Many biological processes are regulated by a cell's ability to sense molecules in its environment and create an intracellular signal to effect an appropriate biological response. One major signaling pathway involves the regulation of cAMP levels, which is a function of cAMP synthesis by adenylate cyclases and cAMP destruction by cAMP phosphodiesterases (PDE). In the fission yeast, Schizosaccharomyces pombe, cAMP levels are regulated by a glucose signaling pathway that includes a single PDE gene. We have developed reporter constructs,which confer growth phenotypes that reflect the cell's intracellular cAMP level. We propose to introduce mammalian PDE genes into our strains, such that the growth behavior will be a function of PDE activity. We will use such strains to carry out the following two aims. 1) We will conduct high throughput screening for chemical inhibitors of specific PDEs. Utilizing strains expressing various murine PDEs (4A, 4B, 8A, 8B), we expect to identify both nonspecific and specific inhibitors. Of note, there are no known PDE8-specific inhibitors, thus making it difficult to determine the relative role of PDES enzymes in various biological processes. 2) We will use these strains to screen a cDNA library for biological activatorsof the target PDE and identify the tissues in which these activatorsare expressed. Strains expressing both the activator and the target PDE will be subjected to chemical library screens for compounds that inhibit the activator, rather than the PDE itself. As these activators may be expressed in a subset of tissues in which the PDE is found, compounds that target the activator may provide a more tissue-specific effect on PDE activity, and thus provide a therapeutic benefit with less of a side-effect than would be possible for compounds that target the PDE directly. There is a broad range of diseases that are currently being treated with PDE inhibitors, or are thought to be amenable to treatment with PDE inhibitors. Therefore, the development of this in vivo platform to identify chemical and biological regulators of PDEs has the potential of identifying the next generation of PDE-related Pharmaceuticalsfor the treatment of cardiac, pulmonary, and renal diseases, as well as certain cancers, cystic fibrosis, multiple sclerosis, rheumatoid arthritis, Huntington's Disease, allergic rhinitis, psoriasis, schizophrenia, Alzheimer's disease and depression.
