The work presented here demonstrates that we have developed an effective fission yeast-based HTS for PDE7 inhibitors. Among the most potent inhibitors identified from over 48,000 compounds screened were two steroids and three podocarpanes, which are structurally related to steroids. This is noteworthy as ICOS Corporation previously identified a steroid-like compound, IC242, as a PDE7-specific inhibitor (23
); however, as with most compounds under development by pharmaceutical companies, this compound is not readily available nor has its structure been revealed. As BRL50481 is the only commercially-available PDE7 inhibitor, we set out to compare it to some compounds identified in our screen, all of which are commercially-available from either Microsource or Maybridge.
BRL50481 was developed based on its ability to inhibit PDE7A as judged by in vitro enzyme assays, and in both the 5FOA cell growth assay and in vitro enzyme assays of PDE7A inhibition, it is superior to our PDE7 inhibitors. In contrast, BRL50481 is not nearly as effective for inhibition of PDE7B as judged by our cell-based assay and by in vitro enzyme assays (, ). While we were initially surprised by this degree of subtype-selectivity, it should be noted that the PDE7A and PDE7B catalytic domains are only 66% identical, compared with PDE4A, PDE4B, PDE4C, and PDE4D, where the catalytic domains are ~85% identical. We have identified several other PDE7 and PDE4/7 inhibitors that produce a growth response in our PDE7B strain at lower concentrations than required for BRL50481 (, data not shown). Further work with BC30 or these other compounds could lead to the development of more potent PDE7 inhibitors, including ones based on a compound in our collection that inhibits PDE7B better than PDE7A (data not shown).
An unusual observation from the in vitro enzyme assays is that several compounds originally identified as inhibitors of PDE7, including BC30, stimulate the activity of the PDE4D catalytic domain (). Although we see no evidence that such stimulation occurs in mammalian cells or in our yeast strains that express full-length PDE4D2 or PDE4D3, this finding is quite intriguing. Because BC30 increases the Vmax of the PDE4D catalytic domain, and because it does not interfere with substrate binding, BC30 appears to bind PDE4D somewhere other than at the substrate-binding site. Given that the PDE4D catalytic domain is ~33% identical and 60% similar to those of the PDE7 enzymes, it is possible that a similar allosteric interaction occurs to inhibit the PDE7 enzymes. BC30 appears to increase the Km of PDE7B (data not shown), however, such a response is not incompatible with allosteric binding. Much remains to be learned about the mechanism of BC30 action, including why the stimulatory effect is not observed with the full-length PDE4D2 enzyme. It may simply be that the N-terminal or C-terminal ends of the protein interfere with either the site or the outcome of binding. We would like to emphasize that it is unlikely that compounds that act through allosteric sites will be found via structure-based drug design approaches that target PDE active sites or through medicinal chemistry programs that are based on previously-identified PDE inhibitors. In contrast, our screening platform identifies PDE inhibitors regardless of the site of action.
Finally, we show that BC30 reduces the inflammatory effect of LPS on activated U937 cells, as judged by TNFα release assays. In particular, we observe a potent synergistic effect upon treatment with BC30 and rolipram (). While we anticipated that combining PDE4 and PDE7 inhibition would produce such synergy, the degree to which TNFα release was inhibited was unexpected, as BRL50481 plus rolipram produces only a modest improvement over rolipram alone. In both our growth assays and in enzyme assays, BRL50481 is superior to BC30 as a PDE7A inhibitor, and is fully effective at 10μM, the concentration used in the TNFα assay. On the other hand, 10μM BRL50481 only partially inhibits PDE7B as judged by either assay, while BC30 is a fully effective PDE7B inhibitor. Therefore, it appears that PDE7B may be more important than PDE7A in this assay, or that dual inhibition of both subtypes is required for the synergistic effect seen with rolipram-mediated inhibition of PDE4. Alternatively, the anti-inflammatory effect of BC30 may be due to a target other than PDE7. 5FOA growth assays using strains expressing members of ten of the eleven mammalian PDE families show BC30 to be a potent PDE7 inhibitor and a weak PDE10A inhibitor. As transcription of PDE10A is induced in rat peritoneal macrophages upon LPS treatment (29
), it is possible that PDE10A inhibition could have an effect on TNFα release.
The yeast growth-based screening method used to discover BC30 as a PDE7A/PDE7B inhibitor with potent anti-inflammatory properties requires that the compounds identified have drug-like characteristics in addition to being effective PDE inhibitors. These compounds must show little toxicity to fission yeast, which should exclude compounds that bind a large number of proteins, as such promiscuous binding would likely reduce cell growth. In addition, these compounds must be cell permeable to fission yeast, and remain active for most or all of the 48 hour incubation period of the assay. Therefore, compounds identified by this method may represent superior starting material with regard to drug development relative to compounds identified by in vitro enzyme assays that prioritize candidates solely on the basis of binding affinity. (At the same time, the relative potencies of the compounds discovered by this method are confirmed by in vitro enzyme assays (, ).) In addition to BC30, podocarpanes could serve as starting material to develop PDE7 inhibitors that might avoid the side-effects associated with treatment with steroids, which also inhibit PDE7. Thus, this work opens up new avenues for drug development in the PDE7 arena.