We have described a novel fission yeast cell-based screening platform, amenable for high throughput screening to identify compounds that alter PDE activity. While a budding yeast system based on heat shock sensitivity of stationary phase cells has been previously reported (30
), cells in that assay had to be exposed to 0.5mM to 2mM rolipram to detect an effect on PDE4B (31
). In contrast, we have successfully screened compound libraries at an average concentration of 20μM to detect both known and previously unidentified PDE inhibitors (). We have gone on to determine ED50
values for rolipram against PDE4A and PDE4B of 25.9μM and 1.5μM, respectively. These values are higher than the IC50
values for rolipram against PDE4 enzymes (generally in the range of 1μM, (2
)), however cell-based assays cannot be as sensitive as in vitro
assays due to additional requirements of cell permeability and the presence of thousands of proteins within a cell, some of which may bind to the compound and reduce its availability to the target protein. In fact, it is even unclear how the intracellular concentration of rolipram relates to the concentation in the growth medium. In addition, we observe a significant difference in the ED50
for rolipram against PDE4A and PDE4B that is not consistent with in vitro
measurements. This may reflect the fact that PDE4B appears to be less active than PDE4A in our strains (), thus less inhibition is required to produce an effect on reporter expression. This is consistent with the lower ED50
of rolipram against PDE4A for repression of β-galactosidase expression (3-4μM) than for 5FOA-resistant growth (25.9μM). For strains that express the reporter genes at a high level due to high initial PDE activity, a greater reduction in reporter expression is required to produce 5FOA-resistant growth.
Using this inexpensive assay, it will be straightforward to develop a large collection of strains expressing either mammalian cAMP-specific or dual-specificity PDEs. This platform can also be used with PDEs from pathogens, whose inhibition may either kill the target pathogen or reduce virulence. Strains representing a broad panel of PDEs could be used to identify compounds possessing desirable specificity profiles to suggest the potential of individual compounds as candidate therapeutics. Moreover, because this platform identifies compounds based on stimulation of cell growth, it will not detect compounds that, while inhibiting PDEs in vitro, are too cytotoxic or cell-impermeable for therapeutic use. The compounds identified in this screen must also remain active within the yeast for much of the 48 hour incubation period in order to promote significant cell growth. This is not the case for the majority of PDE assays, which are carried out in vitro on purified proteins or on protein extracts over a short timecourse. In addition, this cell-based screening platform should be able to detect PDE inhibitors that may not be identified by in vitro screens. For example, compounds that prevent either intermolecular or intramolecular interactions required for enzyme formation would be overlooked in an in vitro assay on purified enzymes or protein extracts, yet should be identifiable in this assay.
This screening platform is not meant to replace in vitro assays that are well-established as a means of identifying and characterizing PDE inhibitors. Clearly, there are disadvantages of not being able to identify compounds that are not soluble in the yeast growth medium or are not permeable to the yeast cells, problems not encountered in an in vitro assay. However, our screen may represent a more holistic approach to drug discovery that identifies compounds with favorable drug-like qualities beyond having a low IC50 for the target enzyme.
Where this approach may show the greatest advantage over the in vitro
assays is in identifying compounds that selectively inhibit PDEs within a single family such as PDE4. While there has been significant interest in PDE4 inhibitors based on the CNS and anti-inflammatory effect of rolipram and other PDE4-specific inhibitors for the past 25 years (33
), no such inhibitor has received FDA approval. This is largely due to an emetic side-effect that is thought to be caused by PDE4D inhibition (35
). Structural approaches to PDE4 inhibition focus on the highly conserved active sites, making it unlikely to find compounds that display a significant difference in IC50
values against the PDE4 subtypes. As this cell-based assay can theoretically detect compounds that work by mechanisms other than binding to PDE active sites, there is the potential for identifying subtype-selective compounds.
The pilot high throughput screens against 3,120 bioactive compounds using strains expressing the yeast PDE Cgs2, or the murine PDEs 2A, 4A, and 4B identified a number of compounds that promote 5FOAR
growth, presumably by inhibiting the target PDEs to raise cAMP levels. These included the known PDE4 inhibitors rolipram and zardaverine, which only affected the PDE4A- and PDE4B-expressing strains (). Other compounds identified include coumarins, furanocoumarins, and furanochromones (), as well as flavonoids and steroids (not shown). Members of these compound classes are known to have PDE inhibitory properties (37
), including the furanocourmarin trioxsalen and the furanochromones khellin, and visnagin (38
), although most of these compounds identified had not been shown to be PDE inhibitors. Interestingly, both rolipram and imperatorin had been previously shown to inhibit HIV replication (40
), though the mechanism of imperatorin action was unknown. We demonstrate here that imperatorin is an effective PDE4B inhibitor, while its effect on PDE4A is very modest (). As with imperatorin, many compounds identified in our screens display therapeutic potential, in the absence of a complete understanding of their mechanism. Therefore, data from these screens will help to partially elucidate the effect of these compounds on mammalian cells.
While, the relative overlap of the compounds identified in each screen further validates this platform, it also alerts us to some caveats. Candidates from the Cgs2 screen display the least overlap with candidates from the other three screens (), consistent with the fact that the murine PDEs are more closely related to each other than to Cgs2. Furthermore, eighteen compounds inhibit both PDE4A and PDE4B, but not Cgs2 or PDE2A, consistent with the pharmacological grouping of PDE4A and PDE4B into the PDE4 family. On the other hand, there was unexpected amount of overlap from the PDE2A and PDE4B screens. As these PDEs appear to be less active in fission yeast than Cgs2 and PDE4A (), some of these candidates may either weakly reduce fbp1-driven transcription by a cAMP-independent manner or raise cAMP levels by stimulating adenylyl cyclase activity. This later hypothesis is consistent with the presence of diterpenoids in this group of compounds. However, we should be able to distinguish among cAMP-independent effects, adenylyl cyclase activation and PDE inhibition by measuring cAMP levels in experimental and control strains as in and . Specificially, compounds that act in a cAMP-independent manner will not affect cAMP levels in any strains, while compounds that activate adenylyl cyclase will elevate cAMP levels in control strains that lack PDE activity. Finally, the fact that a number of compounds were detected in only one of each of the four screens lends support for this platform as a tool for identifying isoenzyme-specific inhibitors, which could lead to the development of highly specific therapeutic compounds.
The ability to identify PDE inhibitors is based on the growth phenotype conferred by the cAMP-repressible fbp1-ura4 reporter. This system can also identify compounds that stimulate PDE activity to lower cAMP levels and increase fbp1-ura4 expression. PDE activators should confer Ura+ growth to strains whose high basal cAMP levels repress fbp1-ura4 expression in the absence of drug exposure (). Finally, as yeast are capable of maintaining autonomously-replicating plasmids, one could screen cDNA libraries for genes that encode biological inhibitors or activators of target PDEs, which could serve as novel targets for high throughput screens. Thus, this screening platform has the potential for identifying novel PDE inhibitors and activators, as well as new ways to moderate cAMP signaling pathways in an effort to improve therapeutic approaches to treating a wide array of human diseases.