A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse Leydig cells via PDE8 and PDE4 inhibition.
A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse Leydig cells via PDE8 and PDE4 inhibition.
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DOI：
10.1371/journal.pone.0071279
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Hoffman CS
中科院分区：
文献类型：
--
作者：
Demirbas D;Wyman AR;Shimizu-Albergine M;Cakici O;Beavo JA;Hoffman CS
建立了一种基于细胞的高通量筛选技术(HTS)来检测磷酸二酯酶8(PDE8)和PDE4/8组合抑制剂。在缺乏腺酰环化酶的菌株中，用小鼠PDE8A1基因替换裂殖酵母的PDE基因，我们获得了在5-氟罗丹酸(5FOA)培养基中蛋白激酶A(PKA)刺激的生长反映PDE8活性的菌株。从我们之前确定的PDE4和PDE7抑制剂中，我们确定了一种PDE4/8抑制剂，使我们能够优化筛选条件。在所筛选的222,711种化合物中，有0.2%的∼显示>；20的复合Z分数。另外，使用最有效的367种化合物的酵母检测确定了30种候选化合物进行进一步表征。其中化合物BC8-15体外抑制PDE4和PDE8的IC50值最低。该化合物对PDE10A和PDE11A也表现出显著的活性。BC8-15作为单一药理药物可促进小鼠间质细胞的类固醇合成。使用BC8-15和两个结构衍生物的分析支持一个模型，在该模型中，PDE8是睾丸间质细胞产生睾酮的主要调节因子，PDE4在这一过程中扮演额外的角色。BC8-15、BC8-15A和BC8-15C是商业上可用的化合物，它们对PDE4、PDE8、PDE10A和PDE11A表现出不同的活性模式，这是一种化学工具箱，可用于研究这些酶在细胞培养系统中的生物学作用。
A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC50 value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.
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DOI：
10.1073/pnas.95.15.8991
发表时间：
1998-07-21
影响因子：
11.1
作者：
Soderling, SH;Bayuga, SJ;Beavo, JA
通讯作者：
Beavo, JA
DOI：
10.1073/pnas.050585197
发表时间：
2000-03-28
影响因子：
11.1
作者：
Fawcett, L;Baxendale, R;Phillips, SC
通讯作者：
Phillips, SC
DOI：
10.1007/978-3-642-17969-3_5
发表时间：
2011-01-01
期刊：
PHOSPHODIESTERASES AS DRUG TARGETS
影响因子：
--
作者：
Demirbas, Didem;Ceyhan, Ozge;Hoffman, Charles S.
通讯作者：
Hoffman, Charles S.
DOI：
10.1073/pnas.86.20.7775
发表时间：
1989-10-01
影响因子：
11.1
作者：
MELLON, SH;VAISSE, C
通讯作者：
VAISSE, C
影响因子：
3.6
作者：
Shimizu-Albergine, Masami;Tsai, Li-Chun Lisa;Beavo, Joseph A.
通讯作者：
Beavo, Joseph A.