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 the 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, transition metal ion Forster resonance energy transfer (tmFRET), chemical crosslinking, directed mutagenesis, and 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, we have been able to obtain by X-ray crystallography a 7- to 8-angstrom resolution structure of mouse Pgp purified from insect cells. 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 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 (Department of 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. In addition, transition metal ion Forster resonance energy transfer (tmFRET) is a novel biophysical method developed to determine short range (5 - 20 angstrom) and small-scale distances within different locations of the protein at very low concentrations. Using this sensitive fluorescence-based method, we have begun to determine the changes in distance associated with the apo and the closed (ATP/Vi trapped) conformations of P-gp. With tmFRET, preliminary results show that there is a small change in the distance of the two NBDs between the apo and closed conformations (less than 20 angstrom). 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. We have docked cyclosporine A, tariquidar, verapamil, valinomycin and FSBA in the drug-binding domain of human Pgp using the structure of mouse Pgp in QZ59RRR-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 clinically important tyrosine kinase inhibitors (TKIs) for their interactions with ABC drug transporters. In collaboration with Dr. Maria Baer (University of Maryland), we demonstrated that both PIM kinase inhibitor SGI-1776 and the FLT3 kinase inhibitor quizartinib modulate the function of ABCG2 at pharmacologically relevant concentrations with implications for chemosensitization and adverse drug interactions. We found that the recently developed TKIs saracatinib and Tandutinib interact potently with Pgp and ABCG2 affecting the chemosensitization of tumor cells (in collaboration with Drs. Zhe-Sheng Chen and Tanaji Talele [St. Johns University], and Li-wu Fu [Sun Yet Sen University, Guangzhou, China]). We have characterized the interaction of vemurafenib with Pgp and ABCG2, which is used for treatment of melanoma cells harboring V600E mutant BRAF kinase and found that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF (V600E) mutant A375 cells. These findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting that combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation (in collaboration with Dr. Chug-Pu Wu, Chang Gung University, Taiwan). In collaboration with Dr. Stuart Yuspa (LG, CCR, NCI), we have developed a high throughput Pgp-mediated efflux assay using the fluorescent and phase-contrast live cell imaging system, the IncuCyteTMFLR (Essen BioScience). This assay will be very useful for assessing drug-drug interactions and for predicting MDR in clinical treatment. 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. 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. Using this preparation, we have obtained by X-ray crystallography a 7- to 8-angstrom resolution structure of mouse Pgp in apo conformation. At this low resolution, the structure in apo conformation is slightly different than previously reported by Aller et al., in 2009. Currently, we are testing various crystallization conditions to improve the quality of crystals to obtain structure at high (less than 3 angstrom) resolution and to obtain a high-resolution structure in apo and closed conformations. We have joined the NIH-FEI living lab program to obtain the high-resolution structure of both human and mouse Pgp by using single particle cryo-electron microscopy studies. 4. Role of intracellular loops 1 and 3 in folding and stability of human Pgp: We investigated the role of residues in intracellular loops 1 and 3 in folding and maturation of human Pgp. The residue D164 in ICL1 and the residue D805 in ICL3 were replaced with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of Pgp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at lower temperature (27C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar) or small corrector molecules in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70 and the treatment with cyclosporine A reduces association of mutant Pgp with Hsp70, thus allowing it to be trafficked to the cell surface. These data demonstrate that the D164 and D805 residues are critical for proper folding of Pgp.
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