Methods to Assess Phosphodiesterase and/or Adenylyl Cyclase Activity Via Heterologous Expression in Fission Yeast.
Methods to Assess Phosphodiesterase and/or Adenylyl Cyclase Activity Via Heterologous Expression in Fission Yeast.
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通过裂殖酵母中的异源表达评估磷酸二酯酶和/或腺苷酸环化酶活性的方法。
DOI：
10.1007/978-1-0716-2245-2_6
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Hoffman,CharlesS
中科院分区：
文献类型：
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作者：
Domin,Marek;Hoffman,CharlesS
裂解酵母中环核苷酸磷酸二酯酶（PDEs）和腺苷酸环化酶（ACs）的异源表达可以与pka抑制的报告基因结合使用，对这些靶酶的小分子抑制剂进行高通量筛选，或从这些筛选中评估命中的化合物及其类似物。在这里，我们描述了测试这种化合物面板的两种方法。第一种方法使用生长试验，在含有嘧啶类似物5-氟糖酸（5FOA）的培养基中进行生长，以抑制PDE活性以激活PKA。第二种方法使用质谱法直接测量化合物处理的影响，以研究调节PDE或AC活性的化合物。
Heterologous expression of cyclic nucleotide phosphodiesterases (PDEs) and adenylyl cyclases (ACs) in the fission yeastSchizosaccharomyces pombecan be used in combination with PKA-repressed reporters to either carry out high throughput screens for small molecule inhibitors of these target enzymes or to assess hit compounds and their analogs from such screens. Here, we describe two methods for testing panels of such compounds. The first uses a growth assay for which growth in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA) occurs in response to inhibiting PDE activity to activate PKA. The second uses mass spectrometry to directly measure the impact of compound treatment to study compounds that modulate either PDE or AC activity.