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. 2011 Jun;7(6):2019-30.
doi: 10.1039/c0mb00298d. Epub 2011 Apr 12.

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

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Free PMC article

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

Craig M Tamble et al. Mol Biosyst. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1. Identification of “pathway hubs” p-hubs based on the synthetic genetic network
Two genes, G1 and G2, are considered as candidate p-hubs to the target pathway P1. Genes (circles) are scored according to how specific their synthetic genetic interactions (solid lines) are to genes from a target pathway, P1 (see Methods). G1 has a higher p-hub score (10) than G2 (1.9) since G1 has interactions more specifically associated with P1. The hypothesis of this work is that a drug that is selectively lethal to a deletion strain G1Δ (relative to wt) is more likely to target P1 than a drug that is selective for G2Δ, since such a drug may target other pathways such as pathway P2.
Figure 2
Figure 2. Pathway Genetic Interaction (PGI) correlates with differential sensitivity (DSS)
For each of 15 positive control pathways, genes were grouped into three categories based on their PGI scores to the pathway – disconnected (D; PGI = 0), moderately connected (M; 0 ≥ PGI ≤ 3), or highly connected (H; PGI > 3). Both positive (top twelve) and negative (bottom three) pathways were included in the analysis. Boxplots represent the distribution of DSS for genes in each group: disconnected (top boxplot marked D), moderately connected (middle boxplot marked M), and highly connected (bottom boxplot marked H). Boxplots represent the distribution of differential sensitivity, where each point represents how sensitive a gene’s mutant is to compounds known to target the pathway compared to those that do not; line is the median level; edges represent the upper- and lower-quartiles; points show extreme values. Vertical column annotates whether genes with medium to high PGI had higher DSS on average than genes with lower PGI. Large upper quartile for genes highly connected to membrane biogenesis not shown in the plot.
Figure 3
Figure 3. Global overview of indicator genes for several pathways
Purple gradient shows increasing PGI score representing the significance of synthetic-lethal overlap of a gene (row) with a target pathway (column). Genes (rows) were clustered according to their PGI scores, and pathways (columns) were clustered according to PGI scores across all genes (left matrix) or binary membership of those genes (right matrix). All genes predicted as an indicator for at least one pathway are included. Inset shows detail of cluster F, a set of genes with specific synthetic lethal interactions to genes of the DNA damage and DNA replication pathways. SOD1 has the highest PGI score for the DNA damage-related pathways and is not a member of one of these pathways. All of the pathways associated with each pathway cluster i through xv are available as Table S3; all of the genes in clusters A through S are available as Table S4.
Figure 4
Figure 4. Identification of screening candidates for the damage pathway
A. Genome-wide scores for screening candidates. Each gene was scored according to how significant its synthetic genetic interaction (SGI) partners overlapped with genes in the damage pathway. Plotted is the number of damage pathway genes that also are linked to the gene against the significance of this overlap computed as −log10(P), where P is the P-value given by the hypergeometric distribution (see Methods). B. SOD1’s synthetic genetic defect neighborhood. The SGI neighborhood of the gene, SOD1 (circles linked to SOD1), was found to have the most significant overlap with genes in the damage pathway (orange circles). Lines depict SGI links; blue circles represent genes that are synthetically defective with SOD1 but are not known to belong to the damage pathway.
Figure 5
Figure 5. Structures of compounds that selectively kill sod1Δ yeast
NSC numbers are given for the 15 compounds.
Figure 6
Figure 6. 1A08 Disrupts Normal DNA Damage Responses in Wild Type Yeast
A. Spot assays with 1A08. 0.5 μl of a 40 mM solution of 1A08 were spotted onto filter discs on top of soft agar containing yeast and the following: i) Untreated plate; ii) 0.01% MMS; iii) 0.02% MMS; iv) 250 μM cisplatin; v) 1 mM cisplatin. B. Liquid culture validation of spot assays. The effect on growth in liquid culture was measured for 1A08 at 10 μM and 25 μM in the presence of 0.02% MMS.
Figure 7
Figure 7
The sensitivities of 4759 homozygous deletion mutants to 17μM 1A08 is shown. The experiments were performed as reported[15]. Sensitivities, relative to the DMSO control, are plotted on the y-axis for each homozygous deletion strain (arranged alphabetically according to gene name on the x-axis).
Figure 8
Figure 8. Growth of wild type and sod1Δ yeast in the presence of 1A08
A. Wild-type. B. sod1Δ.
Figure 9
Figure 9. SGI neighbors of 1A08-sensitive genes enriched for DNA damage checkpoint and repair
Each Gene Ontology (GO) category was overlapped with both 1A08-sensitive deletions (x-axis; −log P-value of overlap), and significant SGI “neighbors” of 1A08-sensitive deletions (y-axis; −log P-value of overlap). Only GO categories with a Bonferroni-corrected P value < 0.05 for either hits or neighbors are shown.
Figure 10
Figure 10. Growth Inhibition of 1A08 on Log Phase Growing HCT-116 Cells
Cells were treated with various concentrations of 1A08, from 250 nM to 20 μM, for 24 h. MTT was added for 3 h before washing cells and dissolving them in DMSO. Absorbance at 570 nm was measured using a plate reader.
Figure 11
Figure 11. 1A08 is a Specific Inhibitor of the Intra-S-Phase DNA Damage Checkpoint
A. HCT-116 cells show a dose dependent response for escape from S-phase in the presence of 50 nM CPT after 24 h. 1A08 was added at the indicated concentrations. Caffeine was added at 2 mM. B. Incorporation of Brd-U in the presence of 50 nM CPT is also dependent upon 1A08 concentration, with the strongest activity near the LD50 value of untreated cells. C. The percentage of cells in G1 does not significantly change when treated with 5 μM 1A08 for 6 h in the presence of 200 nM CPT, black bars show G1, white bars show S-phase, and grey bars show G2/M. D. The percentage of mitotic cells in HCT-116 p53 −/− cells does not change significantly when treated with 5 μM 1A08 in the presence of 10 nM CPT, black bars show interphase cells, grey bars show mitotic cells. Error bars represent standard deviations from the average of three independent experimental replicates.

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