Generation of the hormone-responsive human hepatoma cell line, AH-G6PC, and optimization of assay conditions for siRNA transfection.

<p>A. AH-G6PC cells express both the reporter gene, β-lactamase, under the control of the G6PC promoter and endogenous G6PC. B. Insulin dose-responsively decreases Dex/cAMP activation of β-lactamase activity. C. Changes in endogenous G6PC mRNA levels in AH-G6PC cells treated with vehicle (basal), Dex/cAMP, Dex/cAMP after transfection with control siRNAs (siControl), or Dex/cAMP after transfection with glucocorticoid receptor siRNAs (siGR), and increasing concentrations of insulin. D. Incubation of AH-G6PC cells with metformin (667 µM) for 16 h or rosiglitazone (10 uM) for 6 h reduces G6PC mRNA expression. Data are shown as the means ± SEM fold change relative to basal (no Dex/cAMP or insulin) in a study performed in triplicate, and is representative of multiple experiments. *, <i>P</i><0.05 by Student’s t-test vs. Dex/cAMP-treated samples.</p

Identification of insulin sensitizer and GNG inhibitor genes.

<p>A. Chronic (16 h) 100 nM insulin pretreatment reduces acute (15 min) insulin induction of Akt phosphorylation (Akt-p) in AH-G6PC cells. Akt-p data are presented as the means ± SEM of % Akt-p/total Akt from a representative experiment performed in triplicate. B. Pooled siRNAs targeting JNK1, IKKβ, or PTEN significantly potentiates acute insulin-induced Akt-p after chronic insulin preincubation. Akt-p data are presented as the % Akt-p/total Akt relative to siControl-transfected cells (set as 100%) in response to acute insulin treatment after chronic insulin preincubation and are the means ± SEM of a representative experiment performed in triplicate. C. Differential impact of pooled siRNAs targeting each of the 113 genes described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036384#pone-0036384-g005" target="_blank">figures 5C and 5D</a> in regulating G6PC mRNA expression vs. potentiating insulin-induced Akt-p. Hits were separated into insulin sensitizers and GNG inhibitors that augmented acute insulin induction of Akt-p by more and less than 15% (vertical line), respectively. Akt-p data are presented as the % Akt-p/total Akt relative to siControl-transfected cells (set as 100%) in response to acute insulin treatment after chronic insulin preincubation and are the means of 3 independent experiments. G6PC mRNA expression data were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036384#pone-0036384-g005" target="_blank">figures 5C and 5D</a>. D. Table summarizing the number of insulin sensitizer and GNG inhibitor hits in distinct druggable gene categories.</p

Confirmation of primary screen hits by 2 distinct siRNA assays.

<p>A. Pooled siRNA knockdown of 165 Taqman-detectable primary hits vs. reduction of G6PC mRNA expression as determined by Taqman qPCR analysis. Data are shown as fold change compared to mRNA expression in siControl-transfected cells and are the means of a study performed in triplicate. The square contains 111 primary hits that reduce G6PC and target gene expression by ≤0.7- and ≤0.8-fold, respectively. Red, blue, black, and green dots represent target gene knockdown by <0.25-fold, between 0.25-fold to 0.5-fold, between 0.5-fold to 0.8-fold, and no knockdown, respectively. B. Schematic diagram for the 270 primary screen hits separated into different categories after target gene knockdown study. Red represents the 111 hits identified above in A. Blue, orange, and light blue represent primary hits that were not confirmed due to lack of G6PC knockdown. Sets of 7 distinct single siRNAs to each of the 270 primary screen hits were tested for their ability to diminish G6PC expression using the 4-gene HTG platform to quantify mRNA levels. Any target gene for which ≥2 single siRNAs reduced G6PC mRNA was deemed “rescued”. This assay rescued 9 primary hits whose siRNA knocked down their target gene mRNA but only reduced G6PC mRNA between 0.7- to 0.8-fold (purple) and 29 primary hits that were undetectable by Taqman analysis in the pooled siRNA confirmation assay described in A, but were detectable using enriched cDNA as describe in the “Results” section (green). The blue squares label the gene subsets that were combined to become our final 149 confirmed hits using the confirmation strategy described here. ND: undetectable by Taqman analysis; KD: knockdown.</p

Schematic summary of the siRNA screening process that identified, confirmed, and characterized novel insulin sensitizer and GNG inhibitor target genes.

<p>Schematic summary of the siRNA screening process that identified, confirmed, and characterized novel insulin sensitizer and GNG inhibitor target genes.</p

Screening of a druggable siRNA library with a 4-gene High-Throughput-Genomics (HTG) assay.

<p>A. Layout of genes within each well of a 4-gene HTG 384-well ArrayPlate. B. Insulin dose-responsively inhibits Dex/cAMP induction of G6PC and PDK4 mRNA expression in AH-G6PC cells when assayed by the HTG platform. Data are presented as the means ± SEM of a study performed in triplicate; similar results were obtained in 4 independent experiments. C. Dual-flashlight plot comparing SSMD values vs. average fold change in G6PC mRNA expression by all siRNA pools in the library in order to select hits that regulate G6PC mRNA levels. Glucocorticoid receptor (GR) siRNAs were used as positive controls in each plates (red dots), while non-targeting siRNAs were used as negative controls in each plate (green dots). The blue square and the small rectangle represent the up-regulators with G6PC ≥2 and SSMD ≥1 and G6PC ≥1.3 and SSMD ≥2, respectively. The orange square and the small rectangle represent the down-regulators with G6PC≤1/2 and SSMD≤−1 and G6PC≤0.7 and SSMD≤−2, respectively. D. Major proximal molecular mediators and key negative modulators of hepatic insulin signaling. siRNA knockdown of target genes that lowered and increased G6PC mRNA expression are indicated in orange and blue, respectively.</p

Known mediators and inhibitors of insulin signal transduction identified in the HTG Screen.

*<p>Gene expressios are significantly increased or decreased compared to siControl treated samples with p<0.05.</p

Pharmacological inhibitor of glucocorticoid receptor (GR) led to similar reduction of gluconeogenesis gene expression to GR siRNA in AH-G6PC cell.

<p>AH-G6PC cells were preincubated with or without the GR antagonist RU486 (5 µM) for 18 h. The cells were then incubated with Dex/cAMP alone, Dex/cAMP plus 400 nM insulin, or Dex/cAMP plus RU486 (in the cells treated with RU486 overnight) for 6 h. A. Taqman analysis was performed on mRNA isolated from the cells to assay G6PC mRNA relative to Dex/cAMP treated cells. B. Multiplex HTG ArrayPlate analysis of mRNA isolated from the cells was used to quantify PEPCK, PGC1α, and G6PC mRNA expression. Data are plotted as fold change vs. mRNA levels in cells treated only with Dex/cAMP and are the means ± SEM of 3 independent experiments.</p

Discovery of 5‑Amino‑<i>N</i>‑(1<i>H</i>‑pyrazol-4-yl)pyrazolo[1,5‑<i>a</i>]pyrimidine-3-carboxamide Inhibitors of IRAK4

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-<i>N</i>-(1<i>H</i>-pyrazol-4-yl)­pyrazolo­[1,5-<i>a</i>]­pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing