Src inhibitors are promising therapy molecules for human cervical carcinomas

by Selleckchem | Jan 22 2013

Candida albicans is responsible for approximately 50% of all human life-threatening nosocomial fungal infections . Completion of its diploid genome sequence now provides a foundation for studies on C. albicans biology and SRC Inhibitors pathogenesis, and offers new opportunities for therapeutic intervention. Critical to such activities, however, remains the task of functionally annotating the C. albicans genome. To date, genomic studies reveal significant differences in genomic organization and gene essentiality between C. albicans and Saccharomyces cerevisiae. In part, these differences reflect their evolutionary divergence and distinct lifestyle as an opportunistic fungal pathogen versus a saprophytic yeast, respectively. Unlike S. cerevisiae, a major impediment to large-scale genetic analyses in C. albicans is the limited ability to perform classical genetic screens, due to its natural diploid state and lack of an easily manipulated sexual cycle. Thus, alternative genetic strategies are required. The STA-9090 phenomenon of haploinsufficiency ??that is, growth phenotypes associated with the loss of function of one allele in a diploid??is widespread amongst eukaryotes and has been effectively applied in C.albicans to screen for genes involved in filamentous growth . HI has been studied extensively in S. cerevisiae and offers a way to exploit the diploidy of C. albicans. While only ;3% of the S. cerevisiae genome displays HI under the standard growth conditions , chemically induced HI is more specific. It has been shown in an assay termed the fitness test that target-specific inhibitory molecules typically induce a growth disadvantage of heterozygous deletion strains corresponding to the drug targets and/or other mechanism supply peptide of action ?Crelated genes . If this specificity is also prevalent in C. albicans, chemically induced HI by mechanistically characterized inhibitors could be used to identify essential cellular processes and genetic interactions relevant to this pathogen. Conversely, when a novel inhibitory compound is tested, the response of specific heterozygous strains may provide phenotypic information reflecting the MOA of the compound. Here, we report on the application of chemically induced HI on a genomic scale in C. albicans. Drawing from analogous studies performed in S. cerevisiae, we term this assay the C. albicans fitness test, or CaFT .

We systematically constructed 2,868 heterozygous deletion strains in which two unique barcodes were TAK-700 introduced in the up- and downstream regions of the deleted allele. Gene-specific barcodes differentiate heterozygotes from one another and enable their multiplex screening for HI growth phenotypes when challenged with antifungal agents. To readout growth changes resulting from drug treatment, each unique tag is amplified by PCR using common flanking primer sequences, labeled, and hybridized to a DNA microarray that identifies all barcodes . Statistical analyses enable identification of strains significantly affected in growth rate. With a CaFT of ;45% genome coverage, we have examined mechanistically diverse inhibitory compounds with three objectives: 1) to determine and validate the TAK-875 prevalence and specificity of chemically induced HI in the pathogen, 2) to provide experimental data to functionally annotate genes and their genetic interactions involved in the cellular processes inhibited by these compounds, and 3) to establish the utility of the CaFT in MOA studies of inhibitory compounds for antifungal drug discovery.