RESUMEN
The continuing emergence of antibiotic-resistant microbes highlights the need for the identification of new chemotypes with antimicrobial activity. One of the most prolific sources of antimicrobial molecules has been the systematic screening of natural product samples. The National Institute of Allergy and Infectious Diseases and the National Cancer Institute here report a large screen of 326,656 partially purified natural product fractions against a panel of four microbial pathogens, resulting in the identification of >3000 fractions with antifungal and/or antibacterial activity. A small sample of these active fractions was further purified and the chemical structures responsible for the antimicrobial activity were elucidated. The proof-of-concept study identified many different chemotypes, several of which have not previously been reported to have antimicrobial activity. The results show that there remain many unidentified antibiotic compounds from nature.
Asunto(s)
Antiinfecciosos , Productos Biológicos , Estados Unidos , Productos Biológicos/farmacología , Productos Biológicos/química , National Cancer Institute (U.S.) , Antiinfecciosos/farmacología , Antibacterianos/farmacología , Extractos VegetalesRESUMEN
Reactivation of p53 tumor-suppressor function by small molecules is an attractive strategy to defeat cancer. A potent p53-reactivating molecule RITA, which triggers p53-dependent apoptosis in human tumor cells in vitro and in vivo, exhibits p53-independent cytotoxicity due to modifications by detoxification enzyme Sulfotransferase 1A1 (SULT1A1), producing a reactive carbocation. Several synthetic modifications to RITA's heterocyclic scaffold lead to higher energy barriers for carbocation formation. In this study, we addressed the question whether RITA analogs NSC777196 and NSC782846 can induce p53-dependent apoptosis without SULT1A1-dependent DNA damage. We found that RITA analog NSC782846, but not NSC777196, induced p53-regulated genes, targeted oncogene addiction, and killed cancer cells upon p53 reactivation, but without induction of DNA damage and inhibition RNA pol II. Our results might demonstrate a method for designing more specific and potent RITA analogs to accelerate translation of p53-targeting compounds from laboratory bench to clinic.
Asunto(s)
ARN Polimerasa II , Proteína p53 Supresora de Tumor , Apoptosis , Línea Celular Tumoral , Daño del ADN , Furanos/farmacología , Humanos , Sulfotransferasas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The preclinical antitumor agent RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan, NSC 652287), an analog of the natural product α-terthiophene, failed during the development phase due to acute pulmonary toxicity in animal models. A series of synthetic modifications to RITA's heterocyclic scaffold resulted in activity ranging from broadly cytotoxic to highly selective. In the NCI 60-cell line screen, these "hyperselective" agents (e.g., imatinib) are rare. A selectivity index (SI) was developed to quantify this desirable feature, which is 20 for imatinib, whereas RITA's SI is only 0.10. One of the described hyperselective RITA analogs (SI = 7.9) completely lost activity in the presence of a known SULT1A1 inhibitor. These results, coupled with previous evidence that RITA is a SULT1A1 substrate, suggest that carbinol modification by a sulfate leaving group and subsequent formation of a reactive carbocation may explain RITA's broad cytotoxicity. Although SULT1A1 expression is required for susceptibility, hyperselective analogs exhibited reduced association of activity with SULT1A1 mRNA expression compared with RITA, apparently requiring some additional target(s). In support of this hypothesis, there is a strong correlation (P < 0.01, r = 0.95) between quantum mechanically calculated energy barriers for carbocation formation from sulfonated analogs and SI, indicating that hyperselective RITA analogs generate reactive carbocations less readily after sulfate activation. Importantly, narrowing the cytotoxicity profile of RITA did not eliminate its analogs' in vivo antitumor activity, as several new hyperselective agents, NSC 773097 (1), 773392 (2), and 782846 (6), displayed impressive activity against A498 xenografts in mice.
Asunto(s)
Antineoplásicos/farmacología , Furanos/farmacología , Animales , Antineoplásicos/química , Arilsulfotransferasa/genética , Arilsulfotransferasa/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Furanos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ1 torsion by 150-180° orients the side chain ß-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a > 2000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.
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Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Sitio Alostérico , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Treonina , Secuencias de Aminoácidos , Ligandos , Modelos Moleculares , Unión ProteicaRESUMEN
Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins' proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin's enzymatic components, to antagonize multiple BoNT serotypes in motor neurons.
Asunto(s)
Toxinas Botulínicas/toxicidad , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Transducción de Señal/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo , Células Madre Embrionarias/citología , Humanos , Proteolisis/efectos de los fármacos , SerogrupoRESUMEN
Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Virus de la Fiebre del Valle del Rift/metabolismo , Proteínas Virales/metabolismo , Virión/metabolismo , Silenciador del Gen , Proteínas HSP90 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Proteómica , ARN Interferente Pequeño , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/genética , Espectrometría de Masas en Tándem , Proteínas Virales/genética , Virión/genéticaRESUMEN
The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/inmunología , Linfoma de Células B Grandes Difuso/patología , Pirazoles/farmacología , Pirimidinas/farmacología , Adenina/análogos & derivados , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , PiperidinasRESUMEN
The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.
Asunto(s)
Imagen Molecular , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Yersinia pestis/efectos de los fármacos , Yersinia pestis/fisiología , Animales , Línea Celular , Evaluación Preclínica de Medicamentos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Fagocitosis/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Transporte de Proteínas/efectos de los fármacos , Yersinia pestis/genéticaRESUMEN
An assessment of the total protein composition of filovirus (ebolavirus and marburgvirus) virions is currently lacking. In this study, liquid chromatography-linked tandem mass spectrometry of purified ebola and marburg virions was performed to identify associated cellular proteins. Host proteins involved in cell adhesion, cytoskeleton, cell signaling, intracellular trafficking, membrane organization, and chaperones were identified. Significant overlap exists between this data set and proteomic studies of disparate viruses, including HIV-1 and influenza A, generated in multiple cell types. However, the great majority of proteins identified here have not been previously described to be incorporated within filovirus particles. Host proteins identified by liquid chromatography-linked tandem mass spectrometry could lack biological relevance because they represent protein contaminants in the virus preparation, or because they are incorporated within virions by chance. These issues were addressed using siRNA library-mediated gene knockdown (targeting each identified virion-associated host protein), followed by filovirus infection. Knockdown of several host proteins (e.g. HSPA5 and RPL18) significantly interfered with ebolavirus and marburgvirus infection, suggesting specific and relevant virion incorporation. Notably, select siRNAs inhibited ebolavirus, but enhanced marburgvirus infection, suggesting important differences between the two viruses. The proteomic analysis presented here contributes to a greater understanding of filovirus biology and potentially identifies host factors that can be targeted for antiviral drug development.
Asunto(s)
Filoviridae/metabolismo , Proteómica , Interferencia de ARN , Proteínas Virales/metabolismo , Virión/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Técnicas de Silenciamiento del Gen , Microscopía Fluorescente , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Espectrometría de Masas en Tándem , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Ebola virus (EBOV) infection of humans is a lethal but accidental dead-end event. Understanding resistance to EBOV in other species may help establish the basis of susceptibility differences among its hosts. Although rodents are resistant to EBOV, a murine-adapted variant is lethal when injected intraperitoneally into mice. We find that mice expressing reduced levels of the tyrosine phosphatase CD45 are protected against EBOV, whereas wild-type, CD45-deficient, or enzymatically inactive CD45-expressing mice succumbed to infection. Protection was dependent on CD8(+) T cells and interferon gamma. Reduced CD45-expressing mice retained greater control of gene expression and immune cell proliferation following EBOV infection, which contributed to reduced apoptosis, enhanced viral clearance, and increased protection against the virus. Together, these findings suggest that host susceptibility to EBOV is dependent on the delicate balance of immune homeostasis, which, as demonstrated here, can be determined by the levels of a single regulator.
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Ebolavirus/inmunología , Ebolavirus/patogenicidad , Interacciones Huésped-Patógeno , Antígenos Comunes de Leucocito/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Humanos , Interferón gamma/inmunología , Ganglios Linfáticos/virología , Macrófagos Peritoneales/virología , Ratones , Modelos Biológicos , Bazo/virología , Análisis de SupervivenciaRESUMEN
Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared priming sequences. These features have enabled researchers to handle yeast mutant collections as complex pools of approximately 6000 strains. The presence of any individual mutant within a pool can be assessed indirectly by measuring the relative abundance of its corresponding TAG(s) in genomic DNA prepared from the pool. This is readily accomplished by wholesale polymerase chain reaction (PCR) amplification of the TAGs using fluorescent oligonucleotide primers that recognize the common flanking sequences, followed by hybridization of the labeled PCR products to a TAG oligonucleotide microarray. Here we describe a method-diploid-based synthetic lethality analysis by microarray (dSLAM)-whereby such pools can be manipulated to rapidly construct and assess the fitness of 6000 double-mutant strains in a single experiment. Analysis of double-mutant strains is of growing importance in defining the spectrum of essential cellular functionalities and in understanding how these functionalities interrelate.
Asunto(s)
Regulación Fúngica de la Expresión Génica/fisiología , Genes Letales , Mutación/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Saccharomyces cerevisiae/genética , Eliminación de Gen , Perfilación de la Expresión Génica/métodos , Regulación Fúngica de la Expresión Génica/genética , Genoma FúngicoRESUMEN
TAG, or bar-code, microarrays allow measurement of the oligonucleotide sequences (TAGs) that mark each strain of deletion mutants in the Saccharomyces cerevisiae yeast knockout (YKO) collection. Comparison of genomic DNA from pooled YKO samples allows estimation of relative abundance of TAGs marking each deletion strain. Features of TAG hybridizations create unique challenges for analysis. Analysis is complicated by the presence of two TAGs in most YKO strains and the hybridization behavior of TAGs that may differ in sequence from array probes. The oligonucleotide size of labeled TAGs also results in difficulty with contaminating sequences that cause reduced specificity. We present methods for analysis that approach these unique features of TAG hybridizations.
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Genómica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Saccharomyces cerevisiae/genética , Lugares Marcados de Secuencia , Programas Informáticos , Sondas de ADN , Interpretación Estadística de Datos , Genes Letales , Genoma Fúngico , Internet , Mutación/genética , Eliminación de SecuenciaRESUMEN
The Saccharomyces genome-deletion project created >5900 'molecularly barcoded' yeast knockout mutants (YKO mutants). The YKO mutant collections have facilitated large-scale analyses of a multitude of mutant phenotypes. For example, both synthetic genetic array (SGA) and synthetic-lethality analysis by microarray (SLAM) methods have been used for synthetic-lethality screens. Global analysis of synthetic lethality promises to identify cellular pathways that 'buffer' each other biologically. The combination of global synthetic-lethality analysis, together with global protein-protein interaction analyses, mRNA expression profiling and functional profiling will, in principle, enable construction of a cellular 'wiring diagram' that will help frame a deeper understanding of human biology and disease.
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Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Fúngicos , Genes Letales , Técnicas Genéticas , Genoma Fúngico , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN de Hongos/genética , ARN Mensajero/genéticaRESUMEN
BACKGROUND: In a genetic interaction, the phenotype of a double mutant differs from the combined phenotypes of the underlying single mutants. When the single mutants have no growth defect, but the double mutant is lethal or exhibits slow growth, the interaction is termed synthetic lethality or synthetic fitness. These genetic interactions reveal gene redundancy and compensating pathways. Recently available large-scale data sets of genetic interactions and protein interactions in Saccharomyces cerevisiae provide a unique opportunity to elucidate the topological structure of biological pathways and how genes function in these pathways. RESULTS: We have defined congruent genes as pairs of genes with similar sets of genetic interaction partners and constructed a genetic congruence network by linking congruent genes. By comparing path lengths in three types of networks (genetic interaction, genetic congruence, and protein interaction), we discovered that high genetic congruence not only exhibits correlation with direct protein interaction linkage but also exhibits commensurate distance with the protein interaction network. However, consistent distances were not observed between genetic and protein interaction networks. We also demonstrated that congruence and protein networks are enriched with motifs that indicate network transitivity, while the genetic network has both transitive (triangle) and intransitive (square) types of motifs. These results suggest that robustness of yeast cells to gene deletions is due in part to two complementary pathways (square motif) or three complementary pathways, any two of which are required for viability (triangle motif). CONCLUSION: Genetic congruence is superior to genetic interaction in prediction of protein interactions and function associations. Genetically interacting pairs usually belong to parallel compensatory pathways, which can generate transitive motifs (any two of three pathways needed) or intransitive motifs (either of two pathways needed).
Asunto(s)
Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genéticaRESUMEN
Saccharomyces cerevisiae knockout collection TAG microarrays are an emergent platform for rapid, genome-wide functional characterization of yeast genes. TAG arrays report abundance of unique oligonucleotide 'TAG' sequences incorporated into each deletion mutation of the yeast knockout collection, allowing measurement of relative strain representation across experimental conditions for all knockout mutants simultaneously. One application of TAG arrays is to perform genome-wide synthetic lethality screens, known as synthetic lethality analyzed by microarray (SLAM). We designed a fully defined spike-in pool to resemble typical SLAM experiments and performed TAG microarray hybridizations. We describe a method for analyzing two-color array data to efficiently measure the differential knockout strain representation across two experimental conditions, and use the spike-in pool to show that the sensitivity and specificity of this method exceed typical current approaches.
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Genómica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Saccharomyces cerevisiae/genética , Lugares Marcados de Secuencia , Interpretación Estadística de Datos , Colorantes Fluorescentes , Genes Letales , Genoma Fúngico , Eliminación de SecuenciaRESUMEN
A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3-6% and 15-18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens.
Asunto(s)
Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Saccharomyces cerevisiae/genética , Colorantes , Indicadores y Reactivos , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Reacción en Cadena de la PolimerasaRESUMEN
We predicted gene function using synthetic lethal genetic interactions between null alleles in Saccharomyces cerevisiae. Phenotypic and protein interaction data indicate that synthetic lethal gene pairs function in parallel or compensating pathways. Congruent gene pairs, defined as sharing synthetic lethal partners, are in single pathway branches. We predicted benomyl sensitivity and nuclear migration defects using congruence; these phenotypes were uncorrelated with direct synthetic lethality. We also predicted YLL049W as a new member of the dynein-dynactin pathway and provided new supporting experimental evidence. We performed synthetic lethal screens of the parallel mitotic exit network (MEN) and Cdc14 early anaphase release pathways required for late cell cycle. Synthetic lethal interactions bridged genes in these pathways, and high congruence linked genes within each pathway. Synthetic lethal interactions between MEN and all components of the Sin3/Rpd3 histone deacetylase revealed a novel function for Sin3/Rpd3 in promoting mitotic exit in parallel to MEN. These in silico methods can predict phenotypes and gene functions and are applicable to genomic synthetic lethality screens in yeast and analogous RNA interference screens in metazoans.