A Fission Yeast-Based Platform for Phosphodiesterase Inhibitor HTSs and Analyses of Phosphodiesterase Activity
A Fission Yeast-Based Platform for Phosphodiesterase Inhibitor HTSs and Analyses of Phosphodiesterase Activity
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DOI：
10.1007/978-3-642-17969-3_5
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Hoffman, Charles S.
中科院分区：
文献类型：
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作者：
Demirbas, Didem;Ceyhan, Ozge;Hoffman, Charles S.
对裂解酵母菌株进行了工程改造，使其生长行为反映了异源环核苷酸磷酸二酯酶(PDE)的活性。这些菌株可用于高通量筛选(HTS)，以筛选具有“类药物”特征的PDE抑制剂，在48小时的生长刺激试验中显示活性。经过三代的发展，我们实验室已经产生了一批表达11个哺乳动物PDE家族中10个的适合于小分子抑制剂筛选的菌株。不能合成环核苷酸的菌株可以表征PDE的活性，因为酶的效力反映在必须添加到生长介质中以刺激细胞生长的cAMP或cGMP的数量上。在未来，该系统可用于筛选目标PDE的生物调节因子的cDNA文库，并用于构建共表达PDE及其相关调节蛋白的菌株，以促进对其功能的分子和遗传学研究，特别是鉴定不同的PDE伙伴蛋白复合体是否表现出不同的抑制剂敏感性模式。
Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High-Throughput Screens (HTSs) for PDE inhibitors that possess "drug-like" characteristics, displaying activity in a growth stimulation assay over a 48-h period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme's potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify whether different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.