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 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. 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 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 the NBDs are much closer to each other than found in the published mouse Pgp structure. In related studies, we have characterized the interaction of an ATP analog, 5-fluorosulfonylbenzoyl 5-adenosine (FSBA) with Pgp. For the first time, we observed that FSBA interacts not only at the nucleotide-binding domains (NBDs, ATP sites) but also at the transport-substrate binding sites of Pgp. By HPLC/Mass spec analysis of FSBA labeled Pgp, we found that this ATP analog crosslinks to residues in NBDs, but not in the drug-binding sites in the transmembrane domains of this transporter. We believe that FSBA will be very useful for understanding the polyspecificity of Pgp. 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. Currently, we are characterizing the transport function of these single and double cys mutants in bac-mam baculovirus-transduced HeLa cells. The preliminary results suggest that these mutants are expressed, at a similar level as in the wild-type, at the plasma membrane of HeLa cells. 2. Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: We continue to validate the use of natural product, non-toxic modulators to reverse drug resistance in cancer. In collaboration with Drs. Pornngarm Limtrakul and Mei-Lin Go, we found that Kugacin J, stemofoline as well as functionalized aurones modulate activities of Pgp and ABCG2 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. Susan Bates (Medical Oncology Branch, CCR, NCI) and Zhe-Sheng Chen (St. Johns Univ.), we continue to characterize interaction of TKIs and other small molecules with ABC drug transporters. Interestingly we found that sildenafil (Viagra) is a potent modulator of Pgp and ABCG2, suggesting that this clinically approved drug with known side effects and drug-drug-interactions may be used to increase the efficacy of anticancer drugs. 3. Resolution of 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 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. We are currently testing these conditions and the hits obtained in the initial screening assays will assist us in identifying crystallization conditions optimal for obtaining good quality crystals of human Pgp. We continue to use Pgp-Fab of UIC2 complex for crystallization. To identify the epitope of UIC2 on Pgp, we have sequenced the Fab of UIC2. In addition, we have labeled the FAB with Alexa fluorescent dye and demonstrated that the Fab recognizes human Pgp, similar to UIC2 antibody, at the cell surface in intact HeLa cells. The identification of an epitope of this antibody will be of use to understand the conformation-sensitivity of UIC2. 4. Molecular mechanism of drug resistance in single- and multi-step selection with anticancer agents in cancer cells: Our recent work with the multi-step selected breast cancer cell line MCF-7/ADR, which was generated by continuous exposure to increasing concentrations of doxorubicin, demonstrates that 30 to 50% of these cells are CD44+/CD24-. In addition, these cells have enhanced capacity to self-renew, migrate and proliferate in three-D cultures in vitro and form tumors in vivo. These results suggest that cells with characteristics of breast cancer stem cells (CSCs) are enriched following prolonged drug treatment. From these findings, we speculate that prolonged drug treatment of patients with breast cancer may also result in increased numbers of cells with a highly chemotherapy-resistant cancer stem cell-like phenotype that may even be cancer stem cells. 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 Gottesmans 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. These studies resulted in identification of three gene signatures with a significant correlation with overall survival, response to treatment and progression free survival. These gene signatures may be useful for optimizing the treatment for ovarian cancer patients with effusions.
Showing the most recent 10 out of 89 publications
