A novel fluorescence-based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third-generation P-gp inhibitors and OATP1A2.

Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.

Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitors.

The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5'-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.

Intracellular Doppler Spectroscopy detects altered drug response in SKOV3 tumor spheroids with silenced or inhibited P-glycoprotein.

Intracellular Doppler spectroscopy is a form of low-coherence digital holography based upon Doppler detection of scattered light that measures drug response/resistance in tumor spheroids, xenografts, and clinical biopsies. Multidrug resistance (MDR) is one of the main causes of ineffective cancer treatment. One MDR mechanism is mediated by the MDR1 gene that encodes the drug efflux pump P-glycoprotein (Pgp). Overexpression of Pgp in some cancers is associated with poor chemotherapeutic response. This paper uses intracellular Doppler spectroscopy to detect Pgp-mediated changes to drug response in 3D tissues grown from an ovarian cancer cell line (SKOV3). The SKOV3 cell line was incrementally exposed to cisplatin to create a cell line with increased Pgp expression (SKOV3cis). Subsequently, MDR1 in a subset of these cells was silenced in SKOV3cis using shRNA to create a doxycycline inducible, Pgp-silenced cell line (SKOV3cis-sh). A specific Pgp inhibitor, zosuquidar, was used to study the effects of Pgp inhibition on the Doppler spectra. Increased drug sensitivity was observed with Pgp silencing or inhibition as determined by drug IC50s of paclitaxel-response of silenced Pgp and doxorubicin-response of inhibited Pgp, respectively. These results indicate that intracellular Doppler spectroscopy can detect changes in drug response due to silencing or inhibition of a single protein associated with drug resistance with important consequences for personalized medicine.

Structural insight into substrate and inhibitor discrimination by human P-glycoprotein.

ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo-electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate-hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.

Synthesis and Evaluation of the First Fluorescent Antagonists of the Human P2Y2 Receptor Based on AR-C118925.

The human P2Y2 receptor ( hP2Y2R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y2R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y2R antagonists, based on the known, non-nucleotide hP2Y2R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y2R (p Kd = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y2R. These properties make 98 one of the first tools for studying hP2Y2R distribution and organization.

Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1.

The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.

From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor.

The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions.

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment.

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp.

Dibenzosuberones as p38 mitogen-activated protein kinase inhibitors with low ATP competitiveness and outstanding whole blood activity.

p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a "deep pocket" moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy.

Amurensin G inhibits angiogenesis and tumor growth of tamoxifen-resistant breast cancer via Pin1 inhibition.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem among estrogen-receptor-positive breast cancer patients. We have previously reported that TAM-resistant MCF-7 (TAMR-MCF-7) cells have elevated angiogenic potential via Pin1-dependent vascular endothelial growth factor (VEGF) production. Vitis amurensis grape consumed as wine and fruit contains several resveratrol-like stilbenes or oligostilbenes. In this study, we screened for the most active compound to inhibit VEGF production from V. amurensis. Among the tested compounds, amurensin G most potently suppressed VEGF production in TAMR-MCF-7 cells. The enhanced VEGF gene transcription in TAMR-MCF-7 cells was suppressed by amurensin G. Molecular analyses using reporter genes with hypoxia response elements and activator protein-1 (AP-1) elements, and western blots revealed that the activities and the nuclear levels of hypoxia inducible factor-1 (HIF-1)α and AP-1 in TAMR-MCF-7 cells were decreased by amurensin G. Moreover, amurensin G concentration-dependently inhibited protein expression and gene transcription of Pin1 in TAMR-MCF-7 cells, which was dependent on E2F1 inhibition. Chick chorioallantoic membrane assays confirmed that amurensin G had significant antiangiogenic and antitumor growth effects in TMAR-MCF-7 cells. These results demonstrate for the first time that amurensin G may have therapeutic potential for TAM-resistant breast cancer through blocking of Pin1-mediated VEGF gene transcription.

Skepinone-L is a selective p38 mitogen-activated protein kinase inhibitor.

Until now, a lack of inhibitors with high potency and selectivity in vivo has hampered investigation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We describe the design of skepinone-L, which is, to our knowledge, the first ATP-competitive p38 MAPK inhibitor with excellent in vivo efficacy and selectivity. Therefore, skepinone-L is a valuable probe for chemical biology research, and it may foster the development of a unique class of kinase inhibitors.

Pt-catalyzed hydrative cyclization of 2-enynylbenzaldehydes and its application to faveline synthesis.

Various 2-[6-en-1-ynyl]benzaldehydes and their analogues were successfully cyclized via Huisgen-type [3+2] cycloaddition to the tetracyclic platinum-carbene complex, which would subsequently undergo hydration to afford the tricyclic products in good yields with excellent stereoselectivities. This hydrative cyclization was also applied to the faveline synthesis.

P-glycoprotein-mediated active efflux of the anti-HIV1 nucleoside abacavir limits cellular accumulation and brain distribution.

P-glycoprotein (P-gp)-mediated efflux at the blood-brain barrier has been implicated in limiting the brain distribution of many anti-HIV1 drugs, primarily protease inhibitors, resulting in suboptimal concentrations in this important sanctuary site. The objective of this study was to characterize the interaction of abacavir with P-gp and determine whether P-gp is an important mechanism in limiting abacavir delivery to the central nervous system (CNS). In vitro and in vivo techniques were employed to characterize this interaction. Abacavir stimulated P-gp ATPase activity at high concentrations. The cellular accumulation of abacavir was significantly decreased by approximately 70% in Madin-Darby canine kidney II (MDCKII)-MDR1 monolayers compared with wild-type cells and was completely restored by the P-gp inhibitors ((R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo(a,e)cyclopropa(c)cycloheptan-6-yl)-alpha-((5-quinoloyloxy)methyl)-1-piperazineethanol, trihydrochloride) (LY335979) and N-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide (GF120918). Directional flux experiments indicated that abacavir had greater permeability in the basolateral-to-apical direction (1.58E-05 cm/s) than in the apical-to-basolateral direction (3.44E-06 cm/s) in MDR1-transfected monolayers. The directionality in net flux was abolished by both LY335979 and GF120918. In vivo brain distribution studies showed that the AUC(plasma) in mdr1a(-/-) CF-1 mutant mice was approximately 2-fold greater than the AUC(plasma) in the wild type, whereas the AUC(brain) in the mutant was 20-fold higher than that in the wild type. Therefore, the CNS drug targeting index, defined as the ratio of AUC brain-to-plasma for mutant over wild type, was greater than 10. These data are the first in vitro and in vivo evidence that a nucleoside reverse transcriptase inhibitor is a P-gp substrate. The remarkable increase in abacavir brain distribution in P-gp-deficient mutant mice over wild-type mice suggests that P-gp may play a significant role in restricting the abacavir distribution to the CNS.

New bioactive prenylflavonoids and dibenzocycloheptene derivative from roots of Dendrolobium lanceolatum.

Two new flavanones (1 and 2), a new flavan (3), and a new rare dibenzocycloheptene derivative (4) together with a known flavan, 4'-hydroxy-2' ',2' 'dimethyl-pyranoflavan (5), were isolated from the roots of Dendrolobium lanceolatum. Their structures were established on the basis of spectral evidence, and an X-ray analysis was performed to confirm the structure of 4. Compounds 1-3 exhibited antimalarial activity with IC50 values of 2.6, 3.3, and 3.1 microg/mL, respectively. Compounds 1-5 showed moderate antimycobacterial activity with MIC values of 6.3, 12.5, 25, 25, and 50 microg/mL, respectively. In addition, 1 showed strong cytotoxicity against cancer cell lines KB, BC, and NCI-H187 with IC50 values of 1.2, 1.6, and 0.6 microg/mL, respectively, while 2 showed moderate cytotoxicity against the NCI-H187 cell line with an IC50 value of 8.1 microg/ mL.

Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.

Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.