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 such as continuous wave and pulse double electron-electron resonance ESR spectroscopy, directed mutagenesis, molecular modeling to elucidate molecular mechanisms of 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 tomography. Recently, for resolution of the 3-D structure, we have initiated studies with mouse Pgp (mdr1a). 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, we are using an EPR spectroscopy and spin labeling approach. 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 generated 25 double- and 25 single-cys mutants so far. These mutants, after their expression in High-Five insect cells, were purified and found to retain function to the same level as wild-type protein. We have optimized the conditions for labeling of these mutant proteins in detergent solution with the spin label MTSL for EPR spectroscopy analysis. We have begun to use continuous wave and pulse double electron-electron resonance (DEER) ESR spectroscopy in collaboration with Dr. Jack Freed at an NIH funded facility (Chemistry and Chemical Biology, Cornell University) to monitor conformational changes in the presence and absence of drug-substrate and ATP. The DEER ESR spectroscopy studies with the double cys mutants will also allow us to validate the homology model of human Pgp. Preliminary results of DEER and chemical crosslinking studies suggest 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. To improve the quality of DEER data, we have begun to use Pgp reconstituted into nanodiscs, which allows access to both extra- and intracellular regions without further manipulations. We have docked cyclosporine A, tariquidar, verapamil and FSBA in the drug-binding domain of human Pgp using the structure of mouse Pgp in QZ59SSS-bound form as a template. The residues interacting with these substrates/modulators have been substituted with cysteine to map the drug-binding sites. We found that neither cyclosporine A, tariquidar nor valinomycin were able to inhibit labeling with IAAP in the Y307C/Q725C and V982C triple mutant, indicating that the drugs had lost the ability to bind to the primary drug-binding site. However, these drugs still modulate the ATPase activity and transport function of mutant Pgp by binding at an alternate site. Additional studies suggest that Pgp exhibits exceptional chemical flexibility for interaction with substrates and modulators. 2.Development of potent non-toxic small molecule modulators/inhibitors of ABC transporters: We continue to study tyrosine kinase inhibitors (TKIs) for their potential use as inhibitors of ABC drug transporters. We have demonstrated that the second generation TKI nilotinib (Tasigna) is transported by both Pgp and ABCG2. In addition, for the first time we have synthesized and characterized a fluorescent derivative of Tasigna (bodipy-Tasigna), which may be a useful probe for functional analysis of these transporters in cancer cells and also in preclinical studies. In collaboration with Drs. Zhe-Sheng Chen and Tanaji Talele (St. Johns University), and Li-wu Fu (Sun Yet Sen University, Guangzhou, China) we continue to characterize the interaction of TKIs and other small molecules with ABC drug transporters. Several TKIs that are used in the clinic or are in phase II/III clinical trials including Ponatinib, saracatinib and neratinib interact with Pgp and potently modulate its transport function. These studies strongly suggest that combining one or two of these TKIs conventional chemotherapeutic drugs may help to increase the efficiency of chemotherapy in cancer patients.3. Resolution of the three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of Pgp is an ongoing project and for this we have developed a purification scheme that has yielded total protein of 7.5-10.0 mg of >99% homogeneously pure Pgp at 10-12 mg/ml concentration. The high-throughput screening laboratory at Hauptman Woodward Institute, Buffalo, New York, has designed a crystallization screen specifically for membrane proteins based on the fact that membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the protein-detergent complex. Recently, we have purified mouse Pgp (mdr1a) in large amounts (10-12 mg protein/ml) from insect cells using conditions developed for purification of human Pgp and initiated crystallization studies. We have joined the NIH-FEI living lab program to obtain the high-resolution structure of human Pgp by using single particle cryo-electron microscopy studies. 4. Elucidation of pathways involved in internalization and degradation of cell surface Pgp: We found that the half-life of Pgp at the cell surface in the colon cancer cell line HCT-15 is in the range of 24-26 hrs and treatment with the lysosomal/phagosomal inhibitor bafilomycin results in prolonged retention at the cell surface (half-life 32-36 hrs), indicating that the cell suface Pgp is degraded in lysosomes/phagosomes. When cells were treated with the proteasomal inhibitor MG132, the half-life of the protein was not altered, suggesting that the proteasomal pathway does not play a significant role in the degradation of cell surface Pgp. These studies may provide one or more therapeutic targets for the reversal of drug resistance by accelerating the degradation of cell surface transporters. 5. Evaluation of expression profiles of ABC transporters as well as other genes linked with MDR in patient tumor samples: These studies are carried out in collaboration with Dr. Michael Gottesman?s group in LCB. We have assessed the MDR-linked transcriptome in 32 unpaired ovarian serous carcinoma patients with the state-of-the art microfluidic TLDA chip-based qRT-PCR assay. When gene expression was added to four covariates (age, stage, CA125 level and surgical debulking, we found an 11-gene signature that provides a major improvement in overall survival prediction. This 11-gene signature allows a more precise prognosis for patients with sereous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. In another collaborative study with same group we compared the expression profile of MDR-linked 380 genes in tissue culture cancer cell lines and cells isolated from patient tumor samples. The expression profile of a majority of genes was quite different in tumor samples compared to cell lines grown in tissue culture, suggesting that cell lines grown for extended periods of time in culture may not mimic the in vivo cancer microenvironment.
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