Comprehensive screening of human genes with inhibitory effects on yeast growth and validation of a yeast cell-based system for screening chemicals.
To evaluate yeast as a high-throughput cell-based system for screening chemicals that may lead to drug development, 10,302 full-length human cDNAs (~50% of the total cDNAs) were introduced into yeast. Approximately 5.6% (583 clones) of the cDNAs repressed the growth of yeast. Notably, ~25% of the repressive cDNAs encoded uncharacterized proteins. Small chemicals can be readily surveyed by monitoring their restorative effects on the growth of yeast. The authors focused on protein kinases because protein kinases are involved in various diseases. Among 263 protein kinase cDNAs (~50% of the total) expressed in yeast, 60 cDNAs (~23%), including c-Yes, a member of the Src tyrosine kinase family, inhibited the growth of yeast. Known inhibitors for protein kinases were examined for whether they reversed the c-Yes-induced inhibition of the yeast growth. Among 85 inhibitors tested, 6 compounds (PP2, PP1, SU6656, purvalanol, radicicol, and geldanamycin) reversed the inhibition, indicating a high specificity sufficient for validating this screening system. Human c-Yes was found to interact with Hsc82, one of the yeast chaperones. Radicicol and geldanamycin probably exerted their actions through interactions with Hsc82. These results indicate that when human proteins requiring molecular chaperones for their activities are subjected to the yeast screening system, 2 groups of chemicals may be found. The actions of one group are exerted through direct interactions with the human proteins, whereas those of the other group are mediated through interactions with chaperones.
Sekigawa
Masayuki
M
Nagahama Institute of Bio-Science and Technology, Shiga, Japan.
Kunoh
Tatsuki
T
Wada
Shu-Ichi
S
Mukai
Yukio
Y
Ohshima
Kazuhiko
K
Ohta
Shinji
S
Goshima
Naoki
N
Sasaki
Ryuzo
R
Mizukami
Tamio
T
D016227:Benzoquinones / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004795:Enzyme Stability / Q000187:drug effects; D017353:Gene Deletion; D005796:Genes; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D051575:Proto-Oncogene Proteins c-yes / Q000037:antagonists & inhibitors; D011687:Purines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D013449:Sulfonamides / Q000494:pharmacology; D014170:Transformation, Genetic
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; C471843:6-((3-chloro)anilino)-2-(isopropyl-2-hydroxyethylamino)-9-isopropylpurine; C412373:AG 1879; D016227:Benzoquinones; D018076:DNA, Complementary; D007211:Indoles; D047029:Lactams, Macrocyclic; D018942:Macrolides; D018832:Molecular Chaperones; D047428:Protein Kinase Inhibitors; D011687:Purines; D011720:Pyrazoles; D011743:Pyrimidines; C416927:SU 6656; D013449:Sulfonamides; D011494:Protein Kinases; D051575:Proto-Oncogene Proteins c-yes; C035359:monorden; C001277:geldanamycin