Inhibition of PDE4A, PDE7A and PDE8A by BC8-15 as determined by <i>in vitro</i> enzyme assays.

<p>BC8-15 inhibitory activity was measured by <i>in vitro</i> PDE assays as described in Materials and Methods. Substrate concentrations are 10 nM cAMP for PDE8A and PDE7A and 50 nM cAMP for PDE4A. Each assay was performed at 10 different compound concentrations in duplicate reaction tubes. IC₅₀± SD values are determined by performing non-linear regression analysis on three independent experiments.</p

Development of a PDE8A inhibitor HTS.

<p><b>A)</b> Structure of PDE4/8 inhibitor BC69 used to optimize PDE8 inhibitor assay. <b>B)</b> A yeast-based screen finds no potent PDE8 inhibitors in a Known Bioactives Collection. PDE8A-expressing strain CHP1204 was screened against 2,640 known bioactive compounds in duplicate wells (plate A and B). Scatter plot of absorbance values from plate A against plate B is shown. Negative control wells contained 0.2% DMSO (black circles), positive control wells contained 10 or 20 µM BC69 (dark gray circles). Experimental wells are shown in light gray. The data set has a correlation value of 0.996.</p

Strain list.

<p>Strain list.</p

BC8-15 elevates testosterone release by primary Leydig cells.

<p>Leydig cells isolated from wild-type (Panel A) and PDE8A^−/−/B^−/− knock-out (Panel B) mice were treated with rolipram (20 µM) or BC8-15 (10 µM and 40 µM) for three hours. Testosterone released into the media was assayed in duplicate for each condition. Values represent the mean ± SEM. Data from wild type and knock-out samples were separately analyzed with one-way ANOVA followed by Tukey’s multiple comparison test. Significant differences are indicated in comparison to the DMSO control (* p<0.05, ** p<0.01, *** p<0.001).</p

Summary of the HTS data.

<p>The ability of 220,071 small molecules to promote 5FOA^R growth of a PDE8A-expressing strain was assessed by their composite Z scores (see Materials and Methods). The number of compounds within each indicated composite Z score interval is presented.</p

BC8-15 derivative elevation of progesterone release by MA-10 Leydig tumor cells corresponds with PDE8 inhibitory activity.

<p>MA-10 cells were treated with 20 µM of rolipram, BC8-15, BC8-15A or BC8-15C. Progesterone released into the media after 2 h was measured using a progesterone ELISA kit (Neogen). The data are representative of three independent experiments with 2–4 sample wells for each condition. Values are the mean of four experimental wells ± SEM. Data were analyzed with one-way ANOVA followed by a Tukey’s multiple comparison test (*** p<0.001). The response to BC8-15 is significantly different from that for BC8-15A (p<0.001) and both are significantly different from all other groups (p<0.001).</p

IC₅₀ values for BC8-15 and related compounds.

<p>The structures of BC8-15 and two structurally-related derivatives are shown. The IC₅₀ values of each compound were determined by <i>in vitro</i> PDE assays (see Material and Methods). The values represent mean IC₅₀± SD determined from three independent experiments.</p