High-Content, Image-Based Screening for Drug Targets in Yeast

Background Drug discovery and development are predicated on elucidation of the potential mechanisms of action and cellular targets of candidate chemical compounds. Recent advances in high-content imaging techniques allow simultaneous analysis of a range of cellular events. In this study, we propose a novel strategy to identify drug targets by combining genetic screening and high-content imaging in yeast. Methodology In this approach, we infer the cellular functions affected by candidate drugs by comparing morphologic changes induced by the compounds with the phenotypes of yeast mutants. Conclusions Using this method and four well-characterized reagents, we successfully identified previously known target genes of the compounds as well as other genes involved with functionally related cellular pathways. This is the first demonstration of a genetic high-content assay that can be used to identify drug targets based on morphologic phenotypes of a reference mutant panel.

[1]  Y. Ohya,et al.  Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants , 2010, Genetics.

[2]  Gary D Bader,et al.  The Genetic Landscape of a Cell , 2010, Science.

[3]  B. Sjöberg,et al.  RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank , 2009, BMC Genomics.

[4]  John A. Tallarico,et al.  Multi-parameter phenotypic profiling: using cellular effects to characterize small-molecule compounds , 2009, Nature Reviews Drug Discovery.

[5]  Yolanda T. Chong,et al.  A picture is worth a thousand words: Genomics to phenomics in the yeast Saccharomyces cerevisiae , 2009, FEBS letters.

[6]  Yoshikazu Ohya,et al.  Multidimensional quantification of subcellular morphology of Saccharomyces cerevisiae using CalMorph, the high-throughput image-processing program. , 2009, Journal of biotechnology.

[7]  D. Botstein,et al.  A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds , 2009, Nature Biotechnology.

[8]  Corey Nislow,et al.  Yeast chemical genomics and drug discovery: an update. , 2008, Trends in pharmacological sciences.

[9]  Arthur Wuster,et al.  Chemogenomics and biotechnology. , 2008, Trends in biotechnology.

[10]  Kathryn A. O’Donnell,et al.  Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae. , 2008, Molecular cell.

[11]  Ian Dunham,et al.  A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae , 2008, Nature Methods.

[12]  P. Kane.  The long physiological reach of the yeast vacuolar H+-ATPase , 2007, Journal of bioenergetics and biomembranes.

[13]  E. Krausz,et al.  Cell-based high-content screening of small-molecule libraries. , 2007, Current opinion in chemical biology.

[14]  Y. Ohya,et al.  Diversity of Ca2+-Induced Morphology Revealed by Morphological Phenotyping of Ca2+-Sensitive Mutants of Saccharomyces cerevisiae , 2007, Eukaryotic Cell.

[15]  Inmar E. Givoni,et al.  Exploring the Mode-of-Action of Bioactive Compounds by Chemical-Genetic Profiling in Yeast , 2006, Cell.

[16]  H. Kitano,et al.  In Vivo Robustness Analysis of Cell Division Cycle Genes in Saccharomyces cerevisiae , 2006, PLoS genetics.

[17]  Taro L. Saito,et al.  High-dimensional and large-scale phenotyping of yeast mutants. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Peter G Schultz,et al.  A genome-wide overexpression screen in yeast for small-molecule target identification. , 2005, Chemistry & biology.

[19]  David Botstein,et al.  GO: : TermFinder--open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes , 2004, Bioinform..

[20]  T. Stevens,et al.  The Bafilomycin/Concanamycin Binding Site in Subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[21]  John D. Storey,et al.  Strong control, conservative point estimation and simultaneous conservative consistency of false discovery rates: a unified approach , 2004 .

[22]  Joshua D. Schnell,et al.  Receptor internalization in yeast requires the Tor2-Rho1 signaling pathway. , 2003, Molecular biology of the cell.

[23]  K. Altendorf,et al.  Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c* , 2002, The Journal of Biological Chemistry.

[24]  J. Toyn,et al.  Saccharomyces cerevisiae Is Capable of de Novo Pantothenic Acid Biosynthesis Involving a Novel Pathway of β-Alanine Production from Spermine* , 2001, The Journal of Biological Chemistry.

[25]  Yudong D. He,et al.  Functional Discovery via a Compendium of Expression Profiles , 2000, Cell.

[26]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[27]  N. Sauer,et al.  The Fenpropimorph Resistance Gene FEN2 from Saccharomyces cerevisiae Encodes a Plasma Membrane H+-Pantothenate Symporter* , 1999, The Journal of Biological Chemistry.

[28]  Elizabeth A. Winzeler,et al.  Genomic profiling of drug sensitivities via induced haploinsufficiency , 1999, Nature Genetics.

[29]  David Botstein,et al.  SGD: Saccharomyces Genome Database , 1998, Nucleic Acids Res..

[30]  A. Chabes,et al.  Rnr4p, a novel ribonucleotide reductase small-subunit protein , 1997, Molecular and cellular biology.

[31]  T. Watanabe,et al.  Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae. , 1995, European journal of biochemistry.

[32]  N. Morin,et al.  The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[33]  B. Taillon,et al.  STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p. , 1994, The Journal of biological chemistry.

[34]  K. Altendorf,et al.  Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. , 1993, Biochemistry.

[35]  R. Hector,et al.  Compounds active against cell walls of medically important fungi , 1993, Clinical Microbiology Reviews.

[36]  A. Gräslund,et al.  EPR stopped-flow studies of the reaction of the tyrosyl radical of protein R2 from ribonucleotide reductase with hydroxyurea. , 1992, Biochemical and biophysical research communications.

[37]  Y. Moriyama,et al.  Kinetic studies of chromaffin granule H+-ATPase and effects of bafilomycin A1. , 1990, Biochemical and biophysical research communications.

[38]  R. W. Davis,et al.  Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase. , 1990, Genes & development.

[39]  M. Basson,et al.  Saccharomyces cerevisiae contains two functional genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[40]  D. Kerridge,et al.  Echinocandin inhibition of 1,3‐β‐D‐glucan synthase from Candida albicans , 1984 .

[41]  D. Kerridge,et al.  Echinocandin inhibition of 1,3-beta-D-glucan synthase from Candida albicans. , 1984, FEBS letters.

[42]  H. Kinashi,et al.  Alkaline degradation products of concanamycin a , 1981 .

[43]  R Monaghan,et al.  Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[44]  C. Chang,et al.  Ribonucleotide reductase isolated from human cells. Heterogeneity among the sources. , 1978, Biochemical pharmacology.

[45]  P. Reichard,et al.  Inhibition of ribonucleoside diphosphate reductase by hydroxyurea. , 1968, Cancer research.

[46]  D. Cox,et al.  An Analysis of Transformations , 1964 .

[47]  A. R. Jonckheere,et al.  A DISTRIBUTION-FREE k-SAMPLE TEST AGAINST ORDERED ALTERNATIVES , 1954 .