Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action

Abstract

Methods to systematically test drugs against all possible proteins in a cell are needed to identify the targets underlying their therapeutic action and unwanted effects. Here, we show that a genome-wide drug-induced haploinsufficiency screen by using yeast can reveal drug mode of action in yeast and can be used to predict drug mode of action in human cells. We demonstrate that dihydromotuporamine C, a compound in preclinical development that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism. The systematic, unbiased and genome-wide nature of this technique makes it attractive as a general approach to identify cellular pathways affected by drugs.

Fig. 1.

Sensitivity of yeast to dhMotC. (A) Structural formula of dhMotC. (B) Growth of wild-type (BY4743) strain (OD₆₀₀) as a function of time in hours. BY4743 was grown in YPD liquid culture with or without dhMotC treatment as indicated. Growth curves were performed in triplicate and represent the average of three experiments.

Fig. 2.

dhMotC sensitivity of heterozygous deletion strains identified in the high-throughput screen. (A–D) Growth of wild-type (A), ylr294/YLR294 (B), tsc10/TSC10 (C), and lcb1/LCB1 (D) strains as a function of time in hours in YPD liquid culture under two conditions: ♦, no drug control (DMSO); ▴ 20 μM dhMotC. Growth curves were preformed in triplicate, and OD₆₀₀ was measured by using a Tecan Sunrise plate reader. Growth curves represents the average of three experiments. (E) Plot of the IGCD for 21 heterozygous deletion strains identified in the drug screen are represented by black bars. The IGCD levels for the three additional heterozygous strains in the sphingolipid pathway sensitive to dhMotC are represented by gray bars. The dashed line represents 2-fold IGCD level used to define supersensitive strains.

Fig. 3.

Schematic diagram of sphingolipid biosynthesis and sphingosine-dependent kinase cascade in yeast and humans. Yeast genes are italic, and yeast proteins are Roman. Human proteins are bold. Heterozygous yeast strains tested for sensitivity to dhMotC are indicated by asterisks.

Fig. 4.

dhMotC targets the sphingolipid biosynthesis pathway. (A) Growth of wild-type strain (BY4743, OD₆₀₀) as a function of time in YPD liquid culture with and without treatment of 60 μM dhMotC and increasing amounts of DHS as indicated. Growth curves were performed in triplicate and represent the average of three experiments. Treatment of yeast with DHS does not adversely affect the growth rate of yeast. (B) Actin staining in wild-type cells in the presence or absence of dhMotC. BY4743 cells were incubated at 25°C in the presence or absence of 60 μM dhMotC and 50 μM DHS as indicated for 1.5 h. The cells were then fixed, nuclear staining was visualized by using 4′,6-diamidino-2-phenylindole (Upper), and filamentous actin was visualized using rhodamine-phalloidin (Lower). (C) Growth of wild-type (TDY2037) and csg2Δ (TDY2038) haploid cells as a function of time in YPD liquid culture with and without 10 μM dhMotC treatment. Growth curves were performed in triplicate and represent the average of three experiments. Haploid cells have an increased sensitivity to dhMotC. (D) Incorporation of ³H-serine into ceramide after 8 h was measured in both wild-type (TDY 2037) and csg2Δ (TDY 2038) cells with and without 5 μM dhMotC. (E) Rescue of dhMotC toxicity by ceramide in human cells. MDA-231 cell survival after 24-h exposure to 0 or 10 μM dhMotc and 0, 10, or 50 nM C6-ceramide is expressed as a percentage of untreated controls. The assay was performed in quadruplicate, and error bars represent 1 SD.