Transcriptional Response of Saccharomyces Cerevisiae to DNA-damaging Agents Does Not Identify the Genes That Protect Against These Agents

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2002  |  Pubmed ID: 12077312

The recent completion of the deletion of all of the nonessential genes in budding yeast has provided a powerful new way of determining those genes that affect the sensitivity of this organism to cytotoxic agents. We have used this system to test the hypothesis that genes whose transcription is increased after DNA damage are important for the survival to that damage. We used a pool of 4,627 diploid strains each with homozygous deletion of a nonessential gene to identify those genes that are important for the survival of yeast to four DNA-damaging agents: ionizing radiation, UV radiation, and exposure to cisplatin or to hydrogen peroxide. In addition we measured the transcriptional response of the wild-type parental strain to the same DNA-damaging agents. We found no relationship between the genes necessary for survival to the DNA-damaging agents and those genes whose transcription is increased after exposure. These data show that few, if any, of the genes involved in repairing the DNA lesions produced in this study, including double-strand breaks, pyrimidine dimers, single-strand breaks, base damage, and DNA cross-links, are induced in response to toxic doses of the agents that produce these lesions. This finding suggests that the enzymes necessary for the repair of these lesions are at sufficient levels within the cell. The data also suggest that the nature of the lesions produced by DNA-damaging agents cannot easily be deduced from gene expression profiling.

Systematic Screen for Human Disease Genes in Yeast

Nature Genetics. Aug, 2002  |  Pubmed ID: 12134146

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.

Functional Profiling of the Saccharomyces Cerevisiae Genome

Nature. Jul, 2002  |  Pubmed ID: 12140549

Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

"Chemogenomics: Tools for Protein Families" and "Chemical Genomics: Chemical and Biological Integration"

Pharmacogenomics. Jan, 2003  |  Pubmed ID: 12517281

A Chemical Genomics Approach to Understanding Drug Action

Trends in Pharmacological Sciences. Sep, 2003  |  Pubmed ID: 12967766

The complete collection of yeast deletion strains represents a unique, living biological computer for understanding gene function. The molecular 'barcodes' present in each of the deletion strains allow a quantitative ranking of the importance of any gene under any experimental condition of choice. In this article, some of the recent results generated from experiments that exploit the yeast deletion collection to understand mechanisms of drug action are discussed.

Chemogenomic Profiling: Identifying the Functional Interactions of Small Molecules in Yeast

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2004  |  Pubmed ID: 14718668

We demonstrate the efficacy of a genome-wide protocol in yeast that allows the identification of those gene products that functionally interact with small molecules and result in the inhibition of cellular proliferation. Here we present results from screening 10 diverse compounds in 80 genome-wide experiments against the complete collection of heterozygous yeast deletion strains. These compounds include anticancer and antifungal agents, statins, alverine citrate, and dyclonine. In several cases, we identified previously known interactions; furthermore, in each case, our analysis revealed novel cellular interactions, even when the relationship between a compound and its cellular target had been well established. In addition, we identified a chemical core structure shared among three therapeutically distinct compounds that inhibit the ERG24 heterozygous deletion strain, demonstrating that cells may respond similarly to compounds of related structure. The ability to identify on-and-off target effects in vivo is fundamental to understanding the cellular response to small-molecule perturbants.

Noise Minimization in Eukaryotic Gene Expression

PLoS Biology. Jun, 2004  |  Pubmed ID: 15124029

All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or "noise." Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

Mechanisms of Haploinsufficiency Revealed by Genome-wide Profiling in Yeast

Genetics. Apr, 2005  |  Pubmed ID: 15716499

Haploinsufficiency is defined as a dominant phenotype in diploid organisms that are heterozygous for a loss-of-function allele. Despite its relevance to human disease, neither the extent of haploinsufficiency nor its precise molecular mechanisms are well understood. We used the complete set of Saccharomyces cerevisiae heterozygous deletion strains to survey the genome for haploinsufficiency via fitness profiling in rich (YPD) and minimal media to identify all genes that confer a haploinsufficient growth defect. This assay revealed that approximately 3% of all approximately 5900 genes tested are haploinsufficient for growth in YPD. This class of genes is functionally enriched for metabolic processes carried out by molecular complexes such as the ribosome. Much of the haploinsufficiency in YPD is alleviated by slowing the growth rate of each strain in minimal media, suggesting that certain gene products are rate limiting for growth only in YPD. Overall, our results suggest that the primary mechanism of haploinsufficiency in yeast is due to insufficient protein production. We discuss the relevance of our findings in yeast to human haploinsufficiency disorders.

Functional Genomic Analysis of the Rates of Protein Evolution

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2005  |  Pubmed ID: 15800036

The evolutionary rates of proteins vary over several orders of magnitude. Recent work suggests that analysis of large data sets of evolutionary rates in conjunction with the results from high-throughput functional genomic experiments can identify the factors that cause proteins to evolve at such dramatically different rates. To this end, we estimated the evolutionary rates of >3,000 proteins in four species of the yeast genus Saccharomyces and investigated their relationship with levels of expression and protein dispensability. Each protein's dispensability was estimated by the growth rate of mutants deficient for the protein. Our analyses of these improved evolutionary and functional genomic data sets yield three main results. First, dispensability and expression have independent, significant effects on the rate of protein evolution. Second, measurements of expression levels in the laboratory can be used to filter data sets of dispensability estimates, removing variates that are unlikely to reflect real biological effects. Third, structural equation models show that although we may reasonably infer that dispensability and expression have significant effects on protein evolutionary rate, we cannot yet accurately estimate the relative strengths of these effects.

A Latent Variable Model for Chemogenomic Profiling

Bioinformatics (Oxford, England). Aug, 2005  |  Pubmed ID: 15919724

In haploinsufficiency profiling data, pleiotropic genes are often misclassified by clustering algorithms that impose the constraint that a gene or experiment belong to only one cluster. We have developed a general probabilistic model that clusters genes and experiments without requiring that a given gene or drug only appear in one cluster. The model also incorporates the functional annotation of known genes to guide the clustering procedure.

Genome-wide Requirements for Resistance to Functionally Distinct DNA-damaging Agents

PLoS Genetics. Aug, 2005  |  Pubmed ID: 16121259

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of approximately 4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

A Unique and Universal Molecular Barcode Array

Nature Methods. Aug, 2006  |  Pubmed ID: 16862133

Molecular barcode arrays allow the analysis of thousands of biological samples in parallel through the use of unique 20-base-pair (bp) DNA tags. Here we present a new barcode array, which is unique among microarrays in that it includes at least five replicates of every tag feature. The use of smaller dispersed replicate features dramatically improves performance versus a single larger feature and allows the correction of previously undetectable hybridization defects.

Experimental Approaches to Identify Genetic Networks

Current Opinion in Biotechnology. Oct, 2006  |  Pubmed ID: 16962766

Systems biology offers the promise of a fully integrated view of cellular physiology. To realize this potential requires the analysis of diverse genome-wide datasets and the incorporation of these analyses into integrated networks. In the past decade, the budding yeast Saccharomyces cerevisiae has provided the benchmark for the design of such large-scale experiments. Many of these experimental approaches have been adopted and adapted to study other systems, including worm, fly, fish and mammalian cultured cells, using an ingenious set of molecular tools.

Chemical Genomic Profiling for Identifying Intracellular Targets of Toxicants Producing Parkinson's Disease

Toxicological Sciences : an Official Journal of the Society of Toxicology. Jan, 2007  |  Pubmed ID: 17043098

The yeast deletion collection includes approximately 4700 strains deleted for both copies of every nonessential gene. This collection is a powerful resource for identifying the cellular pathways that functionally interact with drugs. In the present study, the complete pool of approximately 4700 barcoded homozygous deletion strains of Saccharomyces cerevisiae were surveyed to identify genes/pathways interacting with 1-methyl-4-phenylpyridinium (MPP(+)) and N,N-dimethyl-4-4-bipiridinium (paraquat), neurotoxicants that can produce Parkinson's disease. Each yeast mutant is molecularly "barcoded" the collections can be grown competitively and ranked for sensitivity by microarray hybridization. Analysis data from these screens allowed us to determine that the multivesicular body pathway is an important element of toxicity induced by both MPP(+) and paraquat. When yeast genes that when deleted showed sensitivity to MPP(+) and paraquat toxicity were analyzed for their homology to human genes, 80% were found to have highly conserved human homologs (with e < 10(-8)). Future work will address if these human genes may also functionally interact with MPP(+) and paraquat toxicity.

Systematic Pathway Analysis Using High-resolution Fitness Profiling of Combinatorial Gene Deletions

Nature Genetics. Feb, 2007  |  Pubmed ID: 17206143

Systematic genetic interaction studies have illuminated many cellular processes. Here we quantitatively examine genetic interactions among 26 Saccharomyces cerevisiae genes conferring resistance to the DNA-damaging agent methyl methanesulfonate (MMS), as determined by chemogenomic fitness profiling of pooled deletion strains. We constructed 650 double-deletion strains, corresponding to all pairings of these 26 deletions. The fitness of single- and double-deletion strains were measured in the presence and absence of MMS. Genetic interactions were defined by combining principles from both statistical and classical genetics. The resulting network predicts that the Mph1 helicase has a role in resolving homologous recombination-derived DNA intermediates that is similar to (but distinct from) that of the Sgs1 helicase. Our results emphasize the utility of small molecules and multifactorial deletion mutants in uncovering functional relationships and pathway order.

Accelerating the Discovery of Biologically Active Small Molecules Using a High-throughput Yeast Halo Assay

Journal of Natural Products. Mar, 2007  |  Pubmed ID: 17291044

The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.

Examining Protein Protein Interactions Using Endogenously Tagged Yeast Arrays: the Cross-and-capture System

Genome Research. Dec, 2007  |  Pubmed ID: 17989249

Comprehensive approaches to detect protein-protein interactions (PPIs) have been most successful in the yeast model system. Here we present "Cross-and-Capture," a novel assay for rapid, sensitive assessment of PPIs via pulldown of differently tagged yeast strain arrays. About 500 yeast genes that function in DNA replication, repair, and recombination and nuclear proteins of unknown function were chromosomally tagged with six histidine residues or triple VSV epitopes. We demonstrate that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we use "Cross-and-Capture" to identify two novel protein complexes: Rtt101p-Mms1p and Sae2p-Mre11p. The Rtt101p-Mms1p interaction was subsequently characterized by genetic and functional analyses. Our studies establish the "Cross-and-Capture" assay as a novel, versatile tool that provides a valuable complement for the next generation of yeast proteomic studies.

Genome-wide Analysis of Barcoded Saccharomyces Cerevisiae Gene-deletion Mutants in Pooled Cultures

Nature Protocols. 2007  |  Pubmed ID: 18007632

The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.

Defining Genetic Interaction

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2008  |  Pubmed ID: 18305163

Sometimes mutations in two genes produce a phenotype that is surprising in light of each mutation's individual effects. This phenomenon, which defines genetic interaction, can reveal functional relationships between genes and pathways. For example, double mutants with surprisingly slow growth define synergistic interactions that can identify compensatory pathways or protein complexes. Recent studies have used four mathematically distinct definitions of genetic interaction (here termed Product, Additive, Log, and Min). Whether this choice holds practical consequences has not been clear, because the definitions yield identical results under some conditions. Here, we show that the choice among alternative definitions can have profound consequences. Although 52% of known synergistic genetic interactions in Saccharomyces cerevisiae were inferred according to the Min definition, we find that both Product and Log definitions (shown here to be practically equivalent) are better than Min for identifying functional relationships. Additionally, we show that the Additive and Log definitions, each commonly used in population genetics, lead to differing conclusions related to the selective advantages of sexual reproduction.

Chemical-genetic Approaches for Exploring the Mode of Action of Natural Products

Progress in Drug Research. Fortschritte Der Arzneimittelforschung. Progrès Des Recherches Pharmaceutiques. 2008  |  Pubmed ID: 18416308

Determining the mode of action of bioactive compounds, including natural products, is a central problem in chemical biology. Because many genes are conserved from the yeast Saccharomyces cerevisiae to humans and a number of powerful genomics tools and methodologies have been developed for this model system, yeast is making a major contribution to the field of chemical genetics. The set of barcoded yeast deletion mutants, including the set of approximately 5000 viable haploid and homozygous diploid deletion mutants and the complete set of approximately 6000 heterozygous deletion mutants, containing the set of approximately 1000 essential genes, are proving highly informative for identifying chemical-genetic interactions and deciphering compound mode of action. Gene deletions that render cells hypersensitive to a specific drug identify pathways that buffer the cell against the toxic effects of the drug and thereby provide clues about both gene and compound function. Moreover, compounds that show similar chemical-genetic profiles often perturb similar target pathways. Gene dosage can be exploited to discover connections between compounds and their targets. For example, haploinsufficiency profiling of an antifungal compound, in which the set of approximately 6000 heterozygous diploid deletion mutants are scored for hypersensitivity to a compound, may identify the target directly. Creating deletion mutant collections in other fungal species, including the major human fungal pathogen Candida albicans, will expand our chemical genomics tool set, allowing us to screen for antifungal lead drugs directly. The yeast deletion mutant collection is also being exploited to map large-scale genetic interaction data obtained from genome-wide synthetic lethal screens and the integration of this data with chemical genetic data should provide a powerful system for linking compounds to their target pathway. Extensive application of chemical genetics in yeast has the potential to develop a small molecule inhibitor for the majority of all approximately 6000 yeast genes.

The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes

Science (New York, N.Y.). Apr, 2008  |  Pubmed ID: 18420932

Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes ( approximately 80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.

Identification of Small Molecule Inhibitors of Pseudomonas Aeruginosa Exoenzyme S Using a Yeast Phenotypic Screen

PLoS Genetics. Feb, 2008  |  Pubmed ID: 18454192

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

The Extensive and Condition-dependent Nature of Epistasis Among Whole-genome Duplicates in Yeast

Genome Research. Jul, 2008  |  Pubmed ID: 18463300

Since complete redundancy between extant duplicates (paralogs) is evolutionarily unfavorable, some degree of functional congruency is eventually lost. However, in budding yeast, experimental evidence collected for duplicated metabolic enzymes and in global physical interaction surveys had suggested widespread functional overlap between paralogs. While maintained functional overlap is thought to confer robustness against genetic mutation and facilitate environmental adaptability, it has yet to be determined what properties define paralogs that can compensate for the phenotypic consequence of deleting a sister gene, how extensive this epistasis is, and how adaptable it is toward alternate environmental states. To this end, we have performed a comprehensive experimental analysis of epistasis as indicated by aggravating genetic interactions between paralogs resulting from an ancient whole-genome duplication (WGD) event occurring in the budding yeast Saccharomyces cerevisiae, and thus were able to compare properties of large numbers of epistatic and non-epistatic paralogs with identical evolutionary times since divergence. We found that more than one-third (140) of the 399 examinable WGD paralog pairs were epistatic under standard laboratory conditions and that additional cases of epistasis became obvious only under media conditions designed to induce cellular stress. Despite a significant increase in within-species sequence co-conservation, analysis of protein interactions revealed that paralogs epistatic under standard laboratory conditions were not more functionally overlapping than those non-epistatic. As experimental conditions had an impact on the functional categorization of paralogs deemed epistatic and only a fraction of potential stress conditions have been interrogated here, we hypothesize that many epistatic relationships remain unresolved.

An Integrated Platform of Genomic Assays Reveals Small-molecule Bioactivities

Nature Chemical Biology. Aug, 2008  |  Pubmed ID: 18622389

Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.

Yeast Barcoders: a Chemogenomic Application of a Universal Donor-strain Collection Carrying Bar-code Identifiers

Nature Methods. Aug, 2008  |  Pubmed ID: 18622398

The ability to perform complex bioassays in parallel enables experiments that are otherwise impossible because of throughput and cost constraints. For example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is bar-coded with unique DNA sequences. It is, however, time-consuming and expensive to individually bar-code individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique bar codes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of bar-coded 'decreased abundance by mRNA perturbation' (DAmP) loss-of-function strains comprising 87.1% of all essential yeast genes. These experiments validate both the Barcoders and the DAmP strain collection as useful tools for genome-wide chemical-genetic assays.

Off-target Effects of Psychoactive Drugs Revealed by Genome-wide Assays in Yeast

PLoS Genetics. 2008  |  Pubmed ID: 18688276

To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes.

Yeast Chemical Genomics and Drug Discovery: an Update

Trends in Pharmacological Sciences. Oct, 2008  |  Pubmed ID: 18755517

The Saccharomyces cerevisiae sequencing project (the first eukaryotic genome decoded) was completed in 1995 and, subsequently, the first version of the yeast knockout collection was made available in 2002. Since then, many diverse studies have applied these resources to understand drug mechanism of action and to identify novel drug targets and target pathways. In this update of an earlier review, we present a snapshot of the current state of chemical genomic approaches in yeast, propose a set of integrated chemical genomic assays to move the field forward and consider its near-term future.

Combination Chemical Genetics

Nature Chemical Biology. Nov, 2008  |  Pubmed ID: 18936752

Predicting the behavior of living organisms is an enormous challenge given their vast complexity. Efforts to model biological systems require large datasets generated by physical binding experiments and perturbation studies. Genetic perturbations have proven important and are greatly facilitated by the advent of comprehensive mutant libraries in model organisms. Small-molecule chemical perturbagens provide a complementary approach, especially for systems that lack mutant libraries, and can easily probe the function of essential genes. Though single chemical or genetic perturbations provide crucial information associating individual components (for example, genes, proteins or small molecules) with pathways or phenotypes, functional relationships between pathways and modules of components are most effectively obtained from combined perturbation experiments. Here we review the current state of and discuss some future directions for 'combination chemical genetics', the systematic application of multiple chemical or mixed chemical and genetic perturbations, both to gain insight into biological systems and to facilitate medical discoveries.

Chemical-genetic Profiling of Imidazo[1,2-a]pyridines and -pyrimidines Reveals Target Pathways Conserved Between Yeast and Human Cells

PLoS Genetics. Nov, 2008  |  Pubmed ID: 19043571

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical-genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure-activity relationships.

Identification of Genes Involved in the Toxic Response of Saccharomyces Cerevisiae Against Iron and Copper Overload by Parallel Analysis of Deletion Mutants

Toxicological Sciences : an Official Journal of the Society of Toxicology. Jan, 2008  |  Pubmed ID: 17785683

Iron and copper are essential nutrients for life as they are required for the function of many proteins but can be toxic if present in excess. Accumulation of these metals in the human body as a consequence of overload disorders and/or high environmental exposures has detrimental effects on health. The budding yeast Saccharomyces cerevisiae is an accepted cellular model for iron and copper metabolism in humans primarily because of the high degree of conservation between pathways and proteins involved. Here we report a systematic screen using yeast deletion mutants to identify genes involved in the toxic response to growth-inhibitory concentrations of iron and copper sulfate. We aimed to understand the cellular responses to toxic concentrations of these two metals by analyzing the different subnetworks and biological processes significantly enriched with these genes. Our results indicate the presence of two different detoxification pathways for iron and copper that converge toward the vacuole. The product of several of the identified genes in these pathways form molecular complexes that are conserved in mammals and include the retromer, endosomal sorting complex required for transport (ESCRT) and AP-3 complexes, suggesting that the mechanisms involved can be extrapolated to humans. Our data also suggest a disruption in ion homeostasis and, in particular, of iron after copper exposure. Moreover, the identification of treatment-specific genes associated with biological processes such as DNA double-strand break repair for iron and tryptophan biosynthesis for copper suggests differences in the mechanisms by which these two metals are toxic at high concentrations.

Combining Chemical Genomics Screens in Yeast to Reveal Spectrum of Effects of Chemical Inhibition of Sphingolipid Biosynthesis

BMC Microbiology. 2009  |  Pubmed ID: 19144191

Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug.

Knocking Sense into Regulatory Pathways

Nature Biotechnology. Feb, 2009  |  Pubmed ID: 19204694

Computationally Driven, Quantitative Experiments Discover Genes Required for Mitochondrial Biogenesis

PLoS Genetics. Mar, 2009  |  Pubmed ID: 19300474

Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods.

Novel Insights into Iron Metabolism by Integrating Deletome and Transcriptome Analysis in an Iron Deficiency Model of the Yeast Saccharomyces Cerevisiae

BMC Genomics. 2009  |  Pubmed ID: 19321002

Iron-deficiency anemia is the most prevalent form of anemia world-wide. The yeast Saccharomyces cerevisiae has been used as a model of cellular iron deficiency, in part because many of its cellular pathways are conserved. To better understand how cells respond to changes in iron availability, we profiled the yeast genome with a parallel analysis of homozygous deletion mutants to identify essential components and cellular processes required for optimal growth under iron-limited conditions. To complement this analysis, we compared those genes identified as important for fitness to those that were differentially-expressed in the same conditions. The resulting analysis provides a global perspective on the cellular processes involved in iron metabolism.

Precise Gene-dose Alleles for Chemical Genetics

Genetics. Jun, 2009  |  Pubmed ID: 19332878

Modulating gene dose is an effective way to alter protein levels and modify phenotypes to understand gene function. In addition, combining gene-dose alleles with chemical perturbation can provide insight into drug-gene interactions. Here, we present a strategy that combines diverse loss-of-function alleles to systematically modulate gene dose in Saccharomyces cerevisiae. The generated gene dosage allele set expands the genetic toolkit for uncovering novel phenotypes.

A Molecular Barcoded Yeast ORF Library Enables Mode-of-action Analysis of Bioactive Compounds

Nature Biotechnology. Apr, 2009  |  Pubmed ID: 19349972

We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.

Chemogenomic Approaches to Elucidation of Gene Function and Genetic Pathways

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19521822

The approximately 6,000 strains in the yeast deletion collection can be studied in a single culture by using a microarray to detect the 20 bp DNA "barcodes" or "tags" contained in each strain. Barcode intensities measured by microarray are compared across time-points or across conditions to analyze the relative fitness of each strain. The development of this pooled fitness assay has greatly facilitated the functional annotation of the yeast genome by making genome-wide gene-deletion studies faster and easier, and has led to the development of high throughput methods for studying drug action in yeast. Pooled screens can be used for identifying gene functions, measuring the functional relatedness of gene pairs to group genes into pathways, identifying drug targets, and determining a drug's mechanism of action. This process involves five main steps: preparing aliquots of pooled cells, pooled growth, isolation of genomic DNA and PCR amplification of the barcodes, array hybridization, and data analysis. In addition to yeast fitness applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, siRNA vectors, and molecular inversion probes.

Quantitative Phenotyping Via Deep Barcode Sequencing

Genome Research. Oct, 2009  |  Pubmed ID: 19622793

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

A Comparative Analysis of DNA Barcode Microarray Feature Size

BMC Genomics. 2009  |  Pubmed ID: 19825181

Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity.

The Genetic Landscape of a Cell

Science (New York, N.Y.). Jan, 2010  |  Pubmed ID: 20093466

A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.

Systematic Analysis of Genome-wide Fitness Data in Yeast Reveals Novel Gene Function and Drug Action

Genome Biology. 2010  |  Pubmed ID: 20226027

We systematically analyzed the relationships between gene fitness profiles (co-fitness) and drug inhibition profiles (co-inhibition) from several hundred chemogenomic screens in yeast. Co-fitness predicted gene functions distinct from those derived from other assays and identified conditionally dependent protein complexes. Co-inhibitory compounds were weakly correlated by structure and therapeutic class. We developed an algorithm predicting protein targets of chemical compounds and verified its accuracy with experimental testing. Fitness data provide a novel, systems-level perspective on the cell.

Genotype to Phenotype: a Complex Problem

Science (New York, N.Y.). Apr, 2010  |  Pubmed ID: 20413493

We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Sigma1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Sigma1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Sigma1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals.

Highly-multiplexed Barcode Sequencing: an Efficient Method for Parallel Analysis of Pooled Samples

Nucleic Acids Research. Jul, 2010  |  Pubmed ID: 20460461

Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with no increase in data quality. A solution to making next-generation sequencing as efficient and affordable as possible involves assaying multiple samples in a single run. Here, we report the successful 96-plexing of complex pools of DNA barcoded yeast mutants and show that such 'Bar-seq' assessment of these samples is comparable with data provided by barcode microarrays, the current benchmark for this application. The cost reduction and increased throughput permitted by highly multiplexed sequencing will greatly expand the scope of chemogenomics assays and, equally importantly, the approach is suitable for other sequence counting applications that could benefit from massive parallelization.

A Universal TagModule Collection for Parallel Genetic Analysis of Microorganisms

Nucleic Acids Research. Aug, 2010  |  Pubmed ID: 20494978

Systems-level analyses of non-model microorganisms are limited by the existence of numerous uncharacterized genes and a corresponding over-reliance on automated computational annotations. One solution to this challenge is to disrupt gene function using DNA tag technology, which has been highly successful in parallelizing reverse genetics in Saccharomyces cerevisiae and has led to discoveries in gene function, genetic interactions and drug mechanism of action. To extend the yeast DNA tag methodology to a wide variety of microorganisms and applications, we have created a universal, sequence-verified TagModule collection. A hallmark of the 4280 TagModules is that they are cloned into a Gateway entry vector, thus facilitating rapid transfer to any compatible genetic system. Here, we describe the application of the TagModules to rapidly generate tagged mutants by transposon mutagenesis in the metal-reducing bacterium Shewanella oneidensis MR-1 and the pathogenic yeast Candida albicans. Our results demonstrate the optimal hybridization properties of the TagModule collection, the flexibility in applying the strategy to diverse microorganisms and the biological insights that can be gained from fitness profiling tagged mutant collections. The publicly available TagModule collection is a platform-independent resource for the functional genomics of a wide range of microbial systems in the post-genome era.

A Predictive Model for Drug Bioaccumulation and Bioactivity in Caenorhabditis Elegans

Nature Chemical Biology. Jul, 2010  |  Pubmed ID: 20512140

The resistance of Caenorhabditis elegans to pharmacological perturbation limits its use as a screening tool for novel small bioactive molecules. One strategy to improve the hit rate of small-molecule screens is to preselect molecules that have an increased likelihood of reaching their target in the worm. To learn which structures evade the worm's defenses, we performed the first survey of the accumulation and metabolism of over 1,000 commercially available drug-like small molecules in the worm. We discovered that fewer than 10% of these molecules accumulate to concentrations greater than 50% of that present in the worm's environment. Using our dataset, we developed a structure-based accumulation model that identifies compounds with an increased likelihood of bioavailability and bioactivity, and we describe structural features that facilitate small-molecule accumulation in the worm. Preselecting molecules that are more likely to reach a target by first applying our model to the tens of millions of commercially available compounds will undoubtedly increase the success of future small-molecule screens with C. elegans.

A Survey of Yeast Genomic Assays for Drug and Target Discovery

Pharmacology & Therapeutics. Aug, 2010  |  Pubmed ID: 20546776

Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach.

Genome-wide Screen in Saccharomyces Cerevisiae Identifies Vacuolar Protein Sorting, Autophagy, Biosynthetic, and TRNA Methylation Genes Involved in Life Span Regulation

PLoS Genetics. Jul, 2010  |  Pubmed ID: 20657825

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Exploring Gene Function and Drug Action Using Chemogenomic Dosage Assays

Methods in Enzymology. 2010  |  Pubmed ID: 20946813

In this chapter, we describe a series of genome-wide, cell-based assays that provide a solid basis for understanding drug-gene interactions, gene function, and for defining the mechanism of action of small molecules. Each of these assays takes advantage of the ability to grow complex pools competitively and to use high-density microarrays that report the results of such screens. The assays described here take advantage of alterations in gene dosage of Saccharomyces cerevisiae, and include HIP (haploinsufficiency profiling), HOP (homozygous profiling), and MSP (multicopy suppression profiling) as genetic tools to understand gene function and drug mechanism. The common experimental theme is that, in each assay, strains are pooled and screened in parallel to investigate the relative contribution of each gene product to sensitivity or resistance to a drug or environmental perturbation across the genome in a single assay. Further, the compendium of results from these screens can inform large-scale network analysis of genetic function, gene-gene interactions, and mechanism of drug action.

Gene Annotation and Drug Target Discovery in Candida Albicans with a Tagged Transposon Mutant Collection

PLoS Pathogens. 2010  |  Pubmed ID: 20949076

Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.

Knocking out Multigene Redundancies Via Cycles of Sexual Assortment and Fluorescence Selection

Nature Methods. Feb, 2011  |  Pubmed ID: 21217751

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.

Systematic Exploration of Essential Yeast Gene Function with Temperature-sensitive Mutants

Nature Biotechnology. Apr, 2011  |  Pubmed ID: 21441928

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.

The Synthetic Genetic Interaction Network Reveals Small Molecules That Target Specific Pathways in Sacchromyces Cerevisiae

Molecular BioSystems. Jun, 2011  |  Pubmed ID: 21487606

High-throughput elucidation of synthetic genetic interactions (SGIs) has contributed to a systems-level understanding of genetic robustness and fault-tolerance encoded in the genome. Pathway targets of various compounds have been predicted by comparing chemical-genetic synthetic interactions to a network of SGIs. We demonstrate that the SGI network can also be used in a powerful reverse pathway-to-drug approach for identifying compounds that target specific pathways of interest. Using the SGI network, the method identifies an indicator gene that may serve as a good candidate for screening a library of compounds. The indicator gene is selected so that compounds found to produce sensitivity in mutants deleted for the indicator gene are likely to abrogate the target pathway. We tested the utility of the SGI network for pathway-to-drug discovery using the DNA damage checkpoint as the target pathway. An analysis of the compendium of synthetic lethal interactions in yeast showed that superoxide dismutase 1 (SOD1) has significant SGI connectivity with a large subset of DNA damage checkpoint and repair (DDCR) genes in Saccharomyces cerevisiae, and minimal SGIs with non-DDCR genes. We screened a sod1Δ strain against three National Cancer Institute (NCI) compound libraries using a soft agar high-throughput halo assay. Fifteen compounds out of ∼3100 screened showed selective toxicity toward sod1Δ relative to the isogenic wild type (wt) strain. One of these, 1A08, caused a transient increase in growth in the presence of sublethal doses of DNA damaging agents, suggesting that 1A08 inhibits DDCR signaling in yeast. Genome-wide screening of 1A08 against the library of viable homozygous deletion mutants further supported DDCR as the relevant targeted pathway of 1A08. When assayed in human HCT-116 colorectal cancer cells, 1A08 caused DNA-damage resistant DNA synthesis and blocked the DNA-damage checkpoint selectively in S-phase.

A Comprehensive Platform for Highly Multiplexed Mammalian Functional Genetic Screens

BMC Genomics. 2011  |  Pubmed ID: 21548937

Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.

Dosage Suppression Genetic Interaction Networks Enhance Functional Wiring Diagrams of the Cell

Nature Biotechnology. Jun, 2011  |  Pubmed ID: 21572441

Dosage suppression is a genetic interaction in which overproduction of one gene rescues a mutant phenotype of another gene. Although dosage suppression is known to map functional connections among genes, the extent to which it might illuminate global cellular functions is unclear. Here we analyze a network of interactions linking dosage suppressors to 437 essential genes in yeast. For 424 genes, we curated interactions from the literature. Analyses revealed that many dosage suppression interactions occur between functionally related genes and that the majority do not overlap with other types of genetic or physical interactions. To confirm the generality of these network properties, we experimentally identified dosage suppressors for 29 genes from pooled populations of temperature-sensitive mutant cells transformed with a high-copy molecular-barcoded open reading frame library, MoBY-ORF 2.0. We classified 87% of the 1,640 total interactions into four general types of suppression mechanisms, which provided insight into their relative frequencies. This work suggests that integrating the results of dosage suppression studies with other interaction networks could generate insights into the functional wiring diagram of a cell.

Design, Synthesis, and Characterization of a Highly Effective Hog1 Inhibitor: a Powerful Tool for Analyzing MAP Kinase Signaling in Yeast

PloS One. 2011  |  Pubmed ID: 21655328

The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

Design, Synthesis and Characterization of a Highly Effective Inhibitor for Analog-sensitive (as) Kinases

PloS One. 2011  |  Pubmed ID: 21698101

Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1(T100G)). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

New Azole Antifungal Agents with Novel Modes of Action: Synthesis and Biological Studies of New Tridentate Ligands Based on Pyrazole and Triazole

European Journal of Medicinal Chemistry. Sep, 2011  |  Pubmed ID: 21723647

The synthesis and extensive biological study of two new tridentates ligands based on pyrazole and triazole are described. The antifungal activity against the budding yeast cells of the newly synthesized compounds was determined. These compounds were toxic to yeast cells. Cell cycle analysis suggested that treatment with these compounds impairs cell division in G1 of the cell cycle. Using yeast-based functional genomics technologies, we found that these compounds tolerance requires DNA repair pathway and SKI complex function. We have also found that the PKC1 heterozygous deletion strain was the most sensitive to these compounds using HaploInsufficiency Profiling, suggesting that the Pkc1 protein may be the target for these compounds. These results strongly suggest that these compounds induce DNA damage and thus exert a different mechanism of action compared to other azole derivatives. These two compounds might therefore represent promising lead compounds for further development of antifungal drugs for human therapy.

The Automated Cell: Compound and Environment Screening System (ACCESS) for Chemogenomic Screening

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21863492

The automated cell, compound and environment screening system (ACCESS) was developed as an automated platform for chemogenomic research. In the yeast Saccharomyces cerevisiae, a number of genomic screens rely on the modulation of gene dose to determine the mode of action of bioactive compounds or the effects of environmental/compound perturbations. These and other phenotypic experiments have been shown to benefit from high-resolution growth curves and a highly automated controlled environment system that enables a wide range of multi-well assays that can be run over many days without any manual intervention. Furthermore, precise control of drug dosing, timing of drug exposure, and precise timing of cell harvesting at specific generation times are important for optimal results. Some of these benefits include the ability to derive fine distinctions between growth rates of mutant strains (1) and the discovery of novel compounds and drug targets (2). The automation has also enabled large-scale screening projects with over 100,000 unique compounds screened to date including a thousand genome-wide screens (3). The ACCESS system also has a diverse set of software tools to enable users to set up, run, annotate, and evaluate complex screens with minimal training.

Displaying Chemical Information on a Biological Network Using Cytoscape

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21877291

Cytoscape is an open-source software package that is widely used to integrate and visualize diverse data sets in biology. This chapter explains how to use Cytoscape to integrate open-source chemical information with a biological network. By visualizing information about known compound-target interactions in the context of a biological network of interest, one can rapidly identify novel avenues to perturb the system with compounds and, for example, potentially identify therapeutically relevant targets. Herein, two different protocols are explained in detail, with no prior knowledge of Cytoscape assumed, which demonstrate how to incorporate data from the ChEMBL database with either a gene-gene or a protein-protein interaction network. ChEMBL is a very large, open-source repository of compound-target information available from the European Molecular Biology Laboratory.

Curcumin Inhibits Growth of Saccharomyces Cerevisiae Through Iron Chelation

Eukaryotic Cell. Nov, 2011  |  Pubmed ID: 21908599

Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases. In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumin's mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G(1) phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumin's medicinal properties.

A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

PLoS Genetics. Oct, 2011  |  Pubmed ID: 22028670

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.

Compound Prioritization Methods Increase Rates of Chemical Probe Discovery in Model Organisms

Chemistry & Biology. Oct, 2011  |  Pubmed ID: 22035796

Preselection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ~81,000 compounds in Saccharomyces cerevisiae and identified ~7500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. These data were used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes, we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ~7500 growth-inhibitory molecules have been made commercially available and the computational model and filter used are provided.

Dafadine Inhibits DAF-9 to Promote Dauer Formation and Longevity of Caenorhabditis Elegans

Nature Chemical Biology. Dec, 2011  |  Pubmed ID: 22057127

The DAF-9 cytochrome P450 is a key regulator of dauer formation, developmental timing and longevity in the nematode Caenorhabditis elegans. Here we describe the first identified chemical inhibitor of DAF-9 and the first reported small-molecule tool that robustly induces dauer formation in typical culture conditions. This molecule (called dafadine) also inhibits the mammalian ortholog of DAF-9(CYP27A1), suggesting that dafadine can be used to interrogate developmental control and longevity in other animals.

Systematic Exploration of Synergistic Drug Pairs

Molecular Systems Biology. 2011  |  Pubmed ID: 22068327

Drug synergy allows a therapeutic effect to be achieved with lower doses of component drugs. Drug synergy can result when drugs target the products of genes that act in parallel pathways ('specific synergy'). Such cases of drug synergy should tend to correspond to synergistic genetic interaction between the corresponding target genes. Alternatively, 'promiscuous synergy' can arise when one drug non-specifically increases the effects of many other drugs, for example, by increased bioavailability. To assess the relative abundance of these drug synergy types, we examined 200 pairs of antifungal drugs in S. cerevisiae. We found 38 antifungal synergies, 37 of which were novel. While 14 cases of drug synergy corresponded to genetic interaction, 92% of the synergies we discovered involved only six frequently synergistic drugs. Although promiscuity of four drugs can be explained under the bioavailability model, the promiscuity of Tacrolimus and Pentamidine was completely unexpected. While many drug synergies correspond to genetic interactions, the majority of drug synergies appear to result from non-specific promiscuous synergy.

Inhibition of Mitochondrial Translation As a Therapeutic Strategy for Human Acute Myeloid Leukemia

Cancer Cell. Nov, 2011  |  Pubmed ID: 22094260

To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.

Multiple Means to the Same End: the Genetic Basis of Acquired Stress Resistance in Yeast

PLoS Genetics. Nov, 2011  |  Pubmed ID: 22102822

In nature, stressful environments often occur in combination or close succession, and thus the ability to prepare for impending stress likely provides a significant fitness advantage. Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however, the mechanism of this acquired stress tolerance is poorly understood. To explore this, we exposed the yeast gene-deletion libraries, which interrogate all essential and non-essential genes, to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance. Cells were exposed to one of three different mild stress pretreatments (salt, DTT, or heat shock) and then challenged with a severe dose of hydrogen peroxide (H(2)O(2)). Surprisingly, there was little overlap in the genes required for acquisition of H(2)O(2) tolerance after different mild-stress pretreatments, revealing distinct mechanisms of surviving H(2)O(2) in each case. Integrative network analysis of these results with respect to protein-protein interactions, synthetic-genetic interactions, and functional annotations identified many processes not previously linked to H(2)O(2) tolerance. We tested and present several models that explain the lack of overlap in genes required for H(2)O(2) tolerance after each of the three pretreatments. Together, this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences, underscoring the context-dependent nature of stress tolerance.

Bugs, Drugs and Chemical Genomics

Nature Chemical Biology. Jan, 2012  |  Pubmed ID: 22173359

The serendipitous discovery of penicillin inspired intensive research into how small molecules affect basic cellular processes and their potential to treat disease. Biochemical and genetic approaches have been fundamental for clarifying small-molecule modes of action. Genomic technologies have permitted the use of chemical-genetic strategies that comprehensively study compound-target relationships in the context of a living cell, providing a systems biology view of both the cellular targets and the interdependent networks that respond to chemical stress. These studies highlight the fact that in vitro determinations of mechanism rarely translate into a complete understanding of drug behavior in the cell. Here, we review key discoveries that gave rise to the field of chemical genetics, with particular attention to chemical-genetic strategies developed for bakers' yeast, their extension to clinically relevant microbial pathogens, and the potential of these approaches to affect antimicrobial drug discovery.

Mitochondrial Electron Transport is the Cellular Target of the Oncology Drug Elesclomol

PloS One. 2012  |  Pubmed ID: 22253786

Elesclomol is a first-in-class investigational drug currently undergoing clinical evaluation as a novel cancer therapeutic. The potent antitumor activity of the compound results from the elevation of reactive oxygen species (ROS) and oxidative stress to levels incompatible with cellular survival. However, the molecular target(s) and mechanism by which elesclomol generates ROS and subsequent cell death were previously undefined. The cellular cytotoxicity of elesclomol in the yeast S. cerevisiae appears to occur by a mechanism similar, if not identical, to that in cancer cells. Accordingly, here we used a powerful and validated technology only available in yeast that provides critical insights into the mechanism of action, targets and processes that are disrupted by drug treatment. Using this approach we show that elesclomol does not work through a specific cellular protein target. Instead, it targets a biologically coherent set of processes occurring in the mitochondrion. Specifically, the results indicate that elesclomol, driven by its redox chemistry, interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and consequently cell death. Additional experiments in melanoma cells involving drug treatments or cells lacking ETC function confirm that the drug works similarly in human cancer cells. This deeper understanding of elesclomol's mode of action has important implications for the therapeutic application of the drug, including providing a rationale for biomarker-based stratification of patients likely to respond in the clinical setting.

Dafadine Inhibits DAF-9 to Promote Dauer Formation and Longevity of Caenorhabditis Elegans

Nature Chemical Biology. 2012  |  Pubmed ID: 22337098