Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3′-diindolylmethane. 3,3′-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3′-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine172, located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3′-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3′-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3′-diindolylmethane and was not affected adversely by mutation of arginine172. These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings

Discovery of 9-Cyclopropylethynyl-2-((S)-1-[1,4]dioxan-2-ylmethoxy)-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (GLPG1205), a unique GPR84 negative allosteric modulator undergoing evaluation in a phase II clinical trial

GPR84 is a medium chain free fatty acid-binding G-protein-coupled receptor associated with inflammatory and fibrotic diseases. As the only reported antagonist of GPR84 (PBI-4050) that displays relatively low potency and selectivity, a clear need exists for an improved modulator. Structural optimization of GPR84 antagonist hit 1, identified through high-throughput screening, led to the identification of potent and selective GPR84 inhibitor GLPG1205 (36). Compared with the initial hit, 36 showed improved potency in a guanosine 5′-O-[γ-thio]triphosphate assay, exhibited metabolic stability, and lacked activity against phosphodiesterase-4. This novel pharmacological tool allowed investigation of the therapeutic potential of GPR84 inhibition. At once-daily doses of 3 and 10 mg/kg, GLPG1205 reduced disease activity index score and neutrophil infiltration in a mouse dextran sodium sulfate-induced chronic inflammatory bowel disease model, with efficacy similar to positive-control compound sulfasalazine. The drug discovery steps leading to GLPG1205 identification, currently under phase II clinical investigation, are described herein

Enantioselective synthesis of indoloquinolizidines

Asymmetric synthesis of indoloquinolizidine has been accomplished by using acyl pyridinium salt bearing in 3 position a chiral aminal

Regio and enantioselective synthesis of 4-carbomethoxymethy-1,4-dihydropyridines

Regio and enantioselective 1,4 addition of ethoxyvinyl copper reagent on a pyridine bearing in position 3 a chiral aminal is described. This reaction allows the synthesis of various chiral N-acyl-4-methoxymethyl-1,4-dihydropyridines

Chiral aminal templates 5. Preparation of chiral 3-carboxaldehydes-1,4-dihydropyridines

Organocopper reagents react with 1-acylpyridinium salts, bearing in the 3 position a chiral aminal [1-acyl-3-(trans-1,3-dimethyl-4, 5-diphenylimidazolidin-2-yl)pyridinium salts], to give after acidic hydrolysis chiral 4-substituted 1-acyl-1, 4-dihydropyridine-3-carboxaldehydes in high optical purity and in good chemical yield

Cobalt-Catalyzed Carbozincation of Ynamides

International audienceAn original cobalt-catalyzed ynamide carbozincation leading mainly to diverse 3-aryl enamides with mild reaction conditions and good functional-group tolerance has been developed. This reaction displays an excellent regio- and total stereoselectivity and opens the way to appealing synthetic applications. Moreover, this approach allows the selective synthesis of biologically relevant 3,5-disubstituted oxazolone frameworks

BET Inhibition Silences Expression of <i>MYCN</i> and <i>BCL2</i> and Induces Cytotoxicity in Neuroblastoma Tumor Models

<div><p>BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the <i>MYC</i> oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of <i>MYCN</i> amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of <i>MYCN</i> copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of <i>BCL2</i> and <i>MYCN</i>. Reversal of <i>MYCN</i> or <i>BCL2</i> suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation <i>MYCN</i> and <i>BCL2</i> expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.</p> </div

<i>MYCN</i> expression is directly regulated by BRD4 and repressed by treatment with I-BET726.

<p>(<b>a</b>) Left: Concentration response curve for <i>MYCN</i> RNA expression following 24 hour treatment with I-BET726 in the CHP-212 cell line. Data was normalized to GAPDH and is presented as expression relative to DMSO-treated controls. Data presented as the average of two independent curves from a single experiment, and is representative of data from three independent biological replicates. Right: Table of IC₅₀ and percent inhibition values for <i>MYCN</i> suppression following 24 hour treatment with I-BET726 in the indicated cell lines. (<b>b</b>) BRD4 ChIP in the non-<i>MYCN</i>-amplified cell line SK–N–SH. Binding of BRD4 to the <i>MYCN</i> promoter or to an intergenic region on Chromosome 12 following treatment with vehicle or 1µM I-BET726 for six hours. Data is presented as fold enrichment over signal generated from IgG control immunoprecipitations. Data shown is from a single experiment representative of typical results. (<b>c</b>) BRD4 ChIP data at the <i>MYCN</i> promoter, presented as percent of vehicle control signal in the non-<i>MYCN</i>-amplified cell line SK–N–SH (left) and the <i>MYCN</i>-amplified cell line CHP-212 (right). SK–N–SH data represents the mean value ± standard deviation for three independent biological replicates. Asterisk indicates statistical significance as measured by t-test (p= 0.005). CHP-212 data represents the mean value ± standard deviation for two independent biological replicates. (<b>d</b>) Western blot analysis of N-Myc expression in the <i>MYCN</i>-amplified cell lines CHP-212 and IMR32 following 24 or 48 hour treatment with vehicle or 1µM I-BET726. Actin expression included as a loading control. (<b>e</b>) gIC₅₀ values obtained from CHP-212 cells overexpressing <i>GFP</i> or <i>MYCN</i> following treatment with I-BET726 in a 6 day growth-death assay. Data represents the mean value ± standard deviation from four independent experiments. (<b>f</b>) Concentration response curves for <i>GFP</i> or <i>MYCN</i>–overexpressing CHP-212 cells from a 6 day growth-death assay. Horizontal line indicates T₀ measurement (normalized to 100%). Data shown was from a single experiment representative of typical results. (<b>g</b>) Left: Histograms generated from cell cycle analysis in <i>GFP</i>- or <i>MYCN</i>-overexpressing CHP-212 cells following 4 days treatment with 5 µM I-BET726. Right: Percentage of cells in subs G1, G1, S, and G2 phases from the cell cycle experiment.</p