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. Using this defined framework and the Walker B E556Q/E1201Q double mutant, we can precisely attribute the high-to-low affinity switch in the transport substrate binding site to the formation of the ES reaction intermediate. We have provided evidence that ATP-gamma-S, which is a non-hydrolyzable analog of ATP, can be used to generate the ES intermediate in wild-type protein. We have characterized a conserved subdomain 25 residues upstream of the Walker A motif of the ABC, which we named the A-loop (Aromatic residue interacting with the Adenine ring of ATP). In addition, we substituted the conserved H residue with Q, A, E, Y and K in the H-loop in both NBDs individually and together and these data suggest that the H residue in the H-loop most likely interacts with the gamma-P of ATP through H-bonding. 2. Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: screening and validation in mouse model systems: To develop modulator(s) that will inhibit multiple transporters, we screened synthetic compounds as well as natural products. We found that curcumin isolated from turmeric powder, is a potent modulator of all three transporters. Interestingly, curcumin is not very cytotoxic nor is it transported by ABCB1, C1 or G2. Additionally, we showed that tetrahydrocurcumin, which is a major metabolite of curcumin, also inhibits the activity of ABCB1, ABCC1 and ABCG2, suggesting that this metabolite also has inhibitory potential under in vivo conditions. Thus, curcumin, which modulates the function of three major ABC drug transporters, appears to be a promising candidate for development as an effective chemosensitizer, or at least as a nutraceutical adjuvant to enhance chemotherapy. 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. We found that 1, 4-dihydropyridines, nicardipine and nifedipine, which are used clinically as antihypertensive agents, are inhibitors as well as substrates of ABCG2. In a collaborative study with Dr. Maria R. Baer, we showed that the immunosuppressive agents used in solid organ and hematopoietic stem cell transplantation such as cyclosporine A, tacrolimus and sirolimus modulate drug transport by ABCB1, ABCC1 and ABCG2. We are also studying tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. The newly developed tyrosine kinase inhibitor AMN107 (nilotinib), which is an analog of imatinib (Gleevec) inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. We have shown in collaboration with Brendel et al. that it is a high affinity inhibitor of ABCG2. Another class of protein kinase inhibitors includes indolocarbazole (ICZ) and bisindolylmalemide (BIM) compounds. During their clinical development, both ICZs and BIMs were demonstrated to interact with ABC transporters. We have demonstrated that these inhibitors block ABCG2-mediated drug resistance and thus may increase oral bioavailability of ABCG2 substrates. Compounds from the Developmental Therapeutics Programs (DTP, NCI) chemical libraries have been analyzed for the development of inhibitors. Based on structural similarity hits, several compounds were projected to be potential inhibitors, and our group is involved in a joint effort to screen the potential inhibitors of ABC transporters. In addition to plants, marine organisms also provide a rich source of compounds from which novel agents can be developed to overcome multidrug resistance. In collaboration with Dr. Zhe-Sheng Chen, we have characterized the modulatory effect of sipholenol A, which is a sipholane triterpenoid isolated from the Red Sea sponge. Sipholenol A selectively overcomes the resistance to anticancer drugs mediated by Pgp (ABCB1). 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. We have observed that homogeneously pure Pgp at higher concentration stays in solution only in the presence of NaCl, and detergents such as DHPC, DDM. In the absence of salt, Pgp either aggregates or precipitates even in the presence of higher concentrations of DHPC or DDM detergents. This most recent preparation of homogeneously pure Pgp protein has retained the biological function of ATPase activity. In addition to wild-type protein, several mutants including the E556Q/E1201Q double mutant, which is trapped in an ES pre-hydrolysis transition-like state in the presence of ATP, have also been purified. Similarly, the stabilization of nucleotide-binding domains in the ES pre-hydrolysis transition state should help to generate crystals. 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 under clinical conditions, we have begun to examine how treatment regimens affect the expression of ABC drug transporters in single- and multi-step selection with anticancer drugs such as doxorubicin by employing RT-PCR, siRNA and chromatin immunoprecipitation (ChIP) in addition to regularly used biochemical and cell biological techniques. We have found that ABC transporter mRNA expression patterns vary with single- vs. multi-step treatment with doxorubicin in MCF-7 breast cancer cells. We have shown that multi-step selected MCF-7 cells overexpress only ABCB1 at the mRNA and protein levels and that in addition to gene amplification. We also 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. We also observed by using chromatin immunoprecipitation (ChIP) assay that the upregulation of ABCG2 is facilitated by histone hyperacetylation. 5. Characterization of Single nucleotide polymorphisms and haplotypes in ABCB1: In collaboration with Drs. Michael Gottesman and Kimchi-Sarfaty we have recently highlighted the importance of synonymous SNPs in determining protein conformation and function. Our study showed that in MDR1, synonymous SNPs in the context of a haplotype, with two synonymous (3435C>T & 1236C>T) and one non-synonymous (2677G>T) SNP, were associated with altered substrate and in [summary truncated at 7800 characters]
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