A chemical genomic approach towards determining the molecular target of anti-proliferative compounds

Drug target identification is a time consuming stage of the drug discovery process. Chemical genomics offers a solution to this hurdle. In chemical genomics, a target specific chemical ligand is applied on a genomic scale. This technique was used to identify the molecular target of anti-proliferative agents using changes in mRNA transcript levels upon treatment. Whole-genome transcription profiling experiments employed the eukaryotic model organism Saccharomyces cerevisiae for small-molecule perturbation experiments in addition to traditional genetics.; Chemical genomics was used to examine the molecular target of borrelidin, a macrolide with conflicting published biological activities. The initial transcription profiles showed an increase in the transcript ratios of genes involved in amino acid biosynthesis upon treatment with borrelidin. In yeast, the GCN4 pathway regulates general amino acid control. The accumulation of uncharged tRNA activates Gcn2p which prevents the formation of the eIF-2 complex. In turn, this simulates the translation of Gcn4p, which results in the transcription of over 30 genes involved in amino acid biosynthesis. Experiments using GCN2 and GCN4 gene deletions determined that borrelidin targets the amino acid biosynthetic pathway through GCN4p. The profiling data indicates that an alternative mechanism exists for the translational regulation of Gcn4p other than through Gcn2p, which was confirmed using immunoblot analysis with eIF2 alpha and phosphorylated eIF2 alpha antibodies.; In the second application of chemical genomics, a diverse collection of synthetic compounds was evaluated in a cell-based toxicity assay. The screen revealed a subset of cyclic sulfones that inhibited growth of A549, human small lung carcinoma, cells. Within this subset, 4-(1-phenyl-1H-tetrazole-5-sulfonyl)-butyronitrile (PTSB) was the most active compound. PTSB was shown to inhibit growth of both wild-type S. cerevisiae and A549. Whole-genome transcription profiling experiments in S. cerevisiae indicated that PTSB is involved in the cellular response to oxidative stress. Analysis of the profiling data using systems biology predicted the thioredoxin pathway as the target. Biochemical assays with thioredoxin (Trx) and thioredoxin reductase (TrxR) validated that PTSB inhibits TrxR. The structure of PTSB suggests a novel mechanism of inhibition.; This research illustrates the significance of applying chemical genomics to the target validation stage of drug discovery.