Discovery of 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole,FYX-051-a xanthine oxidoreductase inhibitor for the treatment of hyperuricemia

Our previous study identified 2-[2-(2-methoxy-ethoxy)-ethoxy]-5-[5-(2-methyl-4-pyridyl)-1H-[1,2,4]triazol-3-yl]-benzonitrile (2) as a safe and potent xanthine oxidoreductase (XOR) inhibitor for the treatment of hyperuricemia. Here, we synthesized a series of 3,5-dipyridyl-1,2,4-triazole derivatives and, in particular, examined their in vivo activity in lowering the serum uric acid levels in rats. As a result, we identified 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole (FYX-051, compound 39) to be one of the most potent XOR inhibitors; it exhibited an extremely potent in vivo activity, weak CYP3A4-inhibitory activity and a better pharmacokinetic profile than compound 2. Compound 39 is currently being evaluated in a phase 2 clinical trial.     

Miscibility and phase behavior of DPPG and perfluorocarboxylic acids at the air-water interface

The miscibility and phase behavior of two components of phospholipids and perfluorocarboxylic acids [FCn; perfluorododecanoic acid (FC12), perfluorotetradecanoic acid (FC14), perfluorohexadecanoic acid (FC16), and perfluorooctadecanoic acid (FC18)] have been systematically investigated using Langmuir monolayer technique. Dipalmitoylphosphatidylglycerol (DPPG) is utilized as a phospholipid component in biomembranes. Surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms have been measured for the DPPG/FCn systems on 0.15 M NaCl (pH 2.0) at 298.2 K. From the isotherm results, two-dimensional phase diagrams are constructed and classified into miscible and immiscible patterns. Furthermore, the phase behavior of the DPPG/FCn systems has been morphologically examined using fluorescence microscopy (FM) and atomic force microscopy (AFM). These images indicate different phases among the four systems. In particular, specific phase morphology is observed in the middle molar fraction range for the DPPG/FC14 system; FC14 is selectively excluded from mixed DPPG-FC14 monolayers to be concentrated in the phase boundary as surface pressure increases. Then DPPG is refined as a patched film. Moreover, the data obtained here are compared to those in the previous systems in which different kinds of phospholipids were treated. Through a series of the miscibility investigations, it is proposed that combinations of hydrophobic chain lengths and of polar headgroups contribute to the monolayer miscibility between phospholipids and perfluorocarboxylic acids.     

Usual and unusual biochemical properties of ADF/cofilin-like protein Adf73p in ciliate Tetrahymena thermophila

Actin-depolymerizing factor (ADF)/cofilin is a well-conserved actin-modulating protein, which induces reorganization of the actin cytoskeleton by severing and depolymerizing F-actin. ADF/cofilin also binds to G-actin and inhibits nucleotide exchange, and hence, is supposed to regulate the nucleotide-bound state of the cellular G-actin pool cooperating with profilin, another well-conserved G-actin-binding protein that promotes nucleotide exchange. In this report, we investigated the biochemical properties of the ADF/cofilin-like protein Adf73p from ciliate Tetrahymena thermophila. Adf73p also binds to both G- and F-actin and severs and depolymerizes F-actin. Unlike canonical ADF/cofilin, however, Adf73p accelerates nucleotide exchange on actin and allows repolymerization of disassembled actin. These results suggest that the actin cytoskeleton of T. thermophila is regulated by Adf73p in a different way from those of mammals, plants, and yeasts.     

Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

We employed a comparative genomic approach to understand protein phosphatase 2C (PP2C)-mediated abscisic acid (ABA) signaling in the moss Physcomitrella patens. Ectopic expression of Arabidopsis (Arabidopsis thaliana) abi1-1, a dominant mutant allele of ABI1 encoding a PP2C involved in the negative regulation of ABA signaling, caused ABA insensitivity of P. patens both in gene expression of late embryogenesis abundant (LEA) genes and in ABA-induced protonemal growth inhibition. The transgenic abi1-1 plants showed decreased ABA-induced freezing tolerance, and decreased tolerance to osmotic stress. Analyses of the P. patens genome revealed that only two (PpABI1A and PpABI1B) PP2C genes were related to ABI1. In the ppabi1a null mutants, ABA-induced expression of LEA genes was elevated, and protonemal growth was inhibited with lower ABA concentration compared to the wild type. Moreover, ABA-induced freezing tolerance of the ppabi1a mutants was markedly enhanced. We provide the genetic evidence that PP2C-mediated ABA signaling is evolutionarily conserved between Arabidopsis and P. patens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession Numbers: PpABI1A-AB369256, PpABI1B-AB369255, pphn39k21-AB369257.     

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

Background

A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown.

Methodology/Principal Findings

In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate α-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type.

Conclusions/Significance

These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.     

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

Presence of Thrombin-Activatable Fibrinolysis Inhibitor in Helicobacter pylori-Associated Gastroduodenal Disease

Thrombin-activatable fibrinolysis inhibitor (TAFI) plays a role in the regulation of coagulation and inflammation. In addition to inhibiting the fibrinolytic system, TAFI may also regulate the bradykinin and complement systems. We hypothesized that TAFI also plays a role in defense mechanisms of the gastric mucosa during Helicobacter pylori infection. This study comprised 65 patients with gastroduodenal disorders: 41 patients with H. pylori infection, 13 without, and 11 patients with cured H. pylori infection. The gastric intramucosal concentrations of TAFI were measured by enzyme immunoassay. The gastric levels of TAFI and plasminogen activator inhibitor-1 were significantly increased in patients with H. pylori compared to those without infection or cured H. pylori . The presence of TAFI was detected in gastric mucosal epithelial cells. The concentration of TAFI was correlated with the degree of gastric mucosal atrophy, inflammation, and disease activity. These results show that TAFI is present in the gastric mucosa and that it may play a role in the pathogenesis of H. pylori infection-associated gastroduodenal disorders.