| NAD+-dependent deacetylases require the ubiquitous energy cofactor NAD+ for the removal of acetyl groups from substrate lysine residues. The founding member of this family of enzymes, Sir2p, and its closest homologue, Hst1p, are histone deacetylases found in the nuclei of yeast cells, where they transcriptionally repress genes by regulating the chromatin structure. Sir2p, required for silencing of large chromosomal domains, has been implicated in the genetic regulation of aging. Hst1p is a gene-specific repressor that regulates middle sporulation genes. In addition, we have now shown that Hst1p plays a central role in sensing and regulating physiological levels of NAD+. Gene expression analysis revealed that Hst1p specifically repressed genes involved in de novo NAD + biosynthesis, where the repression was readily eliminated in response to NAD+ depletion. The decline in the Hst1p deacetylase activity at low NAD+ levels was caused by its relatively low affinity toward NAD+, as demonstrated by in vitro experiments. Given that the functions of Sir2p depend on the amount of available NAD +, an increase in cellular NAD+ levels induced by the deletion of HST1 augmented silencing and extended lifespan.; Using a chemical genetic approach, we have developed selective inhibitors for Sir2p or Hst1p. The small-molecule inhibitor splitomicin (1) has been previously shown to be active against Sir2p and to a lesser extent Hst1p. We have now defined a critical amino-acid residue within a small helical module of Hst1p that confers relative resistance to splitomicin (1). Parallel cell-based screens of 100 splitomicin analogues led to the identification of compounds that exhibit a higher degree of selectivity toward Sir2p or Hst1p. A series of compounds based on a splitomicin derivative, dehydrosplitomicin (2), effectively phenocopied a yeast strain that lacked Hst1p deacetylase, while having no effect on the silencing activities of Sir2p. In addition, we identified a compound (26) with improved selectivity for Sir2p. 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. |
