(A) Schematic of the fluorescence-based assay used to identify sporulation inhibitors. Meiotic landmark events indicated as Ent (entry), DS (pre-meiotic DNA replication), Rec (recombination), MI and MII (first and second meiotic division), and Spo (spore formation). The normal final product is depicted as an ascus with four spores (green). Compounds that inhibit sporulation or that are cytotoxic in sporulating yeast cells will suppress the expression of the sporulation-specific gene CDA2 and therefore also CDA2 promotor-driven GFP expression. (B) Real-time measurement of fluorescence intensities in cells harboring the GFP-reporter, which were sporulated in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Fluorescence of the sporulation culture was measured every 15 min over the course of 20 hours (see Methods). (C) Schematic of the post-recombination growth assay. The two defective alleles (his4x and his4B) can give rise to a functional HIS4 allele upon meiotic recombination. The histidine-auxotrophic diploid mother cells can therefore produce histidine-prototrophic spores (indicated in blue). If this event is suppressed by a compound, either because it directly inhibits meiotic recombination or because it is cytotoxic to sporulating yeast cells, no histidine-prototrophs are formed. (D) Proof-of-concept of the his4x/his4B assay. Cells were sporulated for 5 hours in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Aliquots of cells were then transferred to agar plates that lack histidine (-his) or leucine (-leu) and incubated for two days at 30°C, prior to measuring colony density (see Methods) of each spot on the -his agar. Agar plates lacking leucine served as a loading control. The barplot in the upper panel shows mean and standard deviation data from 4 independent experiments.

(A) Images of Nomarski microscopy of cells sporulated in the absence of drug (control), in the presence of 2 mM ammonium sulfate (AS), or 100 µM tripelennamine (TA) for 24 hours. Part of the TA image was magnified; arrows indicate granular bodies of unknown origin. (B) 5-fold serial dilution of yeast sporulated for 24 hours in the presence of three different concentrations of TA (indicated on the right) and then transferred to rich media in order to determine the rate of cell survival. Pictures were taken after incubating the rich media agar plates for 2 days at 30°C. (C) FACS analysis of pre-meiotic DNA synthesis of a “no drug” control, and in cells treated with ammonium sulfate (2 mM), or tripelennamine (100 µM). Samples were taken at 2, 3, 4, 6, and 8 hours after induction of sporulation. Staggered histograms show the frequencies (plotted on the y-axis) of relative DNA content, measured as propidium iodide intensity (plotted on the x-axis). (D) Percentage of cells that have completed meiotic M-phase (cells with 2 DAPI foci and cells with 3 or 4 DAPI foci) and those that have formed mature asci over time in sporulation medium (given in hours, x-axis) in the absence (control, circles and solid lines), or the presence of 100 µM tripelennamine (treatment, squares and dashed lines). (E) Expression patterns of representative genes involved in meiotic development (left column), nitrogen catabolite repression (GAT1 and GAP1), glycolysis/gluconeogenesis (CDC19 and PGK1), and stress response (AZR1). Log2-transformed fluorescence signals of RMA-normalized microarray data (see Methods) are plotted on the y-axis and are graphed versus samples taken in rich media and pre-sporulation media (in the absence of tripelennamine), or total time (4 and 8 hours) the cultures spent in sporulation media in the absence (black curve) or presence (red curve) of TA.

(A) A scatterplot of sensitivity scores (calculated as described in the Methods) representing the impact of compounds from the NIH clinical collection in the sporulation screening assays. Compounds that inhibit growth or that interfered with the fluorescence-based assay because of auto-fluorescence were not included in this representation. Compounds with a score of greater than 5 are shown in green. DMSO controls are shown in red. (B) A Venn diagram representing the overlap of inhibitors in the growth assay and the two sporulation assays (colored in yellow and green, respectively). 200 compounds had no effect in any of the three assays, 231 inhibited vegetative growth (of BY4741 and/or AD1-9 strains), and 64 inhibited sporulation. The overlap of between growth and sporulation inhibitors was 49. (C) Chemical structures of 12 sporulation-specific inhibitors that were confirmed to inhibit sporulation by microscopy analysis.

(A) Heatmap and dendrogram depicting hierarchical clustering of homozygous deletion pool data from three independent experiments. Quantile normalized and log2-transformed microarray fluorescence signals were analyzed using the Significance Analysis of Microarrays (SAM) software (see Methods) and identified 49 genes (indicated on the right-hand side) that were significantly depleted in the presence of tripelennamine (TA) compared to a “no drug” control. Darker shades of red indicate higher fluorescence signals and therefore a higher abundance of that strain in the pool (see legend). (B) Growth of the hoΔ/HO (control) and neo1Δ/NEO1 heterozygous deletion strains (see legend) were determined in the presence of various concentrations of (TA) (indicated on the x-axis). Growth rates relative to the “no drug” control (calculated as a ratio of AvgG, see Methods) were determined for each concentration and are plotted on the y-axis. (C) Sporulation efficiency of the hoΔ/HO control strain (black curve) and the neo1Δ/NEO1 heterozygous deletion strain (red curve) sporulated in the presence or absence of TA is indicated as percent spores on the y-axis. Both strains were incubated in sporulation media for 48 hours at various concentrations of TA (indicated on the x-axis) and percentage of spores was determined by microscopy. A total of 100 cells were counted for every condition.