Fevipiprant (QAW039), a slowly dissociating CRTh2 antagonist with the potential for improved clinical efficacy

Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [3H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2 (PGD2). The binding kinetics of QAW039 determined directly using [3H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 107 M-1min-1 and 0.048 minute-1, respectively. Importantly, the koff of QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [35S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2 after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039's effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2 concentrations, which may be clinically relevant

Isoform-Specific Biased Agonism of Histamine H 3 Receptor Agonists s

ABSTRACT The human histamine H 3 receptor (hH 3 R) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the H 3 R; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hH 3 R splice variants (hH 3 R 445 and hH 3 R 365 ) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3b (GSK3b) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former's designation as a "protean" agonist. The 80 amino acid IL3 deleted isoform hH 3 R 365 is more permissive in its signaling than hH 3 R 445 : 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hH 3 R 365 was completely unable to stimulate GSK3b phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G proteincoupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants

Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates

Abstract Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated

Comparative genotypic and phenotypic analysis of human peripheral blood monocytes and surrogate monocyte-like cell lines commonly used in metabolic disease research

<div><p>Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, <i>IDO1</i>, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.</p></div

Significant upregulation or downregulation in the relative gene expression levels compared to the un-differentiated cell of monocyte-like cells lines and human macrophages upon activation by either 20 nM PMA for 24 h for the cell lines, and 10 ng/mL GM-CSF for 6 days with or without polarization using 10 ng/mL LPS and 20 ng/mL IFNγ.

<p>Summary data used to generate the Venn diagrams as shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.g003" target="_blank">Fig 3</a></b>.</p

Relative expression analysis of chemokines, cytokines, receptors, enzymes, and regulatory factors for undifferentiated, differentiated and polarized human peripheral blood monocytes (PBMCs; A and E; white, grey and black bars respectively), and monocyte-like cell lines (MCLCs) including U-937 (B and F; red bars), THP-1 (C and G; blue bars), and HL-60 (D and H; yellow bars).

<p>PBMCs were differentiated using 10 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) for 6 days to give M(GC) and activated using 100 ng/mL LPS and 20 μg/mL IFNγ for 24 h to generate M(GC)LPS/IFNγ. MCLCs were differentiated using 16 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h. Grouped data are shown as mean ± SEM (n = 3–10). Relative expression data including the mean ± SEM are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s003" target="_blank">S3 Table</a></b>. Genes not detected were marked as <i>n/d</i>. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test compared to undifferentiated cells for the PBMCs, or by Student’s t-test for the MCLCs, with *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 and ****<i>P</i><0.0001 deemed significant.</p

Effects of differentiation treatment on cluster of differentiation (CD) markers expression on human peripheral blood monocytes (PBMCs; grey bars) and monocyte-like cell lines (MCLCs; U-937; red bars and THP-1; blue bars) and concentration-response curves to proinflammatory chemoattractants using a transwell migration assay using PBMCs (grey circles), U-937 (red circles) and THP-1 (blue circles) cells.

<p>Data are expressed as % cells expressing (%PE+ population) for (<b>A</b>) CD11c, (<b>B</b>) CD163, (<b>C</b>) CD14, (<b>D</b>) CD68, (<b>E</b>) CD80, (<b>F</b>) CD86 and (<b>G</b>) HLA Class II, or as the relative level of expression (mean PE) for (<b>H</b>) CD11c, (<b>I</b>) CD163, (<b>J</b>) CD14, (<b>K</b>) CD68, (<b>L</b>) CD80, (<b>M</b>) CD86 and (<b>N</b>) HLA Class II. Grouped data are shown as mean ± SEM (n = 3–5). Data are expressed on a Log2 scale as the chemotactic index compared to basal levels following 3–4 h treatment with the various chemoattractants using (<b>O</b>) MCP-1, (<b>P</b>) fMLP, (<b>Q</b>) LTB4, (<b>R</b>) MIP1α and (<b>S</b>) 10% FBS. Grouped data are shown ± SEM (n = 3–22). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test compared to undifferentiated cells for the CD markers, and two-way ANOVA with Dunnett’s multiple comparison tests compared to vehicle control for chemotaxis, with *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 and ****<i>P</i><0.0001 deemed significant.</p

Hierarchical clustered heat map of mean relative expression values for undifferentiated and differentiated CD14⁺ human peripheral blood mononuclear cells (PBMC, M(GC) and M(GC)LPS/IFNγ) and monocyte-like cell lines (MCLCs ± PMA) including THP-1, U-937 and HL-60s.

<p>PBMCs were differentiated using 10 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) for 6 days to give M(GC) and activated using 100 ng/mL LPS and 20 μg/mL IFNγ for 24 h to generate M(GC)LPS/IFNγ. MCLCs were differentiated using 16 ng/mL phorbol-12-myristate-13-acetate (PMA) for 48 h. Data represent n = 3–10; raw data, including the relative expression ± SEM used to generate this heat map, are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s003" target="_blank">S3</a> Tables</b>. All qPCR data were normalized against both housekeeper genes <i>GAPDH</i> and <i>ACTB2</i> and the values calculated as described in the Methods. Relative expression values were then calculated versus the relevant non-differentiated cell type. Where no expression was detected a value of 0 was used. Thirty-five genes that encode for inflammatory chemokines, cytokines, adipokines and their relevant receptors were selected as they are associated with inflammation or have been implicated in the development and/or progression of obesity-induced insulin resistance. In addition, small subsets of genes encoding for regulatory factors and enzymatic processes that have been implicated in the pathogenesis of T2DM were profiled. Differential gene expression data were analysed using R version 3.4.1 and hierarchical clustering was performed using complete linkage method with Euclidean distance measure.</p