Tariquidar, a potent, nontoxic, third-generation P-glycoprotein (P-gp) inhibitor, is a possible reversal agent for central nervous system drug resistance. In animal studies, tariquidar has been shown to increase the delivery of P-gp substrates into the brain by severalfold. The aim of this study was to measure P-gp function at the human blood-brain barrier (BBB) after tariquidar administration using PET and the model P-gp substrate (R)-11 C-verapamil. Methods: Five healthy volunteers underwent paired (R)-11 C-verapamil PET scans and arterial blood sampling before and at 2 h 50 min after intravenous administration of tariquidar (2 mg/kg of body weight). The inhibition of P-gp on CD56-positive peripheral lymphocytes of each volunteer was determined by means of the 123 Rh efflux assay. Tariquidar concentrations in venous plasma were quantified using liquid chromatography/mass spectrometry. Results: Tariquidar administration resulted in significant increases (Wilcoxon test for paired samples) in the distribution volume (DV, 124% 6 15%) and influx rate constant (K 1 , 149% 6 36%) of (R)-11 C-verapamil across the BBB (DV, 0.65 6 0.13 and 0.80 6 0.07, P 5 0.043; K 1 , 0.034 6 0.009 and 0.049 6 0.009, P 5 0.043, before and after tariquidar administration, respectively). A strong correlation was observed between the change in brain DV after administration of tariquidar and tariquidar exposure in plasma (r 5 0.90, P 5 0.037). The mean plasma concentration of tariquidar achieved during the second PET scan (490 6 166 ng/mL) corresponded to 100% inhibition of P-gp function in peripheral lymphocytes. Conclusion: Tariquidar significantly increased brain penetration of (R)-11 C-verapamil-derived activity due to increased influx. As opposed to peripheral P-gp function, central P-gp inhibition appeared to be far from complete after the administered tariquidar dose.

Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.

P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization timeof-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino-and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.Multidrug resistance represents a serious obstacle to successful cancer chemotherapy. Although multifactorial in etiology, one type of multidrug resistance is associated with the overexpression of energy-dependent membrane-bound pumps, which intercept and efflux drugs before they reach their intracellular target structures. P-glycoprotein (ABCB1) represents a paradigm ATP-dependent efflux pump expressed in human cancer cells. In addition to its expression in cancer cells, P-gp is also physiologically expressed in a number of tissues such as intestinal epithelial cells, at the brush border of renal tubule epithelial cells, the canalicular side of hepatocytes, and in capillary endothelial cells forming the blood-brain barrier. It thus interferes with oral drug absorption and drug delivery to the brain, and it enhances renal and biliary excretion. P-gp has therefore attracted considerable attention as a nontarget in the field of drug development, because for a large number of active compounds, interaction with P-glycoprotein might compromise their future development into a drug. Considerable energy has therefore been devoted to the characterization of molecular features that make compounds P-gp substrates and to the definition of the molecular mechanism of drug transport by P-gp. A number of studies have dealt with the kinetics and thermodynamics of the transport process

The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by highresolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.

Recently, we have introduced [tris(1,10-phenanthroline)lanthanum(III)] trithiocyanate (KP772, FFC24) as a new lanthanum compound which has promising anticancer properties in vivo and in vitro. Aim of this study was to investigate the impact of ABC transporter-mediated multidrug resistance (MDR) on the anticancer activity of KP772. Here, we demonstrate that all MDR cell models investigated, overexpressing ABCB1 (P-glycoprotein), ABCC1 (multidrug resistance protein 1), or ABCG2 (breast cancer resistance protein) either due to drug selection or gene transfection, were significantly hypersensitive against KP772. Using ABCB1-overexpressing KBC-1 cells as MDR model, KP772 hypersensitivity was demonstrated to be based on stronger apoptosis induction and/or cell cycle arrest at unaltered cellular drug accumulation. KP772 did neither stimulate ABCB1 ATPase activity nor alter rhodamine 123 accumulation arguing against a direct interaction with ABCB1. Accordingly, several drug resistance modulators did not sensitize but rather protect MDR cells against KP772-induced cytotoxicity. Moreover, long-term KP772 treatment of KBC-1 cells at subtoxic concentrations led within 20 passages to a complete loss of drug resistance based on blocked MDR1 gene expression. When exposing parental KB-3-1 cells to subtoxic, stepwise increasing KP772 concentrations, we observed, in contrast to several other metallo-drugs, no acquisition of KP772 resistance. Summarizing, our data demonstrate that KP772 is hyperactive in MDR cells and might have chemosensitizing properties by blocking ABCB1 expression. Together with the disability of tumor cells to acquire KP772 resistance, our data suggest that KP772 should be especially active against notoriously drug-resistant tumor types and as second line treatment after standard chemotherapy failure.

KP1019 [indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] (FFC14A) is a metal complex with promising anticancer activity. Since chemoresistance is a major obstacle in chemotherapy, this study investigated the influence of several drug resistance mechanisms on the anticancer activity of KP1019. Here we demonstrate that the cytotoxic effects of KP1019 are neither substantially hampered by overexpression of the drug resistance proteins multidrug resistance-related protein 1, breast cancer resistance protein, and lung resistance protein nor the transferrin receptor and only marginally by the cellular p53 status. In contrast, P-glycoprotein overexpression weakly but significantly (up to 2-fold) reduced KP1019 activity. P-glycoprotein-related resistance was based on reduced intracellular KP1019 accumulation and reversible by known P-glycoprotein modulators. KP1019 dose dependently inhibited ATPase activity of P-glycoprotein with a K(i) of approximately 31 microM. Furthermore, it potently blocked P-glycoprotein-mediated rhodamine 123 efflux under serum-free conditions (EC(50), approximately 8 microM), however, with reduced activity at increased serum concentrations (EC(50) at 10% serum, approximately 35 microM). Moreover, P-glycoprotein-mediated daunomycin resistance could only be marginally restored by KP1019 in serum-containing medium, also indicating an influence of serum proteins on the interaction between KP1019 and P-glycoprotein. Acquired KP1019 resistance was investigated by selecting KB-3-1 cells against KP1019 for more than 1 year. Only an approximately 2-fold KP1019 resistance could be induced, which unexpectedly was not due to overexpression of P-glycoprotein or other efflux pumps. Accordingly, KP1019-resistant cells did not display reduced drug accumulation. Their unique cross-resistance pattern confirmed an ABC transporter-independent resistance phenotype. In summary, the likeliness of acquiring insensitivity to KP1019 during therapy is expected to be low, and resistance should not be based on overexpression of drug efflux transporters.

Terminal erythropoiesis is accompanied by extreme demand for iron to ensure proper hemoglobinization. Thus, erythroblasts must modify the "standard" posttranscriptional feedback regulation, balancing expression of ferritin (Fer; iron storage) versus transferrin receptor (TfR1; iron uptake) via specific mRNA binding of iron regulatory proteins (IRPs). Although erythroid differentiation involves high levels of incoming iron, TfR1 mRNA stability must be sustained and Fer mRNA translation must not be activated because iron storage would counteract hemoglobinization. Furthermore, translation of the erythroid-specific form of aminolevulinic acid synthase (ALAS-E) mRNA, catalyzing the first step of heme biosynthesis and regulated similarly as Fer mRNA by IRPs, must be ensured. We addressed these questions using mass cultures of primary murine erythroid progenitors from fetal liver, either undergoing sustained proliferation or highly synchronous differentiation. We indeed observed strong inhibition of Fer mRNA translation and efficient ALAS-E mRNA translation in differentiating erythroblasts. Moreover, in contrast to self-renewing cells, TfR1 stability and IRP mRNA binding were no longer modulated by iron supply. These and additional data stemming from inhibition of heme synthesis with succinylacetone or from iron overload suggest that highly efficient utilization of iron in mitochondrial heme synthesis during normal erythropoiesis alters the regulation of iron metabolism via the IRE/IRP system. (Blood. 2006;107: 4159-4167)

Overexpression of the xenotoxin transporter P-glycoprotein (P-gp) represents one major reason for the development of multidrug resistance (MDR), leading to the failure of antibiotic and cancer therapies. Inhibitors of P-gp have thus been advocated as promising candidates for overcoming the problem of MDR. However, due to lack of a high-resolution structure the concrete mode of interaction of both substrates and inhibitors is still not known. Therefore, structure-based design studies have to rely on protein homology models. In order to identify binding hypotheses for propafenone-type P-gp inhibitors, five different propafenone derivatives with known structure-activity relationship (SAR) pattern were docked into homology models of the apo and the nucleotide-bound conformation of the transporter. To circumvent the uncertainty of scoring functions, we exhaustively sampled the pose space and analyzed the poses by combining information retrieved from SAR studies with common scaffold clustering. The results suggest propafenone binding at the transmembrane helices 5, 6, 7 and 8 in both models, with the amino acid residue Y307 playing a crucial role. The identified binding site in the non-energized state is overlapping with, but not identical to, known binding areas of cyclic P-gp inhibitors and verapamil. These findings support the idea of several small binding sites forming one large binding cavity. Furthermore, the binding hypotheses for both catalytic states were analyzed and showed only small differences in their protein-ligand interaction fingerprints, which indicates only small movements of the ligand during the catalytic cycle.