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

描述(申请人提供)：许多生物过程由细胞感知其环境中的分子并产生细胞内信号以实现适当的生物反应的能力来调节。一个主要的信号通路涉及cAMP水平的调节，这是腺苷环化酶合成cAMP和cAMP磷酸二酯酶(PDE)破坏cAMP的函数。在裂解酵母中，cAMP水平受包括单个PDE基因的葡萄糖信号通路的调节。我们已经开发了报告结构，它提供了反映细胞内cAMP水平的生长表型。我们建议将哺乳动物的PDE基因引入我们的菌株中，这样生长行为将成为PDE活性的函数。我们将利用这些压力来实现以下两个目标。1)我们将对特定PDE的化学抑制剂进行高通量筛选。利用表达不同小鼠PDE(4A、4B、8A、8B)的菌株，我们希望识别非特异性和特异性抑制剂。值得注意的是，目前还没有已知的PDE8特异性抑制剂，因此很难确定PDEs酶在各种生物过程中的相对作用。2)我们将使用这些菌株来筛选目标PDE的生物激活物的cDNA文库，并鉴定这些激活物在哪些组织中表达。同时表达激活剂和目标PDE的菌株将接受化学文库筛选，以寻找抑制激活剂的化合物，而不是PDE本身。由于这些激活剂可以在发现PDE的组织的子集中表达，因此靶向该激活剂的化合物可以对PDE活性提供更具组织特异性的效应，从而提供副作用比直接靶向PDE的化合物更少的治疗益处。目前有一系列疾病正在用PDE抑制剂治疗，或者被认为可以用PDE抑制剂治疗。因此，开发这一用于识别PDE的化学和生物调节因子的体内平台具有确定下一代PDE相关药物的潜力，这些药物可用于治疗心脏、肺部和肾脏疾病，以及某些癌症、囊性纤维化、多发性硬化症、类风湿性关节炎、亨廷顿病、变应性鼻炎、牛皮癣、精神分裂症、阿尔茨海默病和抑郁症。

DESCRIPTION (provided by applicant): 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 activators of the target PDE and identify the tissues in which these activators are 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 Pharmaceuticals for 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.