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. Based on the thermodynamic and kinetic properties, we have identified the ES and EP stable reaction intermediates of the Pgp-mediated ATPase reaction. To monitor the conformational changes occurring during ATP hydrolysis and drug transport, we have begun to use the EPR spectroscopy and spin labeling approach. In collaboration with Drs. Stuart Durrell and Di Xia we have constructed homology model of human Pgp using Sav1866 and mouse mdr1b structure as a template. Based on this model we have introduced either single cys residue or two cys residues at various locations in the cys-less Pgp including regions from extracellular loops, transmembrane domains, intracellular loops, and NBDs. We have generated thirty single and twenty five double cys mutants so far. Most of these mutants after their expression in High-Five insect cells have been characterized. These mutants will be purified, labeled with spin label, MTSL and subjected to EPR spectroscopy analysis. In collaboration with John Golin (The Catholic University of America), we are using a genetic approach to identify second site mutations in a suppressor mutant which results in recovery of the drug transport activity of the yeast Pdr5p transporter that effluxes a variety of xenobiotic compounds. We observed that suppressor mutation S558Y in TM2 resulted in complete loss of drug transport activity without affecting the ability of Pdr5p to either bind drug substrates or to bind and hydrolyze ATP indicating that the drug transport and ATPase activities in this mutant are uncoupled. Interestingly the second site mutations that recover function are all localized to NBD region. These studies demonstrate that the use of combination of genetic and biochemical approach will allow us to map the signaling pathway between drug substrate binding sites and the ATP sites in Pdr5p transporter. 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 modulator curcumin to reverse the drug resistance in cancer. We in collaboration with Drs. Bjorn Bauer and Anika Hartz (University of Minnesota, MN) use rat brain capillaries as an ex vivo model of blood-brain barrier to demonstrate that curcumin at nanomolar concentration blocks the function of both Abcg2 and Pgp. In addition, by using Abcg2 knockout mice we further confirmed that oral curcumin increased C-max and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function. We propose that non-toxic concentrations of curcumin may be used to enhance drug exposure when the rate-limiting step of drug absorption and/or tissue distributions impacted by ABC drug transporter. In collaboration with Dr. Rajendra Prasad (Jawaharlal Nehru University, India), we also demonstrated that curcumin also modulates the activity of Cdr1p of Candida albicans, a fungal pathogen. Thus, curcumin may be used in combination with certain conventional antifungal drugs to reverse drug resistance in pathogenic yeast. We continue to study the tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. We found that Sunitinib (Sutent, SU11248), which is used in the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal tumors blocks the function of Pgp and ABCG2 and this may affect the bioavailability of drugs co-administered with sunitinib. In collaboration with Drs. Zhe-Sheng Chen (St. Johns Univ.), and L-W Fu (Sun Yat-Sen Univ., China) we observed that another small molecule, FG020326 (triaryl-substituted imidazole derivative) is a potent modulator of Pgp in both cell culture and xenograft model in mice. The ABCG2 transporter confers resistance to multiple chemotherapeutic agents. One approach to combat MDR mediated by this transporter is the development of inhibitors/modulators that block its function at non-toxic concentrations. In collaboration with Drs. Susan Bates, Curtis Henrich, Michael Dean and James McMahon (CCR and Molecular Targets Development Program, NCI) we have screened with a high-throughput assay a library of botryllamides, which are for their ability to inhibit ABCG2 function. In addition, we also screened the National Cancer Institute drug data screen data base (in collaboration with Drs. John Deeken and Susan Bates, NCI) and identified several novel ABCG2 substrates and other compounds, which interact possibly as modulators with this transporter. 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 7.5-10.0 mg of >99% homogeneously pure Pgp at 10-12 mg/ml concentration. For improving the crystallization of P-gp, we have initiated another approach where in the Fab of the conformation-sensitive monoclonal antibody, UIC2 is incubated along with the purified Pgp during crystallization. We have optimized the conditions to generate Fab of UIC2 with protein concentration in the range of 7-10 mg/ml. Additional experiments demonstrate that Fab of UIC2 binds to Pgp in detergent solution under similar conditions that are used for generations of crystals indicating that it is feasible to generate co-crystals of Pgp and UIC2-Fab. We have been successful in obtaining 900-1000 mg of purified UIC2 antibody from the hybridoma cell line HB1287. This will allow us to prepare 250--300 mg of Fab for testing new crystallization conditions. 4. Molecular mechanism of drug resistance in single- and multi-step selection with anticancer agents in cancer cells: To understand the mechanism of multidrug resistance (MDR) under clinical conditions, we established single-step doxorubicin-selected MCF-7 sublines using very low concentrations, 14 or 21 nM. We have found that ABCC2, ABCC4 and ABCG2 were overexpressed at the mRNA level in these single-step selected sublines. Yet, only ABCC4 and ABCG2 were overexpressed at the protein level. Both 14 and 21 nM single-step doxorubicin-selected sublines exhibit nearly 5-fold resistance to doxorubicin compared to parental MCF-7 cells. However, as ABCC4 does not confer resistance to doxorubicin it is most likely that ABCG2 is the major transporter responsible for the development of resistance, which is confirmed by using ABCG2-SiRNA. The stem cell markers including CD44 and CD24 are not enriched in single-step clones of MCF-7 selected with low concentrations of doxorubicin. 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 can be selected by chemotherapy (Calcagno et al., manuscript submitted;work was done in collaboration with Drs. Lyuba Varticovski and Michael M. Gottesman). 5. Evaluation of expression profiles of ABC transporters as well as other genes linked with multidrug resistance in patient tumor samples: These studies are carried out in collaboration with Dr. [summary truncated at 7800 characters]
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