Comparative chemogenomics to examine the mechanism of action of dna-targeted platinum-acridine anticancer agents

Abstract

Platinum-based drugs have been used to successfully treat diverse cancers for several decades. Cisplatin, the original compound of this class, cross-links DNA, resulting in cell cycle arrest and cell death via apoptosis. Cisplatin is effective against several tumor types, yet it exhibits toxic side effects and tumors often develop resistance. To mitigate these liabilities while maintaining potency, we generated a library of non-classical platinum-acridine hybrid agents and assessed their mechanisms of action using a validated genome-wide screening approach in Saccharomyces cerevisiae and in the distantly related yeast Schizosaccharomyces pombe. Chemogenomic profiles from both S. cerevisiae and S. pombe demonstrate that several of the platinum-acridines damage DNA differently than cisplatin based on their requirement for distinct modules of DNA repair.

Figure 1

Chemical structures of the platinum-acridine compounds and carriers screened.

Figure 2

Two-dimensional hierarchical clustering of all 14 compounds based on the profile similarity scores of their genome-wide S. cerevisiae profiles. Red indicates high similarity between compound profiles and grey indicates low similarity. Profiles from experiments repeated under the same conditions cluster together with few outliers, indicating the reproducibility and resolution of the genome-wide assay. The dendrogram reveals structure-function relationships between compounds.

Figure 3

S. cerevisiaeand S. pombe profiles for the platinum-acridine compounds that require DNA-damage response pathways. The top 30 sensitive strains are marked and labeled. Each graph represents the average of at least 3 replicate sensitivity screens in S. cerevisiae or 2 replicates in S. pombe. The x-axis represents gene names in alphabetical order and the y-axis is the log₂ ratio of barcode intensity for drug vs control. Red indicates essential genes, blue indicates non-essential genes and purple indicates marked genes. Strains that often appear as hits in yeast chemogenomic assays are listed in Table S3.

Figure 4

Four platinum-acridine compounds damage DNA, interfere with cell cycle progression and disrupt mitochondrial function. a) Relative importance of DNA-damage repair modules in the resistance to the DNA-damaging platinum compounds in S. cerevisiae and S. pombe. Each bar represents the median rank for genes in each of the DNA repair modules listed in the top 30 or top 250 most sensitive strains. The DNA-repair modules in S. cerevisiae were defined as follows: cross-linking genes (PSO2), NER (RAD1, RAD2, RAD4, RAD10, RAD14), PRR (RAD5, RAD6, RAD18), TLS (REV1, REV3, REV7), HRR (RAD51, RAD52, RAD54, RAD55, RAD57, RAD59), stalled replication fork repair (MUS81, MMS4). The DNA-repair modules in S. pombe were defined as follows: NER (RHP14, RHP41, RHP42, RAD13, RAD16, SWI10), PRR (RHP6, RHP18, RAD8), TLS (REV1, REV3, REV7), HRR (RHP51, RHP54, RHP55, RHP57, RAD22, RTI1), stalled replication fork repair (MUS81). b) DNA content analysis pro-files. Haploid BY4741 cells were synchronized at G1 prior to the addition of compound at IC₅₀. Samples were taken every 30 min after compound addition. The positions of the 1N and 2N DNA content peaks are indicated. c) Mitochondrial function is disrupted by platinum-acridine compounds. Wildtype BY4743 cells were grown in the presence of four platinum-acridine compounds under fermenting (blue) or respiring (red) conditions. The concentration to inhibit growth by 20% (IC₂₀) is plotted.

Figure 5

PT-AMIDINE(EN) treated cells show defects in replication fork progression by DNA combing. Logarithmically growing cultures of S. cerevisiae cells (log) were arrested in G1 with α-factor (αF) and released in the presence of 400μg/mL BrdU and either PBS or PT-AMIDINE(EN) (PT-AM) at IC₅₀. Samples were collected at 30 min and used in subsequent analysis. a) DNA content analysis. Samples were fixed and DNA contents were analyzed using flow cytometry. The positions of cells with 1C and 2C DNA contents are indicated. b) DNA combing. Representative chromosome fibers used for replication fork progression analysis. The image is assembled from fibers on different micrographs following extraction of fibers from the non-fiber background using Adobe Photoshop. A 50 kbp scale bar is indicated in the upper right corner. c) Distributions of BrdU track lengths in PBS or PT-AMIDINE(EN) treated cells, presented as a boxplot. Median BrdU track lengths are shown. The p-value was determined using a two-tailed Mann-Whitney U test.