Identification of selective inhibitors of NAD+-dependent deacetylases using phenotypic screens in yeast.
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.
Hirao
Maki
M
Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Posakony
Jeffrey
J
Nelson
Melisa
M
Hruby
Henning
H
Jung
Manfred
M
Simon
Julian A
JA
Bedalov
Antonio
A
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D004305:Dose-Response Relationship, Drug; D025141:Drug Resistance, Fungal; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005800:Genes, Fungal; D017930:Genes, Reporter; D016681:Genome, Fungal; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000037:antagonists & inhibitors* / Q000737:chemistry; D056565:Sirtuin 2; D037761:Sirtuins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016615:Telomere / Q000648:ultrastructure; D001616:beta-Galactosidase / Q000378:metabolism
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D009281:Naphthalenes; D011753:Pyrones; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D009243:NAD; C444426:splitomicin; D001616:beta-Galactosidase; C103934:HST1 protein, S cerevisiae; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases