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The pregnane X receptor (PXR) regulates drug metabolism by regulating the expression of drug-metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which is involved in the metabolism of >50% of clinically prescribed drugs. The activity of PXR can be controlled by the binding of small molecule agonists or antagonists. Because of its unique ligand binding pocket, PXR binds promiscuously to structurally diverse chemicals. To study the structure-activity relationship, novel modulators for PXR are needed. Here we report the virtual screening of ∼25,000 natural product derivatives from the ZINC database using the Molecular Operating Environment docking software tool against the PXR-rifampicin complex X-ray crystal structure. Our screening resulted in identification of compounds based on the lowest S score, which measures Gibbs free energy. Interestingly, we found that the compounds that bind directly to PXR, as revealed in an intrinsic tryptophan fluorescence assay, modulate CYP3A4 promoter activity differentially in HepG2 cells. Mutational analysis and docking studies showed that these compounds bind broadly in the ligand binding pocket but interact with different amino acid residues. We further investigated the mechanism of binding by analyzing the functional groups that are important for distinguishing agonists from antagonists. The approach we used to identify novel modulators that bind to PXR can be useful for finding novel modulators of PXR., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras-GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras-GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.

DGKs (diacylglycerol kinases) catalyse the conversion of diacylglycerol into PA (phosphatidic acid), a positive modulator of mTOR (mammalian target of rapamycin). We have found that chenodeoxycholic acid and the synthetic FXR (farnesoid X receptor) ligand GW4064 induce the mRNA and protein expression of DGKθ in the HepG2 cell line and in primary human hepatocytes. Reporter gene studies using 1.5 kB of the DGKθ promoter fused to the luciferase gene revealed that bile acids increase DGKθ transcriptional activity. Mutation of putative FXR-binding sites attenuated the ability of GW4046 to increase DGKθ luciferase activity. Consistent with this finding, ChIP (chromatin immunoprecipitation) assays demonstrated that bile acid signalling increased the recruitment of FXR to the DGKθ promoter. Furthermore, GW4064 evoked a time-dependent increase in the cellular concentration of PA. We also found that GW4064 and PA promote the phosphorylation of mTOR, Akt and FoxO1 (forkhead box O1), and that silencing DGKθ expression significantly abrogated the ability of GW4046 to promote the phosphorylation of these PA-regulated targets. DGKθ was also required for bile-acid-dependent decreased glucose production. Taken together, our results establish DGKθ as a key mediator of bile-acid-stimulated modulation of mTORC2 (mTOR complex 2), the Akt pathway and glucose homoeostasis.

The atherogenic cytokine IL-6 (interleukin-6) induces pro-inflammatory gene expression in VECs (vascular endothelial cells) by activating the JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3) signalling pathway, which is normally down-regulated by the STAT3-dependent induction of the E3 ubiquitin ligase component SOCS3 (suppressor of cytokine signalling 3). Novel treatments based on the regulation of SOCS3 protein levels could therefore have value in the treatment of diseases with an inflammatory component, such as atherosclerosis. To this end we carried out a screen of 1031 existing medicinal compounds to identify inducers of SOCS3 gene expression and identified the flavanoids naringenin and flavone as effective inducers of SOCS3 protein, mRNA and promoter activity. This was in contrast with the action of traditional JAK/STAT3 inhibitors and the polyphenol resveratrol, which effectively suppress SOCS3 gene expression. Both naringenin and flavone also effectively suppressed IL-6-stimulated phosphorylation of STAT3 (Tyr⁷⁰⁵) which led to suppression of IL-6-induction of the atherogenic STAT3 target gene MCP1 (monocyte chemotactic protein-1), suggesting that their ability to induce SOCS3 gene expression is STAT3-independent. Supporting this idea was the observation that the general kinase inhibitor compound C inhibits flavone- and cAMP-dependent, but not JAK-dependent, SOCS3 induction in VECs. Indeed, the ability of flavanoids to induce SOCS3 expression requires activation of the ERK (extracellular-signal-regulated kinase)-dependent transcription factor SP3, and not STAT3. In the present paper we therefore describe novel molecular actions of flavanoids, which control SOCS3 gene induction and suppression of STAT3 signalling in VECs. These mechanisms could potentially be exploited to develop novel anti-atherogenic therapies.

The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a regulatory factor of the cell cycle. Its transcriptional activation and protein stability are tightly controlled by several distinct mechanisms. S100A11 is a member of the S100 family of Ca²⁺-binding proteins involved in several biological processes, including cell cycle progression and signal transduction. In the present study, we show that down-regulation of S100A11 results in the reduction of p21 protein in human HaCaT keratinocytes. It appears that a ubiquitin-independent proteasomal degradation process is involved in p21 degradation in S100A11 down-regulated cells. The application of a proteasome inhibitor stabilized p21 protein in these cells. Analysis of distinct signal transduction pathways revealed a disturbed phosphatidylinositol-3-kinase/Akt pathway after S100A11 knockdown. We determined that the glycogen synthase kinase-3, which is negatively regulated by phosphatidylinositol 3-kinase/Akt, was activated in cells possessing knocked-down S100A11 and appears to be involved in p21 protein destabilization. The application of a specific inhibitor of glycogen synthase kinase 3 resulted in an increase of the p21 protein level in S100A11 down-regulated HaCaT cells. Glycogen synthase kinase 3 is able to phosphorylate p21 at T57, which induces p21 proteasomal turnover. Mutation of the glycogen synthase kinase 3 site threonine 57 into alanine (T57A) stabilizes p21 in HaCaT cells lacking S100A11. Beside decreased p21 protein, down-regulation of S100A11 triggered the induction of apoptosis in HaCaT cells. These observations suggest that S100A11 is involved in the maintenance of p21 protein stability and appears to function as an inhibitor of apoptosis in human HaCaT keratinocyte cells. Thus, the data shed light on a novel pathway regulating p21 protein stability., (© 2013 FEBS.)

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu(7.39) that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a "trigger" for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu(7.39), whereas the role of this residue was less clear for chemically distinct agonists.

Purpose: Specific tyrosine kinase inhibitors were recently reported to modulate the activity of ABC transporters, leading to an increase in the intracellular concentration of their substrate drugs. In this study, we determine whether PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could reverse ABC transporter-mediated multidrug resistance., Methods: 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapatinibrazolium bromide assay was used to determine the effect of PD173074 on reversal of ABC transporter-mediated multidrug resistance (MDR). In addition, [³H]-paclitaxel accumulation/efflux assay, western blotting analysis, ATPase, and photoaffinity labeling assays were done to study the interaction of PD173074 on ABC transporters., Results: PD173074 significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to substrate anticancer drugs colchicine, paclitaxel, and vincristine. This effect of PD173074 is specific to ABCB1, as no significant interaction was detected with other ABC transporters such as ABCC1 and ABCG2. The observed reversal effect seems to be primarily due to the decreased active efflux of [³H]-paclitaxel in ABCB1 overexpressing cells observed in efflux assay. In addition, no significant change in the ABCB1 expression was observed when ABCB1 overexpressing cells were exposed to 5 μM PD173074 for up to 3 days, thereby further suggesting its role in modulating the function of the transporter. In addition, PD173074 stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Interestingly, PD173074 did not inhibit photolabeling of ABCB1 with [¹²⁵I]-iodoarylazidoprazosin (IAAP), showing that it binds at a site different from that of IAAP in the drug-binding pocket., Conclusions: Here, we report for the first time, PD173074, an inhibitor of the FGFR, to selectively reverse ABCB1 transporter-mediated MDR by directly blocking the efflux function of the transporter.

Context: The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of ¹³¹I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient., Objective: Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors., Results: Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells., Conclusions: We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.

Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of fibroblast growth factor 21 (FGF21). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF21 within hours. Functional relevance was evidenced in mice with genetic deletion of FGF21, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF21-dependent pathways.

Type 2 diabetes (T2D) has emerged as a major threat to human health in most parts of the world. Therapeutic strategies aimed at improving pancreatic β cell function are predicted to prove beneficial for the treatment of T2D. In the present study, we demonstrate that drug-mediated, chronic, and selective activation of β cell G(q) signaling greatly improve β cell function and glucose homeostasis in mice. These beneficial metabolic effects were accompanied by the enhanced expression of many genes critical for β cell function, maintenance, and differentiation. By employing a combination of in vivo and in vitro approaches, we identified a novel β cell pathway through which receptor-activated G(q) leads to the sequential activation of ERK1/2 and IRS2 signaling, thus triggering a series of events that greatly improve β cell function. Importantly, we found that chronic stimulation of a designer G(q)-coupled receptor selectively expressed in β cells prevented both streptozotocin-induced diabetes and the metabolic deficits associated with the consumption of a high-fat diet in mice. Since β cells are endowed with numerous receptors that mediate their cellular effects via activation of G(q)-type G proteins, our findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.

Selective CB2 agonists have the potential for treating pain without central CB1-mediated adverse effects. Screening efforts identified 1,2-dihydro-3-isoquinolone 1; however, this compound has the drawbacks of being difficult to synthesize with two asymmetric carbons on an isoquinolone scaffold and of having a highly lipophilic physicochemical property. To address these two major problems, we designed the 2-pyridone-based lead 15a, which showed moderate affinity for CB2. Optimization of 15a led to identification of 39f with high affinity for CB2 and selectivity over CB1. Prediction of the binding mode of 39f in complex with an active-state CB2 homology model provided structural insights into its high affinity for CB2., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The anti-diabetic drug glibenclamide inhibits K(ATP) channels in pancreatic β-cells and stimulates insulin release. It also causes adverse effects, among which are abdominal pain, gastrointestinal disturbances and nocturia. We report that glibenclamide activates human TRPA1 in a concentration range that is commonly used to induce inhibition of K(ATP) channels in vitro. Glibenclamide generates calcium transients in HEK293t cells transiently transfected with human TRPA1, which are inhibited by the selective TRPA1 antagonist HC030031 and also evokes outwardly rectifying currents mediated by recombinant TRPA1. Glibenclamide activates a subpopulation of mouse primary sensory neurons, most of which are also sensitive to the selective TRPA1 agonist mustard oil. This glibenclamide sensitivity is completely abolished by genetic ablation of TRPA1. Taken together, our data demonstrate that glibenclamide is an agonist of human TRPA1, which may explain some of the adverse effects of the drug., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background and Purpose: Previous work in our laboratory showed opioid agents inhibit cytokine expression in astrocytes. Recently, Watkins and colleagues hypothesized that opioid agonists activate toll-like receptor 4 (TLR4) signalling, which leads to neuroinflammation. To test this hypothesis, we characterized LPS and opioid effects on TLR4 signalling in reporter cells., Experimental Approach: NF-κB reporter cells expressing high levels of TLR4 were used to compare LPS and opioid effects on NF-κB activation, a pathway activated by TLR4 stimulation., Key Results: LPS increased TLR4 signalling in a concentration-dependent manner and was antagonized by LPS antagonist (LPS-RS, from Rhodobacter sphaeroides). A concentration ratio analysis showed that LPS-RS was a competitive antagonist. The opioid agonists, morphine and fentanyl, produced minor activation of TLR4 signalling when given alone. When tested following LPS stimulation, opioid agonists inhibited NF-κB activation but this inhibition was not blocked by the general opioid antagonist, naloxone, nor by the selective μ opioid receptor antagonist, β-FNA. Indeed, both naloxone and β-FNA also inhibited NF-κB activation in reporter cells. Further examination of fentanyl and β-FNA effects revealed that both opioid agents inhibited LPS signalling in a non-competitive fashion., Conclusions and Implications: These results show that LPS-RS is a competitive antagonist at the TLR4 complex, and that both opioid agonists and antagonists inhibit LPS signalling in a non-competitive fashion through a non-GPCR, opioid site(s) in the TLR4 signalling pathway. If confirmed, existing opioid agents or other drug molecules more selective at this novel site may provide a new therapeutic approach to the treatment of neuroinflammation., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. To investigate the molecular mechanism responsible for these effects, the ability of PEA and of pain-inducing stimuli such as capsaicin (CAP) or bradykinin (BK) to influence intracellular calcium concentrations ([Ca²⁺](i)) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARα and of TRPV1 channels in PEA-induced effects was also studied., Experimental Approach: [Ca²⁺](i) was evaluated by single-cell microfluorimetry in differentiated F11 cells. Activation of TRPV1 channels was assessed by imaging and patch-clamp techniques in CHO cells transiently-transfected with rat TRPV1 cDNA., Key Results: In F11 cells, PEA (1-30 μM) dose-dependently increased [Ca²⁺](i). The TRPV1 antagonists capsazepine (1 μM) and SB-366791 (1 μM), as well as the PPARα antagonist GW-6471 (10 μM), inhibited PEA-induced [Ca²⁺](i) increase; blockers of cannabinoid receptors were ineffective. PEA activated TRPV1 channels heterologously expressed in CHO cells; this effect appeared to be mediated at least in part by PPARα. When compared with CAP, PEA showed similar potency and lower efficacy, and caused stronger TRPV1 currents desensitization. Sub-effective PEA concentrations, closer to those found in vivo, counteracted CAP- and BK-induced [Ca²⁺](i) transients, as well as CAP-induced TRPV1 activation., Conclusions and Implications: Activation of PPARα and TRPV1 channels, rather than of cannabinoid receptors, largely mediate PEA-induced [Ca²⁺](i) transients in sensory neurons. Differential TRPV1 activation and desensitization by CAP and PEA might contribute to their distinct pharmacological profile, possibly translating into potentially relevant clinical differences., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Cinacalcet is predominantly used to treat secondary hyperparathyroidism due to end-stage renal failure, but, more recently, its potential clinical efficacy in treating patients with loss-of-function mutations in the calcium-sensing receptor (CaSR) has been recognized. Many clinically relevant CaSR mutations are located in the heptahelical membrane spanning and extracellular loop regions of the receptor, where allosteric modulators are predicted to bind. The aim of the present study was to investigate the impact of such mutations on the pharmacoregulation of the CaSR by the positive and negative allosteric modulators, cinacalcet and NPS-2143, respectively. Both cinacalcet and NPS-2143 effectively rescued mutants whose cell surface expression was substantially impaired, suggesting that both classes of drug can stabilize a receptor conformation that is trafficked more effectively to the cell surface. In addition, functional impairments in almost all mutant CaSRs were rescued by either cinacalcet or NPS-2143 via restoration of intracellular signaling. There was a significantly greater ability of both compounds to modulate agonist-stimulated intracellular Ca(2+) mobilization than ERK1/2 phosphorylation, indicating that the allosteric modulators engender bias in agonist-stimulated CaSR signaling to different pathways. Three mutations (G(670)R, P(748)R, and L(773)R) altered the binding affinity of allosteric modulators to the CaSR, and 3 mutations (V(817)I, L(773)R, and E(767)K) altered the cooperativity between the allosteric modulator and Ca(2+)(o). These findings have important implications for the treatment of diseases associated with CaSR mutations using allosteric CaSR modulators and for analyzing the effects of mutations on the function and pharmacoregulation of the CaSR.

Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Induction of an effective immune response that can target and eliminate malignant cells or virus-infected cells requires the stimulation of antigen-specific effector T cells. A productive and long-lasting memory response requires 2 signals: a specific signal provided by antigen recognition through engagement of the T cell receptor and a secondary signal via engagement of costimulatory molecules (such as OX40) on these newly activated T cells. The OX40-OX40-ligand interaction is critical for the generation of productive effector and memory T cell functions. Thus agonistic antibodies that stimulate OX40 on activated T cells have been used as adjuvants to augment immune responses. We previously demonstrated that an aptamer modified to stimulate murine OX40 enhanced vaccine-mediated immune responses in a murine melanoma model. In this study, we describe the development of an agonistic aptamer that targets human OX40 (hOX40). This hOX40 aptamer was isolated using systematic evolution of ligands by exponential enrichment and binds the target purified protein with high affinity [dissociation constants (K(d))<10 nM]. Moreover, the hOX40 aptamer-streptavidin complex has an apparent binding affinity of ~50 nM for hOX40 on activated T cells as determined by flow cytometry and specifically binds activated human T cells. A multivalent version of the aptamer, but not a mutant version of the aptamer, was able to stimulate OX40 on T cells and enhance cell proliferation and interferon-gamma production. Future studies will assess the therapeutic potential of hOX40 aptamers for ex vivo stimulation of antigen specific T cells in conjunction with dendritic cell-based vaccines for adoptive cellular therapy.

A series of 2-hydrazinyladenosine derivatives was synthesized and investigated in radioligand binding studies for their affinity at the adenosine receptor subtypes with the goal to obtain potent and A(2A)AR selective agonists and to explore the structure-activity relationships of this class of compounds at A(2A)AR. Modifications included introduction of a second sugar moiety at position 2 of adenosine to form new bis-sugar nucleosides and/or modifications of the 2-position linker in different ways. The performed modifications were found to produce compounds with relatively high A(2A)AR affinity and very high selectivity toward A(2A)AR. The most potent bis-sugar nucleoside was obtained with the D-galactose derivative 16 which exhibited a K(i) value of 329 nM at A(2A)AR with marked selectivity against the other AR subtypes. In another set of compounds, compound 3 was modified via replacement of its cyclic structure with mono- and disubstituted phenyl moieties and the resulting hydrazones 10-14 were found to have low nanomolar affinity for A(2A)AR. In addition to 3, compounds 10, 11 and 13 have been identified as the most potent compounds in the present series with K(i) values of 16.1, 24.4, and 12.0 nM, respectively, at rat A(2A)AR. Species differences were tested and found to exist in different rates. Functional properties of the most potent compounds 10, 11, 13 and 16 were assessed showing that the compounds acted as agonists at A(2A)AR., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Endocrine disrupting chemicals (EDCs) have emerged as a major public health issue because of their potentially disruptive effects on physiological hormonal actions. SXR (steroid xenobiotic receptor), also known as NR1I2, regulates CYP3A expression in response to exogenous chemicals, such as EDCs, after binding to SXRE (SXR response element). In our study, luciferase assay showed that 14 out of 55 EDCs could enhance SXR-mediated rat or human CYP3A gene transcription nearly evenly, and could also activate rat CYP7A1 gene transcription by cross-interaction of SXR and LXRE (LXRα response element). SXR diffused in the nucleus without ligand, whereas intranuclear foci of liganded SXR were produced. Furthermore, endogenous mRNA expression of CYP3A4 gene was enhanced by the 14 positive EDCs. Our results suggested a probable mechanism of EDCs disrupting the steroid or xenobiotic metabolism homeostasis via SXR., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The demonstrated functional interaction of metabotropic glutamate 5 (mGlu₅) receptors with N-methyl-d-aspartate (NMDA) receptors has prompted speculation that their activation may offer a potential treatment for aspects of schizophrenia. Development of selective mGlu₅ agonists has been difficult, but several different positive allosteric modulator (PAM) molecules have now been identified. This study describes two novel mGlu₅ PAMs, LSN2463359 (N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide) and LSN2814617 [(7S)-3-tert-butyl-7-[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-A]pyridine], which are useful tools for this field of research. Both compounds are potent and selective potentiators of human and rat mGlu₅ receptors in vitro, displaying curve shift ratios of two to three fold in the concentration-response relationship to glutamate or the glutamate receptor agonist, DHPG, with no detectable intrinsic agonist properties. Both compounds displaced the mGlu₅ receptor antagonist radioligand, [³H]MPEP in vitro and, following oral administration reached brain concentrations sufficient to occupy hippocampal mGlu₅ receptors as measured in vivo by dose-dependent displacement from the hippocampus of intravenously administered MPEPy. In vivo EEG studies demonstrated that these mGlu₅ PAMs have marked wake-promoting properties but little in the way of rebound hypersomnolence. In contrast, the previously described mGlu₅ PAMs CDPPB and ADX47273 showed relatively poor evidence of in vivo target engagement in either receptor occupancy assays or EEG disturbance. Wake-promoting doses of LSN2463359 and LSN2814617 attenuated deficits in performance induced by the competitive NMDA receptor antagonist SDZ 220,581 in two tests of operant behaviour: the variable interval 30 s task and the DMTP task. These effects were lost if the dose of either compound extended into the range which disrupted performance in the baseline DMTP task. However, the improvements in response accuracy induced by the mGlu₅ potentiators in SDZ 220,581-treated rats were not delay-dependent and, therefore, perhaps more likely reflected optimization of general arousal than specific beneficial effects on discrete cognitive processes. The systematic profiling of LSN2463359 and LSN2814617 alongside other previously described molecules will help determine more precisely how mGlu₅ potentiator pharmacology might provide therapeutic benefit. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Calcium signaling plays a major role in the function of cells. Measurement of intracellular calcium mobilization is a robust assay that can be performed in a high-throughput manner to study the effect of compounds on potential drug targets. Pharmaceutical companies frequently use calcium signaling assays to screen compound libraries on G-protein-coupled receptors (GPCRs). In this chapter we describe the application of FLIPR technology to the evaluation of GPCR-induced calcium mobilization. We also include the implications of GPCR hetero-oligomerization and the identification of heteromeric receptors as novel drug targets on high-throughput calcium screening.

The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake.

Somatostatin-14 (SST) inhibits insulin and glucagon secretion by activating G protein-coupled somatostatin receptors (SSTRs), of which five isoforms exist (SSTR1-5). In mice, the effects on pancreatic β-cells are mediated by SSTR5, whereas α-cells express SSTR2. In both cell types, SSTR activation results in membrane hyperpolarization and suppression of exocytosis. Here, we examined the mechanisms by which SST inhibits secretion from human β- and α-cells and the SSTR isoforms mediating these effects. Quantitative PCR revealed high expression of SSTR2, with lower levels of SSTR1, SSTR3, and SSTR5, in human islets. Immunohistochemistry showed expression of SSTR2 in both β- and α-cells. SST application hyperpolarized human β-cells and inhibited action potential firing. The membrane hyperpolarization was unaffected by tolbutamide but antagonized by tertiapin-Q, a blocker of G protein-gated inwardly rectifying K⁺ channels (GIRK). The effect of SST was mimicked by an SSTR2-selective agonist, whereas a SSTR5 agonist was marginally effective. SST strongly (>70%) reduced depolarization-evoked exocytosis in both β- and α-cells. A slightly weaker inhibition was observed in both cell types after SSTR2 activation. SSTR3- and SSTR1-selective agonists moderately reduced the exocytotic responses in β- and α-cells, respectively, whereas SSTR4- and SSTR5-specific agonists were ineffective. SST also reduced voltage-gated P/Q-type Ca²⁺ currents in β-cells, but normalization of Ca²⁺ influx to control levels by prolonged depolarizations only partially restored exocytosis. We conclude that SST inhibits secretion from both human β- and α-cells by activating GIRK and suppressing electrical activity, reducing P/Q-type Ca²⁺ currents, and directly inhibiting exocytosis. These effects are mediated predominantly by SSTR2 in both cell types.

Background and Purpose: Antidepressants are known to interact with the opioid system through mechanisms not completely understood. We previously reported that tricyclic antidepressants act as agonists at distinct opioid receptors. Here, we investigated the effect of the atypical antidepressant mianserin at cloned and native opioid receptors., Experimental Approach: Effects of mianserin were examined in CHO cells transfected with human opioid receptors, C6 glioma cells and rat brain membranes by the use of radioligand binding and functional assays including the stimulation of [(35)S]GTPγS binding and MAPK phosphorylation., Key Results: Mianserin displayed 12- and 18-fold higher affinity for κ- than µ- and δ-opioid receptors respectively. In [(35)S]GTPγS assays, mianserin selectively activated κ-opioid receptors. The agonist activity was antagonized by the selective κ-opioid blocker nor-binaltorphimine (nor-BNI). The mianserin analogue mirtazapine also displayed κ-opioid agonist activity. Mianserin and mirtazapine increased ERK1/2 phosphorylation in CHO cells expressing κ-opioid receptors and C6 cells, and these effects were antagonized by nor-BNI. In rat striatum and nucleus accumbens, mianserin stimulated [35S]GTPγS binding in a nor-BNI-sensitive manner with maximal effects lower than those of the full κ-opioid agonists (-)-U50,488 and dynorphin A. When combined, mianserin antagonized the effects of the full κ-opioid receptor agonists in [(35)S]GTPγS assays and reduced the stimulation of p38 MAPK and ERK1/2 phosphorylation by dynorphin A., Conclusions and Implications: In different cell systems, mianserin directly activates κ-opioid receptors, displaying partial agonist activity at brain receptors. Thus, this property appears to be a common feature of different classes of antidepressants., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Mu-opioid and CB1-cannabinoid agonists produce analgesia; however, adverse effects limit use of drugs in both classes. Additive or synergistic effects resulting from concurrent administration of low doses of mu- and CB1-agonists may produce analgesia with fewer side effects. Synergism potentially results from interaction between mu-opioid receptors (MORs) and CB1 receptors (CB1Rs). AM-251 and rimonabant are CB1R antagonist/inverse agonists employed to validate opioid-cannabinoid interactions, presumed to act selectively at CB1Rs. Therefore, the potential for direct action of these antagonists at MORs is rarely considered. This study determined if AM-251 and/or rimonabant directly bind and modulate the function of MORs. Surprisingly, AM-251 and rimonabant, but not a third CB1R inverse agonist AM-281, bind with mid-nanomolar affinity to human MORs with a rank order of affinity (K(i)) of AM-251 (251 nM) > rimonabant (652 nM) > AM281 (2135 nM). AM-251 and rimonabant, but not AM-281, also competitively antagonize morphine induced G-protein activation in CHO-hMOR cell homogenates (K(b) = 719 or 1310 nM, respectively). AM-251 and rimonabant block morphine inhibition of cAMP production, while only AM-251 elicits cAMP rebound in CHO-hMOR cells chronically exposed to morphine. AM-251 and rimonabant (10 mg/kg) attenuate morphine analgesia, whereas the same dose of AM-281 produces little effect. Therefore, in addition to high CB1R affinity, AM-251 and rimonabant bind to MORs with mid-nanomolar affinity and at higher doses may affect morphine analgesia via direct antagonism at MORs. Such CB1-independent of these antagonists effects may contribute to reported inconsistencies when CB1/MOR interactions are examined via pharmacological methods in CB1-knockout versus wild-type mice., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Analogs of SLV-319 (Ibipinibant), a CB1 receptor inverse agonist, were synthesized with functionality intended to limit brain exposure while maintaining the receptor affinity and selectivity of the parent compound. Structure activity relationships of this series, and pharmacology of two lead compounds, 16 (JD-5006) and 23 (JD-5037) showing little brain presence as indicated by tissue distribution and receptor occupancy studies, are described. Effects with one of these compounds on plasma triglyceride levels, liver weight and enzymes, glucose tolerance and insulin sensitivity support the approach that blockade of peripheral CB(1) receptors is sufficient to produce many of the beneficial metabolic effects of globally active CB(1) blockers. Thus, PR CB(1) inverse agonists may indeed represent a safer alternative to highly brain-penetrant agents for the treatment of metabolic disorders, including diabetes, liver diseases, dyslipidemias, and obesity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

The metabotropic glutamate receptors (mGluRs) are evolutionarily conserved from nematodes to vertebrates. The Caenorhabditis elegans (C. elegans) genome contains three mGluR genes referred to as mgl-1, mgl-2, and mgl-3. The aim of this study was to characterize the pharmacological profiles of orthosteric and allosteric mGluR ligands on mgl-2. A phylogenetic analysis revealed that mgl-2 is closely associated with the mammalian Group 1 mGluRs (mGluR1 and mGluR5) and is distinct from Group 2 and 3 mGluRs. The ligand binding domain of mgl-2 displayed higher homology to the rat Group 1 mGluRs binding domains compared to the level of homology in the heptahelical transmembrane domain regions. We found that, when transiently expressed in human embryonic kidney 293 cells, mgl-2 can be activated by glutamate and couples to human G-proteins to induce the release of intracellular calcium. Dose-response analyses revealed that mgl-2 has approximately a 15-20-fold lower affinity for glutamate and quisqualate compared to rat mGluR5. In contrast to orthosteric agonists, Group 1 negative allosteric modulators that target the transmembrane domain were ineffective at mgl-2. Surprisingly, CDPPB, an mGluR5 positive allosteric modulator, potentiated glutamate mediated activation of mgl-2, although MPEP and fenobam, two mGluR5 antagonists that share similar binding residues with CDPPB were ineffective at mgl-2. These findings indicate that selective pressures on mGluR protein structures have resulted in conservation of the glutamate binding site, whereas the allosteric modulator sites have been subjected to greater divergent evolutionary changes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate Cys-loop ligand-gated ion channels. It is activated by GABA, and this property, combined with its structural similarity to GABA(A) and other Cys-loop receptors, makes it potentially an excellent model to probe their structure and function. Here we characterise the pharmacological profile of ELIC, examining the effects of compounds that could activate or inhibit the receptor. We confirm that a range of amino acids and classic GABA(A) receptor agonists do not elicit responses in ELIC, and we show the receptor can be at least partially activated by 5-aminovaleric acid and γ-hydroxybutyric acid, which are weak agonists. A range of GABA(A) receptor non-competitive antagonists inhibit GABA-elicited ELIC responses including α-endosulfan (IC₅₀ = 17 μM), dieldrin (IC₅₀ = 66 μM), and picrotoxinin (IC₅₀ = 96 μM) which were the most potent. Docking suggested possible interactions at the 2' and 6' pore-lining residues, and mutagenesis of these residues supports this hypothesis for α-endosulfan. A selection of compounds that act at Cys-loop and other receptors also showed some efficacy at blocking ELIC responses, but most were of low potency (IC₅₀ > 100 μM). Overall our data show that a number of compounds can inhibit ELIC, but it has limited pharmacological similarity to GLIC and to Cys-loop receptors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

Herein we describe the diastereoselective synthesis of glutamic acid analogs and the evaluation of their agonist activity towards metabotropic glutamate receptor subtype 4 (mGluR4). These analogs are based on a monofluorinated cyclopropane core substituted with an α-aminoacid function. The potential of this new building block as a tool for the development of a novel class of drugs is demonstrated with racemic analog 11a that displayed the best agonist activity with an EC50 of 340 nM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Purpose: The aim of this study was to characterize the functional impact of KCNQ1-encoded voltage-dependent potassium channels (K(v)7.1) in the vasculature., Experimental Approach: Mesenteric arteries, intrapulmonary arteries and thoracic aortae were isolated from adult rats. K(v)7.1 channel expression was established by fluorescence immunocytochemistry. Wire myography determined functionality of these channels in response to selective blockers and activators. Xenopus oocytes expressing K(v)7.1 channels were used to assess the effectiveness of selective K(v)7.1 channel blockers., Key Results: K(v)7.1 channels were identified in arterial myocytes by immunocytochemistry. K(v)7.1 blockers HMR1556, L-768,673 (10 µM) and JNJ39490282 (JNJ282; 1 µM) had no contractile effects in arteries, whereas the pan-K(v)7 channel blocker linopirdine (10 µM) evoked robust contractions. Application of two compounds purported to activate K(v)7.1 channels, L-364 373 (R-L3) and mefenamic acid, relaxed mesenteric arteries preconstricted by methoxamine. These responses were reversed by HMR1556 or L-768,673 but not JNJ282. Similar effects were observed in the thoracic aorta and intrapulmonary arteries., Conclusions and Implications: In contrast to previous assumptions, K(v)7.1 channels expressed in arterial myocytes are functional ion channels. Although these channels do not appear to contribute to resting vascular tone, K(v)7.1 activators were effective vasorelaxants., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: Sensory neuronal and epidermal transient receptor potential ion channels (TRPs) serve an important role as pain sensor molecules. While many natural and synthetic ligands for sensory TRPs have been identified, little is known about the endogenous activator for TRPV4. Recently, we reported that endogenous metabolites produced by the mevalonate pathway regulate the activities of sensory neuronal TRPs. Here, we show that dimethylallyl pyrophosphate (DMAPP), a substance produced by the same pathway is an activator of TRPV4., Experimental Approach: We examined the effects of DMAPP on sensory TRPs using Ca²⁺ imaging and whole-cell electrophysiology experiments with a heterologous expression system (HEK293T cells transfected with individual TRP channels), cultured sensory neurons and keratinocytes. We then evaluated nociceptive behavioural and inflammatory changes upon DMAPP administration in mice in vivo., Key Results: In the HEK cell heterologous expression system, cultured sensory neurons and keratinocytes, µM concentrations of DMAPP activated TRPV4. Agonistic and antagonistic potencies of DMAPP for other sensory TRP channels were examined and activation of TRPV3 by camphor was found to be inhibited by DMAPP. In vivo assays, intraplantar injection of DMAPP acutely elicited nociceptive flinches that were prevented by pretreatment with TRPV4 blockers, indicating that DMAPP is a novel pain-producing molecule through TRPV4 activation. Further, DMAPP induced acute inflammation and noxious mechanical hypersensitivities in a TRPV4-dependent manner., Conclusions and Implications: Overall, we found a novel sensory TRP acting metabolite and suggest that its use may help to elucidate the physiological role of TRPV4 in nociception and associated inflammation., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

An orphan G-protein-coupled receptor, T cell death-associated gene 8 (TDAG8) which has been reported to be a proton sensor, inhibits the production of pro-inflammatory cytokines induced by extracellular acidification. Recently, we have found that TDAG8 knockout mice showed significant exacerbation in various immune-mediated inflammation disease models. To elucidate the role of TDAG8, we screened an in-house library to find compounds which have a profile as a TDAG8 agonist using a cyclic adenosine 5'-monophosphate assay. Among the screening hits, we focused on (3-[(2,4-dichlorobenzyl)thio]-1,6-dimethyl-5,6-dihydro-1H-pyridazino[4,5-e][1,3,4]thiadiazin-5-one) (named BTB09089). BTB09089 did not act on other proton sensing G-protein-coupled receptors such as G-protein-coupled receptor 4 (GPR4) nor ovarian cancer G-protein-coupled receptor 1 (OGR1). Moreover, BTB09089 increased cAMP level in the splenocytes from wild-type littermates but not from TDAG8-deficient mice. Thus, BTB09089 was found to be a TDAG8 specific agonist. We then investigated the effects of BTB09089 on T cells and macrophages in vitro. In splenocytes, BTB09089 suppressed the production of IL-2 stimulated with anti-CD3 and anti-CD28 antibodies. In peritoneal exuded macrophages induced by thioglycollate, BTB09089 suppressed the production of TNF-α and IL-6 while it increased that of IL-10 when stimulated with lipopolysaccharide. These effects were observed in cells from wild type mice, but not those from TDAG8 knockout mice. These results indicate that activation of TDAG8 attenuates immune-mediated inflammation by regulating the cytokine production of T cells and macrophages., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A group of sixteen arylpiperazines had been previously synthesized and evaluated for atypical antipsychotic activity. Here we examined these compounds for their neuroprotective capacity. The affinity and agonist/antagonist action of the arylpiperazines at dopamine hD(2S) receptors were determined in vitro on membranes from stably transfected CHO-hD(2S) cell line. The assays for cell viability and antioxidative capacity (total glutathione and total superoxide dismutase activity), amount of nitric oxide and superoxide radicals, as well as influence on prosurvival pathways (Akt and ERK), were performed on the human neuroblastoma cell line SH-SY5Y. Cell death was induced by oxidative or nitrosative stress, or by growing cells in the medium deprived of serum. Only four of the arylpiperazines exhibited notable neuroprotection against cell death induced by sodium nitroprusside. Two of these arylpiperazines induced elevations of pAkt, while two other compounds reduced the levels of pErk, whereas these actions are considered to support the cell survival. The benzimidazole heteroaryl-group, that mimics catechol moiety of the dopamine molecule, might be the prerequisite structure for the neuroprotective action of these ligands. It is postulated that neuroprotection was acquired also by elevation of endogenous glutathione or total superoxide dismutase activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Activation of adenosine A₁ receptors was reported to promote fatty acid synthesis in AML-12 cells, by increasing the expression of SREBP-(1c) (sterol regulatory binding protein 1c) and FAS (fatty acid synthase). Since these findings have important therapeutic implications for the discovery of adenosine A₁ receptor agonists, further studies were undertaken to determine the expression and functional relevance of adenosine A₁ receptor in the liver. To that end, we used two classes of distinct adenosine A₁ receptor agonists: CPA (N⁶-cyclopentyl-adenosine), a full agonist and GS-9667 (2-{6-[((1R,2R)-2-hydroxycyclopentyl)-amino]purin-9-yl}(4S,5S,2R,3R)-5-[(2-fluorophenylthio)methyl]-oxolane-3,4-diol), a partial agonist. Treatment of AML-12 cells, HepG2 cells and primary human hepatocytes with either CPA or GS-9667 did not increase the gene expression of SREBP-(1c) or FAS. Furthermore, in AML-12 and HepG2 cells, CPA did not antagonize forskolin-stimulated cAMP production, a characteristic of adenosine A₁ receptor activation, indicating that these cells lack adenosine A₁ receptor function. Consistent with this finding, adenosine A₁ receptor gene expression was found to be very low and adenosine A₁ receptor protein levels were hardly detectable by radioligand binding assays in hepatic cell lines such as AML-12 and HepG2 as well as in both mouse and human liver tissues. Finally, acute treatment with adenosine A₁ receptor agonist GS-9667 had no significant effect on gene expression of both SREBP-(1c) and FAS in livers of Sprague Dawley rats. Taken together, our data suggest that the expression of adenosine A₁ receptor is too low to play a major role in the regulation of lipogenic gene expression in hepatocytes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.

The cysteine peptidase cathepsin K is a major player in extracellular proteolysis. Here we describe the identification of the multifunctional extracellular chaperone clusterin as a cathepsin K-binding protein. Clusterin increases the stability of cathepsin K in dilute solution and in the presence of high protein concentration. It does not alter the activity of the enzyme but acts as a liberator by preventing substrate inhibition. Kinetic measurements show that clusterin binds cathepsin K with high affinity (K(d) = 0.5-0.6 nM). Altogether these results provide novel insights into the mechanisms involved in the fine-tuning of cysteine cathepsin activity in the extracellular space., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 μM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.

Although adenophostin A (AdA), the most potent agonist of d-myo-inositol 1,4,5-trisphosphate receptors (IP(3)R), is thought to mimic IP(3), the relative roles of the different phosphate groups and the adenosine motif have not been established. We synthesized all three possible bisphosphate analogues of AdA and glucose 3,4-bisphosphate (7, AdA lacking the 2'-AMP). 2'-Dephospho-AdA (6) was prepared via a novel regioselective dephosphorylation strategy. Assessment of the abilities of these bisphosphates to stimulate intracellular Ca(2+) release using recombinant rat type 1 IP(3)R (IP(3)R1) revealed that 6, a mimic of Ins(4,5)P(2), is only 4-fold less potent than IP(3), while 7 is some 400-fold weaker and even 3″-dephospho-AdA (5) is measurably active, despite missing one of the vicinal bisphosphate groups normally thought to be crucial for IP(3)-like activity. Compound 6 is the most potent bisphosphate yet discovered with activity at IP(3)R. Thus, adenosine has a direct role independent of the 2'-phosphate group in contributing toward the potency of adenophostins, the vicinal bisphosphate motif is not essential for activity at the IP(3)R, as always thought, and it is possible to design potent agonists with just two of the three phosphates. A model with a possible adenine-R504 interaction supports the activity of 5 and 6 and also allows a reappraisal of the unexpected activity previously reported for the AdA regioisomer 2″-phospho-3″-dephospho-AdA 40.

Adenosine derivatives were modified with alkynyl groups on N(6) substituents for linkage to carriers using Cu(I)-catalyzed click chemistry. Two parallel series, both containing a rigid North-methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose, behaved as A(3) adenosine receptor (AR) agonists: (5'-methyluronamides) or partial agonists (4'-truncated). Terminal alkynyl groups on a chain at the 3 position of a N(6)-benzyl group or simply through a N(6)-propargyl group were coupled to azido derivatives, which included both small molecules and G4 (fourth-generation) multivalent poly(amidoamine) (PAMAM) dendrimers, to form 1,2,3-triazolyl linkers. The small molecular triazoles probed the tolerance in A(3)AR binding of distal, sterically bulky groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and (18)F radiolabeling. N(6)-(4-Fluoro-3-nitrophenyl)-triazolylmethyl derivative 32 displayed a K(i) of 9.1 nM at A(3)AR with ∼1000-fold subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups potently activated A(3)AR in the 5'-methyluronamide, but not 4' truncated series. N(6)-Benzyl nucleoside conjugate 43 (apparent K(i) 24 nM) maintained binding affinity of the monomer better than a N(6)-triazolylmethyl derivative. Thus, the N(6) region of 5'-methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to achieve high A(3)AR agonist affinity and enhanced selectivity.

Mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of xenobiotics as well as numerous endogenous molecules. The major aryl (phenol) SULT in rat liver, rSULT1A1, has been used extensively as a model enzyme for understanding the catalytic function of SULTs. Previous studies showed that purified rSULT1A1 displays significant catalytic changes in the presence of GSSG and other oxidants. In the present study, the effects of diamide [1,1'-azobis(N,N-dimethylformamide)] and tert-butyl hydroperoxide (TBHP) on the activity of rSULT1A1 in rat hepatic slices were compared with the effects of these oxidants on a homogeneous preparation of the enzyme. Precision-cut hepatic slices were incubated with 10 μM 7-hydroxycoumarin (7-HC) in the presence of varied concentrations of either diamide or TBHP. Analysis of the 7-hydroxycoumarin sulfate released into the incubation medium indicated that both oxidants significantly increased the sulfation of 7-HC, and this occurred at optimal concentrations of 5 and 10 μM, respectively. Cellular GSH and GSSG levels in the hepatic slices were not significantly altered from control values at these concentrations of diamide and TBHP. Exposure of homogeneous rSULT1A1 to diamide or TBHP also increased the rate of sulfation of 7-HC, although the optimal concentrations of diamide and TBHP were lower (50- and 100-fold, respectively) than those required for effects with the hepatic slices. These results indicate that both diamide and TBHP may modify the rSULT1A1 in intact cells in a manner similar to that observed with the homogeneous purified enzyme.

The protein bCblC (bCblCpro) is a bovine homolog of a human B₁₂ trafficking chaperone that is responsible for the processing of vitamin B₁₂ and its escorted delivery in intracellular B₁₂ metabolism. In this study, we found that bCblCpro is highly thermolabile with a T(m) = 42.0 ± 0.2 °C as shown for the human homolog, suggesting thermal regulation of these proteins. Binding of the reduced form of glutathione (GSH) that is a predominant cellular thiol increased the T(m) of bCblCpro from 42 °C to ~45 °C (ΔT(m max) = 3.1 ± 0.2 °C and AC₅₀ = 2.1 ± 0.5 mM). Binding of vitamin B₁₂ and its derivatives also stabilized bCblCpro increasing the T(m) to a different extent and vitamin B₁₂ (cyanocobalamin, CNCbl) was the least efficient (ΔT(m max) = 4.3 ± 0.3 °C and AC₅₀ = 291 ± 36 μM). However, the stabilizing effect of CNCbl was significantly greater for GSH-bound bCblCpro (ΔT(m max) = 12.8 ± 0.6 °C and AC₅₀ = 9.3 ± 1.6 μM) than for GSH-free bCblCpro. In addition, the stabilizing effect of GSH was also greater for CNCbl-bound bCblCpro (ΔT(m max) = 9.3 ± 0.3 °C and AC₅₀ = 57.0 ± 6.8 μM). Limited proteolysis revealed that thermal stabilization of bCblCpro is derived from conformational changes of the protein induced by binding of the ligands. The results in this study indicate that GSH cooperates with vitamin B₁₂ in thermal stabilization of bCblCpro and is a positive regulator of the protein.

Human peroxisome proliferator-activated receptors (hPPARs) are ligand-dependent transcription factors that control various biological responses, and there are three subtypes: hPPARα, hPPARδ, and hPPARγ. We report here that α-substituted phenylpropanoic acid-type hPPAR agonists with similar structure bind to the hPPAR ligand binding domain (LBD) in different conformations, depending on the receptor subtype. These results might indicate that hPPAR ligand binding pockets have multiple binding points that can be utilized to accommodate structurally flexible hPPAR ligands.

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.

More than 20 years after the description of the two IR (insulin receptor) isoforms, designated IR-A (lacking exon 11) and IR-B (with exon 11), nearly every functional aspect of the alternative splicing both in vitro and in vivo remains controversial. In particular, there is no consensus on the precise ligand-binding properties of the isoforms. Increased affinity and dissociation kinetics have been reported for IR-A in comparison with IR-B, but the opposite results have also been reported. These are not trivial issues considering the reported possible increased mitogenic potency of IR-A, and the reported link between slower dissociation and increased mitogenesis. We have re-examined the ligand-binding properties of the two isoforms using a novel rigorous mathematical analysis based on the concept of a harmonic oscillator. We found that insulin has 1.5-fold higher apparent affinity towards IR-A and a 2-fold higher overall dissociation rate. Analysis based on the model showed increased association (3-fold) and dissociation (2-fold) rate constants for binding site 1 of IR in comparison with IR-B. We also provide a structural interpretation of these findings on the basis of the structure of the IR ectodomain and the proximity of the sequence encoded by exon 11 to the C-terminal peptide that is a critical trans-component of site 1.

GPR119 is one of the G-protein-coupled receptors expressed in pancreatic β-cells and intestinal endocrine cells. Since agonists to GPR119 stimulate glucose-dependent insulin secretion, GPR119 agonists are anticipated to promote anti-diabetic effects and control of glucose homeostasis. Here, we reported that an omega-3 unsaturated fatty acid metabolite, 5-hydroxy-eicosapentaenoic acid (5-HEPE), was a potent agonist for GPR119 and enhanced glucose-dependent insulin secretion. 5-HEPE stimulated cAMP accumulation in mouse MIN6 insulinoma cells and human HuTu80 intestinal adenocarcinoma cells. These effects were blunted by GPR119-specific siRNA. Recombinant GPR119 also responded to 5-HEPE as well as authentic agonists. Several previous reports have indicated the beneficial biological effects of omega-3 unsaturated fatty acids, and epidemiological studies have suggested that these fatty acids plays a protective role against diabetes. However, the molecular pharmacology and receptor identifications of omega-3 unsaturated fatty acids and their metabolites have not yet been well investigated. It is hoped that our findings will encourage novel investigations into the molecular relationships between omega-3 fatty acids and diabetes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rivaroxaban and other direct factor Xa inhibitors are used at fixed doses without drug monitoring and dose adjustment. Patients may require determination of the anticoagulant effect during treatment. The aim of this study was to develop a method to reduce the differences between thromboplastin reagents and coagulation analysers for determination of the anticoagulant effect of rivaroxaban in human plasma. Purity of rivaroxaban extracted from commercially available drug was confirmed by mass spectrometry, elemental analysis and 1H-NMR spectroscopy. Coagulation times of pooled human plasma spiked with 50-900  ng/ml rivaroxaban were analysed. Thromboplastin reagents, WHO RBT/90, Innovin, RecombiPlasTin 2G, STA Neoplastin Plus, Technoclot PT Plus and Thromborel S, the manual Kolle-Hook method and the KC10 analyser were used. An international sensitivity index (ISI) was determined for each reagent and coagulation method using the RBT/90 thromboplastin reagent as reference. The orthogonal, used for the determination of the ISI of coumarin plasmas, and ordinary regression analyses were compared. The results showed than increasing concentrations of rivaroxaban prolonged coagulation values of all thromboplastin assays linearly (r (2)= 0.96 and r(2) = 0.99, respectively). The coefficient of variation between the slopes of the dilution curves and the ratios of the thromboplastin reagents were reduced using the international normalized ratio (INR) and ISI calculated for rivaroxaban. The ISIs of the thromboplastin reagents ranged from 0.73 to 1.67 as compared with the WHO reagent using the manual technique. The coefficient of variations between the thromboplastin reagents comparing the orthogonal and the ordinary regression analysis were 6.8 versus 3.7% (Kolle-Hook method, P = 0.0011) and 8.5 versus 4.8% (KC10 method, P < 0.0001). Using ISI for vitamin-K antagonist and rivaroxaban, the INRs for the rivaroxaban-containing samples were significantly different for one of five commercial thromboplastin reagents. In conclusion, the determination of an ISI for rivaroxaban using a WHO thromboplastin reagent is required for commercial thromboplastin reagents. The manual Kolle-Hook method and an ordinary linear regression analysis should be adopted.

Objective: Previous studies show that polyunsaturated fatty acids (PUFAs) increase the insulin secretory capacity of pancreatic β-cells. We aimed at identifying PUFA-derived mediators and their cellular targets that are involved in the amplification of insulin release from β-cells preexposed to high glucose levels., Research Design and Methods: The content of fatty acids in phospholipids of INS-1E β-cells was determined by lipidomics analysis. High-performance liquid chromatography was used to identify peroxidation products in β-cell cultures. Static and dynamic glucose-stimulated insulin secretion (GSIS) assays were performed on isolated rat islets and/or INS-1E cells. The function of peroxisome proliferator-activated receptor-δ (PPAR-δ) in regulating insulin secretion was investigated using pharmacological agents and gene expression manipulations., Results: High glucose activated cPLA(2) and, subsequently, the hydrolysis of arachidonic and linoleic acid (AA and LA, respectively) from phospholipids in INS-1E cells. Glucose also increased the level of reactive oxygen species, which promoted the peroxidation of these PUFAs to generate 4-hydroxy-2E-nonenal (4-HNE). The latter mimicked the GSIS-amplifying effect of high glucose preexposure and of the PPAR-δ agonist GW501516 in INS-1E cells and isolated rat islets. These effects were blocked with GSK0660, a selective PPAR-δ antagonist, and the antioxidant N-acetylcysteine or by silencing PPAR-δ expression. High glucose, 4-HNE, and GW501516 also induced luciferase expression in a PPAR-δ-mediated transactivation assay. Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range., Conclusions: Elevated glucose levels augment the release of AA and LA from phospholipids and their peroxidation to 4-HNE in β-cells. This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells.