Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.Hepatitis C virus (HCV) causes liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (52). The HCV genome is a single-stranded RNA molecule where both the 5Ј and the 3Ј untranslated region (UTR) contain highly conserved RNA structures necessary for polyprotein translation and genome replication (43). The processed polyprotein yields at least three structural proteins and six nonstructural proteins. The structural proteins include the core, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The viral proteins processed by signal peptidases form viral particles that assemble at the endoplasmic reticulum (ER) and/or Golgi bodies and are released from the host cell by viral budding. The structural protein coding regions are separated from nonstructural proteins by the short membrane peptide p7, thought to function as an ion channel (43, 53). The nonstructural proteins NS2, NS3/4A, NS5A, and NS5B are involved in coordinating the intracellular processes of the virus life cycle, including polyprotein processing and viral RNA replication (34).The Luc-1b cell is a human hepatoma cell line (Huh7) that contains a genotype 1b HCV subgenomic replicon, a luciferase reporter, and a neomycin selection marker, allowing HCV replication to be studied both in vitro and in vivo (8,36). This subgenomic replicon lacks the coding regions for NS2 and the structural proteins but contains the nonstructural proteins in cis, which are required for replicat...

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4−/− crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled ␤ 2 adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human ␤ 2 adrenoceptor (E max ϭ 73 Ϯ 1% of the maximal effect of isoprenaline; pEC 50 ϭ 8.06 Ϯ 0.02), whereas salmeterol displays only partial efficacy (38 Ϯ 1%). The functional selectivity profile of indacaterol over ␤ 1 human adrenoceptors is similar to that of formoterol, whereas its ␤ 3 adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 Ϯ 4 min) similar to formoterol and salbutamol, and a long duration of action (529 Ϯ 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled ␤ 2 adrenoceptor agonists.Agents that act as agonists of the ␤ 2 adrenoceptor are effective in the management of asthma and chronic obstructive pulmonary disease (COPD), primarily through their bronchodilatating properties. These drugs induce bronchodilatation by causing direct relaxation of airway smooth muscle through activation of adenylate cyclase, which in turn increases intracellular cAMP levels.Salbutamol is an inhaled ␤ 2 adrenoceptor agonist that provides rapid bronchodilatation and has been widely used over the past 30 years. However, its major drawback is its short duration of action (4 -6 h), requiring the drug to be administered several times a day. Two longer acting inhaled ␤ 2 adrenoceptor agonists, formoterol and salmeterol, are now available and are used in the management of asthma and COPD (Sutherland, 2004). These two drugs have a bronchodilating effect lasting for 12 h after a single inhalation and are therefore given twice daily. Despite the decrease in dosing frequency with the longer acting inhaled ␤ 2 adrenoceptor agonists, patient compliance is still an issue (Ying et al., 1999). In addition, the recent launch of tiotropium bromide, a once-daily inhaled muscarinic antagonist for the treatment of COPD (Gross, 2004), and the development of once-daily inhaled corticosteroids for the...

Background: TLQP-21 is a bioactive peptide for which the receptor(s) are unknown. Results: We demonstrate that C3AR1 is a receptor for TLQP-21. Conclusion: Many of the effects of TLQP-21 can be explained by C3AR1 activation. Significance: These results provide a bridge linking the regulation of metabolism and the activation of complement in rodents.

Studies under nonphysiological conditions suggest that long receptor residency time is responsible for the 24-h duration of action of the long-acting muscarinic antagonist (LAMA) tiotropium. Our aim was to determine how clinically relevant dissociation rates under more physiological conditions influence the differences in onset of action between tiotropium and 3-[(cyclopentylhydroxyphenylacetyl oxy]-1,1-dimethyl-pyrrolidinium bromide (NVA237), a once-daily dry-powder formulation of the LAMA glycopyrronium bromide in development for chronic obstructive pulmonary disease. In addition, we have investigated kinetic selectivity at each of the muscarinic receptor subtypes to determine whether the improved cardiovascular therapeutic index obtained with NVA237 in animal models is attributable to differences in kinetic rate constants. The binding of radioligand [ 3 H]N-methyl-scopolamine was measured in the presence/absence of several concentrations of unlabeled competitors, and data were analyzed using a competition kinetic model to provide on/off rates for the competitor. We found shorter dissociation half-lives for NVA237 and tiotropium under physiological (11.4 and 46.2 min, respectively) versus nonphysiological conditions (173 and 462 min, respectively). NVA237 had a more rapid onset of action (3-4.8 times) versus tiotropium, determined in an vitro calcium and rat tracheal strip assay. Simulations suggested that the more rapid onset of NVA237 action could be explained by differences in kinetic parameters. NVA237 had greater equilibrium binding and kinetic selectivity for muscarinic type 3 (M 3 ) versus muscarinic type 2 (M 2 ) receptors, with a faster off rate from M 2 versus M 3 receptors than tiotropium, potentially affording it a more favorable therapeutic index. This study suggests that the 24-h duration of action of NVA237 and tiotropium is not solely the result of their slow dissociation from the M 3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.

The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M(3) receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.

Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo. We found that concomitant abrogation of metallothioneins 1 and 2 results in activation of the Akt pathway and increases in myotube size, in type IIb fiber hypertrophy, and ultimately in muscle strength. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function was also observed in the setting of glucocorticoid addition, which is a strong atrophy-inducing stimulus. Given the blockade of atrophy and the preservation of strength in atrophy-inducing settings, these results suggest that blockade of metallothioneins 1 and 2 constitutes a promising approach for the treatment of conditions which result in muscle atrophy.KEYWORDS muscle metabolism S keletal muscle hypertrophy is characterized in the adult mammal by an increase in the size of preexisting myofibers. The induction of hypertrophy involves an activation of the pathways that increase protein synthesis and inhibition of cellular signaling, which induces protein degradation. Hypertrophy can be induced by the activation of Akt, through multiple potential inputs (1). Akt induces hypertrophy in part by activating the mTOR/70S6 kinase pathway. In addition, Akt inhibits protein degradation, by phosphorylating and therefore blocking Foxo1 and Foxo3-transcription factors which are required for the upregulation of the E3 ubiquitin ligases MuRF1 and MAFbx, which help mediate protein turnover during muscle atrophy (2-4). Therefore, activation of Akt constitutes a critical signaling node to increase muscle hypertrophy and block muscle atrophy (1).Mammalian metallothioneins (MTs) belong to a family of cysteine-rich, metalbinding proteins. In rodents, four MT isoforms have been identified: the two major isoforms, MT-1 and MT-2, are ubiquitously expressed, while MT-3 and MT-4 show tissue specific expression in the central nervous system and squamous epithelia, respectively. In humans, multiple isoforms have been reported for MT-1 (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1M, and MT-1X), while no splice variants are documented for MT-2, MT-3, or MT-4 (5; for a review, see reference 6).MTs play a role in cellular zinc homeostasis, mitochondrial function (7), defense against oxidative stress (8), and defense against inflammation (5). Moreover, several reports and a recent review highlight a role of metallothioneins in cancer (9), aging (10), and the onset of particular central nervous system diseases (11).

BackgroundHearing loss is the most common sensory defect afflicting several hundred million people worldwide. In most cases, regardless of the original cause, hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. Despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration, thereby providing new avenues for the treatment of hearing loss.ResultsIn order to identify new genetic elements enabling regeneration of inner ear sensory hair cells, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent miRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on these miRSeq results eight most differentially expressed miRNAs were selected for further characterization. In UB/OC-1, miR-210 silencing in vitro resulted in hair cell marker expression, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. Using a lineage tracing mouse model, transdifferentiation of supporting epithelial cells was identified as the likely mechanism for this new hair cell formation. Potential miR-210 targets were predicted in silico and validated experimentally using a miR-trap approach.ConclusionMiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells suggesting that miR-210 might be a potential new factor for hearing loss therapy. In addition, identification of inner ear pathways regulated by miR-210 identified potential new drug targets for the treatment of hearing loss.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2620-7) contains supplementary material, which is available to authorized users.