Identification and Characterization of PDE8 Inhibitors Using a Fission Yeast Based High-throughput Screening Platform

Thesis advisor: Charles S. HoffmanIn this thesis, I describe the development of a screening platform for detecting PDE8A inhibitors using the cAMP-dependent glucose sensing pathway of the fission yeast Schizosaccharomyces pombe, which led us to discover several PDE8A selective inhibitors. In this system, the only PDE of the fission yeast is replaced with mammalian PDE8A1 in strains that have been engineered such that PDE inhibition is required to allow cell growth. Using this system, I screened 56 compounds obtained from PDE4 and PDE7 high throughput screens (HTSs) and identified a PDE4-PDE8 dual specificity inhibitor. Using this as a positive control, I developed a robust high-throughput screen (HTS) for PDE8A inhibitors and screened 240,267 compounds at the Harvard Medical School ICCB Screening Facility. Approximately 0.2 % of the screened compounds were potential PDE8A inhibitors with 0.03% displaying significant potency. Secondary assays of 367 of the most effective compounds against strains expressing PDE8A (both full length and catalytic domain), PDE4A and PDE7A or PDE7B led to the selection of structurally diverse compounds for further testing. To profile the selectivity of twenty-eight of these compounds, dose response assays were conducted using 16 yeast strains that express different PDE isoforms (representing all PDE families with the exception of the PDE6 family). These assays identified compounds with different patterns of inhibition, including structurally-distinct PDE8A-specific inhibitors. By evaluating the effects of these compounds for steroid production in mouse Leydig cells, biologically active compounds that can elevate steroid production were identified.Thesis (PhD) — Boston College, 2011.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse Leydig cells via PDE8 and PDE4 inhibition.

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems

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 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

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 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

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