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Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action.

ABSTRACT: We present a cross-species chemogenomic screening platform using libraries of haploid deletion mutants from two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We screened a set of compounds of known and unknown mode of action (MoA) and derived quantitative drug scores (or D-scores), identifying mutants that are either sensitive or resistant to particular compounds. We found that compound-functional module relationships are more conserved than individual compound-gene interactions between these two species. Furthermore, we observed that combining data from both species allows for more accurate prediction of MoA. Finally, using this platform, we identified a novel small molecule that acts as a DNA damaging agent and demonstrate that its MoA is conserved in human cells.

SUBMITTER: Kapitzky L

PROVIDER: S-EPMC3018166 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action.

Kapitzky Laura L Beltrao Pedro P Berens Theresa J TJ Gassner Nadine N Zhou Chunshui C Wüster Arthur A Wu Julie J Babu M Madan MM Elledge Stephen J SJ Toczyski David D Lokey R Scott RS Krogan Nevan J NJ

Molecular systems biology 20101201

We present a cross-species chemogenomic screening platform using libraries of haploid deletion mutants from two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We screened a set of compounds of known and unknown mode of action (MoA) and derived quantitative drug scores (or D-scores), identifying mutants that are either sensitive or resistant to particular compounds. We found that compound-functional module relationships are more conserved than individual compound-gene inte ...[more]

PMID: 21179023

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Project description:Every year, more than 250,000 invasive candidiasis infections are reported with 50,000 deaths worldwide. The limited number of antifungal agents necessitates the need for alternative antifungals with potential novel targets. The 2-benzylidenebenzofuran-3-(2H)-ones have become an attractive scaffold for antifungal drug design. This study aimed to determine the antifungal activity of a synthetic aurone compound and characterize its mode of action. Using the broth microdilution method, aurone SH1009 exhibited inhibition against C. albicans, including resistant isolates, as well as C. glabrata, and C. tropicalis with IC50 values of 4-29 μM. Cytotoxicity assays using human THP-1, HepG2, and A549 human cell lines showed selective toxicity toward fungal cells. The mode of action for SH1009 was characterized using chemical-genetic interaction via haploinsufficiency (HIP) and homozygous (HOP) profiling of a uniquely barcoded Saccharomyces cerevisiae mutant collection. Approximately 5300 mutants were competitively treated with SH1009 followed by DNA extraction, amplification of unique barcodes, and quantification of each mutant using multiplexed next-generation sequencing. Barcode post-sequencing analysis revealed 238 sensitive and resistant mutants that significantly (FDR P values ≤ 0.05) responded to aurone SH1009. The enrichment analysis of KEGG pathways and gene ontology demonstrated the cell cycle pathway as the most significantly enriched pathway along with DNA replication, cell division, actin cytoskeleton organization, and endocytosis. Phenotypic studies of these significantly enriched responses were validated in C. albicans. Flow cytometric analysis of SH1009-treated C. albicans revealed a significant accumulation of cells in G1 phase, indicating cell cycle arrest. Fluorescence microscopy detected abnormally interrupted actin dynamics, resulting in enlarged, unbudded cells. RT-qPCR confirmed the effects of SH1009 in differentially expressed cell cycle, actin polymerization, and signal transduction genes. These findings indicate the target of SH1009 as a cell cycle-dependent organization of the actin cytoskeleton, suggesting a novel mode of action of the aurone compound as an antifungal inhibitor.

Project description:BACKGROUND: Comparative genomic analysis using cDNA microarray is a new approach and a useful tool to identify important genetic sequences or genes that are conserved throughout evolution. Identification of these conserved sequences will help elucidate important molecular mechanisms or pathways common to many species. For example, the stockpiled transcripts in the oocyte necessary for successful fertilization and early embryonic development still remain relatively unknown. The objective of this study was to identify genes expressed in oocytes and conserved in three evolutionarily distant species. RESULTS: In this study we report the construction of a multi-species cDNA microarray containing 3,456 transcripts from three distinct oocyte-libraries from bovine, mouse and Xenopus laevis. Following the cross-species hybridizations, data analysis revealed that 1,541 positive hybridization signals were generated by oocytes of all three species, and 268 of these are preferentially expressed in the oocyte. Data reproducibility analyses comparing same-species to cross-species hybridization indicates that cross-species hybridizations are highly reproducible, thus increasing the confidence level in their specificity. A validation by RT-PCR using gene- and species-specific primers confirmed that cross-species hybridization allows the production of specific and reliable data. Finally, a second validation step through gene-specific microarray hybridizations further supported the validity of our cross-species microarray results. Results from these cross-species hybridizations on our multi-species cDNA microarray revealed that SMFN (Small fragment nuclease), Spin (Spindlin), and PRMT1 (Protein arginine methyltransferase 1) are transcripts present in oocytes and conserved in three evolutionarily distant species. CONCLUSION: Cross-species hybridization using a multi-species cDNA microarray is a powerful tool for the discovery of genes involved in evolutionarily conserved molecular mechanisms. The present study identified conserved genes in the oocytes of three distant species that will help understand the unique role of maternal transcripts in early embryonic development.

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Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action.

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Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action.

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