An in vivo screen for biological and chemical regulators of mammalial PDEs

许多生物学过程都受到细胞感知环境中分子并创造 细胞内信号以实现适当的生物反应。一个主要的信号通路涉及 调节cAMP水平，这是腺苷酸环化酶和cAMP合成的功能 cAMP磷酸二酯酶（PDE）的破坏。在裂殖酵母，粟酒裂殖酵母，cAMP 水平由包括单个PDE基因的葡萄糖信号传导途径调节。我们已经开发 报告构建体，其赋予反映细胞的细胞内cAMP水平的生长表型。我们 我建议将哺乳动物PDE基因引入我们的菌株，这样生长行为将是一个功能， PDE活动。我们将使用这些菌株来实现以下两个目标。1)我们将进行高 用于特定PDE的化学抑制剂的通量筛选。利用表达各种鼠源性 PDE（4A，4B，8A，8B），我们希望能够识别非特异性和特异性抑制剂。值得注意的是， 已知的PDE8特异性抑制剂，因此难以确定PDES酶在 各种生物过程。2)我们将利用这些菌株筛选一个cDNA文库， 靶PDE并鉴定表达这些激活剂的组织。同时表达两种蛋白的菌株 激活剂和靶PDE将进行化学文库筛选，以寻找抑制PDE的化合物。 激活剂，而不是PDE本身。因为这些激活剂可以在其中表达激活剂的组织亚组中表达， 当发现PDE时，靶向激活剂的化合物可以对PDE活性提供更组织特异性的作用， 因此提供了治疗益处，而副作用比 直接瞄准PDE目前有广泛的疾病正在用PDE治疗 抑制剂，或被认为适合于用PDE抑制剂治疗。因此发展 这种鉴定PDE的化学和生物调节剂的体内平台具有鉴定 用于治疗心脏、肺和肾脏疾病的下一代PDE相关药物， 以及某些癌症、囊性纤维化、多发性硬化、类风湿性关节炎、亨廷顿氏病， 过敏性鼻炎、牛皮癣、精神分裂症、阿尔茨海默病和抑郁症。

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.