Identification of selective inhibitors of NAD+-dependent deacetylases using phenotypic screens in yeast
Identification of selective inhibitors of NAD+-dependent deacetylases using phenotypic screens in yeast
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DOI：
10.1074/jbc.m308966200
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发表时间：
2003-12-26
影响因子：
4.8
通讯作者：
Bedalov, A
中科院分区：
文献类型：
--
作者：
Hirao, M;Posakony, J;Bedalov, A
Sir2 和 Hst1 是 NAD(+) 依赖性脱乙酰酶，参与酵母的转录抑制。这两种酶高度同源，但对小分子抑制剂 splitomicin（化合物 1）具有不同的敏感性（Bedalov, A.、Gatbonton, T.、Irvine, W. P.、Gottschling, D. E. 和 Simon, J. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15113-15118）。我们现在已经在 Hst1 的一个小螺旋模块内定义了一个关键氨基酸残基，它赋予了对 splitomicin 的相对抗性。对 100 种分裂霉素类似物进行基于细胞的平行筛选，鉴定出对 Sir2 或 Hst1 表现出更高选择性的化合物。一系列基于斯普利托米星衍生物、脱氢斯普利托米星（化合物 2）的化合物，有效地对缺乏 Hst1 脱乙酰酶的酵母菌株进行表型复制，同时对 Sir2 的沉默活性没有影响。此外，我们还发现了一种对 Sir2 具有更高选择性的化合物。使用全基因组 DNA 微阵列分析确认了选择性。这项研究强调了表型筛选在酶功能选择性抑制剂的开发和表征中的力量。
Sir2 and Hst1 are NAD(+)-dependent deacetylases involved in transcriptional repression in yeast. The two enzymes are highly homologous yet have different sensitivity to the small-molecule inhibitor splitomicin (compound 1) (Bedalov, A., Gatbonton, T., Irvine, W. P., Gottschling, D. E., and Simon, J. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15113-15118). We have now defined a critical amino acid residue within a small helical module of Hst1 that confers relative resistance to splitomicin. Parallel cell-based screens of 100 splitomicin analogues led to the identification of compounds that exhibit a higher degree of selectivity toward Sir2 or Hst1. A series of compounds based on a splitomicin derivative, dehydrosplitomicin (compound 2), effectively phenocopied a yeast strain that lacked Hst1 deacetylase while having no effect on the silencing activities of Sir2. In addition, we identified a compound with improved selectivity for Sir2. Selectivity was affirmed using whole-genome DNA microarray analysis. This study underscores the power of phenotypic screens in the development and characterization of selective inhibitors of enzyme functions.