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 | $753,084 | Project Dates | null - null |
Fiscal Year | 2018 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Chemoprevention (30.0%) Digestive Diseases (20.0%) Inflammatory Bowel Disease (10.0%) Childhood Cancers (5.0%) |
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 (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%) 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 continue to focus 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 to P-gp and ABCG2; v) the use of single particle cryo-EM for the analysis of the conformational landscape of human P-gp during its catalytic cycle and vi) identification of the epitopes for three human P-gp-specific monoclonal antibodies (MRK-16, UIC2 and 4E3). 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. One goal is to understand the role of TMH1 and TMH7 in binding and transport of substrates. TMH1 and TMH7 are topologically identical helices in the two halves of P-gp and may together regulate substrate transport. We selected six conserved residues each from TMH1 (V53, I59, I60, L65, M68 and F72) and TMH7 (V713, I719, I720, Q725, F728 and F732) that appear to be part of the drug-binding pocket. These residues were substituted with alanine by gene synthesis to generate TMH1,7 mutant P-gp. We found that TMH1,7 mutant P-gp with twelve substitutions was expressed normally in HeLa cells, but was only able to transport three of the 25 tested substrates. We also observed partial loss of ATPase activity and lack of stimulation by the substrates. Based on these results, we propose that TMH1 and TMH7 play a critical role in the translocation step of the transport cycle of P-gp. 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 the 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. Last year we demonstrated that the high-affinity drug-binding site is inaccessible either due to a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. Thus, the structural and functional aspects of ligand interactions with purified P-gp need to be studied in a detergent-free native or artificial membrane environment. We have optimized the conditions to reconstitute purified protein in nanodiscs. We found that inclusion of 0.1% cholesteryl hemisuccinate during purification and preparation of nanodiscs using bacterial polar lipid mixture and MSP1 belt protein stabilizes the P-gp. The interaction of substrates and modulators with purified P-gp in nanodiscs is similar to that observed in native membrane vesicles. Currently, we are using P-gp reconstituted in nanodiscs to determine the affinities of substrates with surface plasmon resonance and |