lining the drug-binding pocket exhibit a significantly lower level of basal ATPase activity even though the expression levels are normal. These findings suggest that the membrane lipids or peptides act as pseudo substrates and contribute to the basal activity. To develop fourth generation non-toxic and potent modulators of Pgp and ABCG2, we synthesized (S)-valine thiazole-derived cyclic and noncyclic peptidomimetic oligomers as well as other compounds by peptide coupling of diverse chemical scaffolds. With these approaches we have identified compound 28 as the most potent modulator (these studies were carried out in collaboration with Dr. Tanaji Talele at St. John's University, NY). Compound 28, similar to its parent compound QZ59SSS, might be useful for co-crystallization of human or mouse Pgp. 2. Development of potent non-toxic small molecule modulators of ABC transporters: We continue to study " |