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, directed mutagenesis, molecular modeling to elucidate molecular mechanisms of polyspecificity, 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 microscopy. 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, 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 the transition metal ion Forster resonance energy transfer (tmFRET) technique, which is a novel biophysical method developed to determine short range (5 - 20 angstroms) 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/vanadate trapped) conformations of Pgp. 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 angstroms). Similarly, results of chemical crosslinking studies with bi-functional sulfhydryl group reagents indicate 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 are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. Our studies with a triple mutant Pgp in a cys-less background demonstrate that when the primary binding site for cyclosporine A, tariquidar or valinomycin is disabled by mutagenesis, these drugs bind to alternate sites, which are capable of transport. Collectively, these data demonstrate that each substrate can bind to more than one site and all sites are capable of transport function, indicating that Pgp exhibits exceptional chemical flexibility for interaction with substrates and modulators. We also observed that certain point mutations in residues lining the drug-binding pocket exhibit a significantly lower level of basal ATPase activity even though the expression levels are normal. These findings suggest that the membrane lipids or peptides act as pseudo substrates and contribute to the basal activity. To develop fourth generation non-toxic and potent modulators of Pgp and ABCG2, we synthesized (S)-valine thiazole-derived cyclic and noncyclic peptidomimetic oligomers as well as other compounds by peptide coupling of diverse chemical scaffolds. With these approaches we have identified compound 28 as the most potent modulator (these studies were carried out in collaboration with Dr. Tanaji Talele at St. John's University, NY). Compound 28, similar to its parent compound QZ59SSS, might be useful for co-crystallization of human or mouse Pgp. 2. Development of potent non-toxic small molecule modulators of ABC transporters: We continue to study clinically important tyrosine kinase inhibitors (TKIs) for their interactions with ABC drug transporters. By employing molecular docking, mutational mapping and synthesizing derivatives, we have identified the binding site for nilotinib on Pgp. Nilotinib binds to the primary site-1 in the drug-binding pocket and residues Y307, M949 and A985 are critical for its interaction with Pgp. We synthesized 25 derivatives of nilotinib and used a three-dimensional quantitative structure-activity relationship method to determine pharmacophore features required for binding of nilotinib to Pgp and ABCG2. A seven-point pharmacophore for Pgp and a six-point pharmacophore for ABCG2 inhibitory activity were generated. In addition, the comparison of pharmacophore features of nilotinib for its interaction with Pgp, ABCG2 and BCR-ABL kinase help to understand the difference in affinity of imatinib and nilotinib for these target proteins. The derived models clearly demonstrated high predictive power for test sets of Pgp and ABCG2 inhibitors. These findings can be exploited to design novel TKIs that would not be recognized by ABC drug transporters. In collaboration with several groups, we have characterized interaction of recently developed TKIs including WHI-P154, icotinib, ARRY-334543 and motesanib (AMG706) with ABC drug transporters. Of these, WHI-P154, icotinib, and ARRY-334543 specifically interact with ABCG2 and motesanib with Pgp. 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. Due to the flexible nature of human Pgp and the difficulty of generating crystals of good diffraction quality, we are also using single particle analysis by the cryo-electron microscopy technique. The current studies indicate that the structural features of human Pgp in the presence and absence of a Fab of conformation-sensitive monoclonal antibody can be observed at 15- to 20-angstrom resolution. We are optimizing conditions to reach a sub-nanometer resolution to obtain the structure of human Pgp in at least three different (apo, ADP-vanadate trapped and Fab-bound) conformations. The structural studies are carried out in collaboration with Drs. Di Xia and Sriram Subramanian. 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. Residue D164 in ICL1 and 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, modulators or small corrector molecules in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70 and treatment with cyclosporine A reduces association of mutant Pgp with Hsp70, thus allowing it to be trafficked to the cell surface. These findings suggest that a similar strategy might be useful for the rescue of other disease-linked ABC transporters such as CFTR and ABCB11
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