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 continue our studies on the catalytic cycle and transport pathway of Pgp. To monitor the conformational changes occurring during ATP hydrolysis and drug transport, we use EPR spectroscopy and spin labeling. Based on a homology model, we have introduced either a 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 25 double- and 15 single-cys mutants so far. These mutants, after their expression in High-Five insect cells, were purified and found to retain function at the same level as wild-type protein. We have optimized the conditions for labeling 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 (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. In collaboration with John Golin (Catholic University), 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. With this approach, we found that the non-canonical and canonical Q-loop residues are functionally overlapping and equivalent in Pdr5p with asymmetric ATP sites. 2) Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: We continue to validate the use of the natural product, non-toxic modulator curcumin to reverse drug resistance in cancer. By screening compounds from the Developmental Therapeutic Program at NCI, we have identified a dual-target multifunctional modulator that interacts with both Pgp and ABCG2 and reverses the drug resistance mediated by these transporters. In addition, this compound selectively kills only drug-selected Pgp-expressing cells. It has no cytotoxic effect on Pgp (ABCB1)-transfected cells that are not exposed to any anticancer drug or to those cells that endogenously express high levels of this transporter. The toxicity in multidrug-resistant cancer cells expressing Pgp appears to be due to preferential induction of both apoptosis and autophagy (Wu et al., manuscript submitted). We continue to study the tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. In collaboration with Drs. Li-Wu Fu (Sun Yat-Sen University) and Zhe-Sheng Chen (St. Johns University), we demonstrated that apatinib, a small molecule multi-targeted tyrosine kinase inhibitor, reverses Pgp- and ABCG2-mediated multidrug resistance by inhibiting their transport function. This anticancer drug is in phase III clinical trial for treatment of non-small cell lung carcinoma and gastric cancer in China, and may improve chemotherapy in these patients. Furthermore, we have compared the interaction of the tyrosine kinase inhibitors imatinib, nilotinib and dasatinib with ABC drug transporters. These inhibitors were tested in murine and human hematopoietic stem cells in ex vivo assays. Our results show that these 3 inhibitors are substrates for Pgp and ABCG2, and we suggest that therapeutic doses of imatinib and nilotinib may help to minimize the role of Pgp and ABCG2 in oral absorption or confer drug resistance (collaboration with Dr. Susan Bates, Medical Oncology Branch, CCR). We have synthesized a fluorescent bodipy derivative of Tasigna (nilotinib) in collaboration with Dr. Craig Thomas (NCGC, NHGRI) that can be used to demonstrate efflux of Tasigna by both Pgp and ABCG2 (Shukla et al., manuscript submitted). These studies suggest that Bodipy-FL-Tasigna can potentially be used as an in vivo probe for imaging Pgp- and/or ABCG2-expressing cancer cells. In addition, we have synthesized 26 derivatives of nilotinib with the goal of identifying compounds that inhibit only BCR-ABL kinase or those that interact only with Pgp or ABCG2. In a collaborative study with Dr. Stuart Yuspa (Laboratory of Cancer Biology and Genetics, CCR), we observed that Pgp in human skin may be exploited for delivery of chemotherapeutic agents that target tumor vasculature. The protein kinase C activator, ingenol-3-angelate (Ing3A), when applied topically, was found to inhibit the growth of subcutaneous tumors derived from PAM212 and B16 (mouse melanoma). Our results demonstrated that Ing3A is a transport substrate for Pgp and the absorptive transport, dermal penetration and vascular damage contribute to the anticancer activity of Ing3A. 3) Resolution of three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of Pgp is an ongoing project. We have developed a purification scheme that has yielded total protein of 7.5-10.0 mg of greater than 99% homogeneously pure Pgp at 10-12 mg/ml concentration. The high-throughput screening laboratory at Hauptman Woodward Institute (Buffalo, NY) has designed a crystallization screen specifically for membrane proteins based on the fact that these proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the protein-detergent complex. This screen employs a combination of 10 polyethylene glycols, 11 salts, and 11 detergents to generate a 1536-cocktail crystallization screen. 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. To complement these studies, in collaboration with Dr. Di Xia (Crystallography Section, LCB), we have resolved the structure of purified Fab of the monoclonal antibody UIC2 at 1.6A resolution. This will be useful for co-crystallization of Pgp with UIC2-Fab. 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 3D cultures in vitro and form tumors in vivo. These results suggest that cells with the characteristics of breast cancer stem cells 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 a collaboration with Dr. Michael Gottesman (LCB). We assessed the expression profile of 380 MDR-linked genes in 140 ovarian cancer patient samples through analysis with a microfluidic TLDA chip. These studies resulted in identification of a 13-gene signature, which may be useful for prediction of clinical response to treatment of ovarian cancer patients (Gillet, et al. manuscript submitted).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010030-15
Application #
8175296
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2010
Total Cost
$1,012,470
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Patel, Bhargav A; Abel, Biebele; Barbuti, Anna Maria et al. (2018) Comprehensive Synthesis of Amino Acid-Derived Thiazole Peptidomimetic Analogues to Understand the Enigmatic Drug/Substrate-Binding Site of P-Glycoprotein. J Med Chem 61:834-864
Wu, Chung-Pu; Hsieh, Ya-Ju; Murakami, Megumi et al. (2018) Human ATP-binding cassette transporters ABCB1 and ABCG2 confer resistance to histone deacetylase 6 inhibitor ricolinostat (ACY-1215) in cancer cell lines. Biochem Pharmacol 155:316-325
Hsiao, Sung-Han; Murakami, Megumi; Yeh, Ni et al. (2018) The positive inotropic agent DPI-201106 selectively reverses ABCB1-mediated multidrug resistance in cancer cell lines. Cancer Lett 434:81-90
Wu, Chung-Pu; Murakami, Megumi; Hsiao, Sung-Han et al. (2018) SIS3, a specific inhibitor of Smad3 reverses ABCB1- and ABCG2-mediated multidrug resistance in cancer cell lines. Cancer Lett 433:259-272
Wu, Chung-Pu; Hsiao, Sung-Han; Murakami, Megumi et al. (2017) Tyrphostin RG14620 selectively reverses ABCG2-mediated multidrug resistance in cancer cell lines. Cancer Lett 409:56-65
Shukla, Suneet; Abel, Biebele; Chufan, Eduardo E et al. (2017) Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions. J Biol Chem 292:7066-7076
Zhang, Yun-Kai; Zhang, Xiao-Yu; Zhang, Guan-Nan et al. (2017) Selective reversal of BCRP-mediated MDR by VEGFR-2 inhibitor ZM323881. Biochem Pharmacol 132:29-37
Wang, Yi-Jun; Patel, Bhargav A; Anreddy, Nagaraju et al. (2017) Thiazole-valine peptidomimetic (TTT-28) antagonizes multidrug resistance in vitro and in vivo by selectively inhibiting the efflux activity of ABCB1. Sci Rep 7:42106
Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V (2017) Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency. Biochem Pharmacol 143:53-64
Wang, Yi-Jun; Zhang, Yun-Kai; Zhang, Guan-Nan et al. (2017) Regorafenib overcomes chemotherapeutic multidrug resistance mediated by ABCB1 transporter in colorectal cancer: In vitro and in vivo study. Cancer Lett 396:145-154

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