Identification and characterization of a potent and biologically-active PDE4/7 inhibitor via fission yeast-based assays

Abstract

We previously constructed a collection of fission yeast strains that express various mammalian cyclic nucleotide phosphodiesterases (PDEs) and developed a cell-based high throughput screen (HTS) for small molecule PDE inhibitors. Here we describe a compound, BC54, that is a selective inhibitor of enzymes from the cAMP-specific PDE4 and PDE7 families. Consistent with the biological effect of other PDE4 and PDE7 inhibitors, BC54 displays potent anti-inflammatory properties and is superior to a combination of rolipram (a PDE4 inhibitor) and BRL50481 (a PDE7A inhibitor) for inducing apoptosis in chronic lymphocytic leukemia (CLL) cells. We further exploited PKA-regulated growth phenotypes in fission yeast to isolate two mutant alleles of the human PDE4B2 gene that encode enzymes possessing single amino acid changes that confer partial resistance to BC54. We confirm this resistance to both BC54 and rolipram via yeast-based assays and, for PDE4B2T407A, in vitro enzyme assays. Thus, we are able to use this system for both chemical screens to identify biologically-active PDE inhibitors and molecular genetic studies to characterize the interaction of these molecules with their target enzymes. Based on its potency, selectivity, and effectiveness in cell culture, BC54 should be a useful tool to study biological processes regulated by PDE4 and PDE7 enzymes.

The future of bronchodilation: looking for new classes of bronchodilators

“…ASB16165, which inhibits PDE4 and PDE7A with IC 50 values of 2.1 nM and 15 nM, inhibited CD3/CD28-stimulated T-cell proliferation and cytokine release [28]. BC54 induced a larger anti-inflammatory effect on tumour necrosis factor-α production by macrophages and interleukin (IL)-2 production by T-lymphocytes as compared to rolipram alone or in combination with BRL50481 [29], and combined antisense inhibition of the expression of PDE4B, PDE4D and PDE7A produced a much greater anti-inflammatory effect than roflumilast alone in an in vivo mouse model of smoke-induced lung inflammation [22].…”

Yeast: bridging the gap between phenotypic and biochemical assays for high-throughput screening

“…However, given the extreme genetic tractability of both S. cerevisiae and S. pombe it is possible to engineer these model yeast to provide an indirect HTS readout through transactivation. Several examples have been reported [22,23] (Table 2), but the most extensively described and HTS utilized example of a yeast transactivation platform has been directed towards the discovery of mammalian phosphodiesterase (PDE) inhibitors [24,25,26,27,28]. The approximately 100, tissue specific, mammalian PDE isoforms all catalyze the conversion of cAMP and cGMP to the cyclic secondary messengers 5'AMP and 5'GMP.…”

“…Inhibition of PDE function rescued growth and the system has been formatted into a positive selection HTS used to screen libraries >200k in size [24,25,26,27,28]. To ensure specificity against single or small numbers of PDE isoforms screening against yeast expressing different isoforms can be undertaken, so called counter screening.…”

“…To ensure specificity against single or small numbers of PDE isoforms screening against yeast expressing different isoforms can be undertaken, so called counter screening. Using this approach, many of the hits were demonstrated to be specific for one or more PDE isoforms [24,25,26,27,28].…”

“…For example, some hits identified in the multiple HTS (3k to >200k compounds) using PDE transactivation 384-well platforms, demonstrated selective PDE inhibition and physiological activity (e.g. cAMP elevation) in secondary assays [24,25,26,27,28]. Similarly, the two (from 40,000 compounds) p38a hits from the lethal expression assay both demonstrated activity in in vitro and in mammalian cell secondary assays [17], and two cyclic peptide hits identified as active against a-syn in yeast prevented dopaminergic neuron loss in a Caenorhabditis elegans Parkinson's model [19].…”