Novel p53 Pathway Activators Figure 6. Connectivity map analysis identifies drugs with similar cellular responses. A) Hierarchical clustering of connectivity scores. Gene expression signatures (1000 genes from each compound profile) were queried against Connectivity Map Database 2.0. Dendrogram of the ranking order expression signatures is presented. B) Top-ranking connectivity signatures. C) Classification of top-ranking connectivities. doi:10.1371/journal.pone.0012996.g006 The lead compounds identified in the chemical screen were structurally diverse, although were represented by two closely related pairs. Though all contained polycyclic heteroaromatic rings, these varied from two –to four-ringed structures. Based on the structural characteristics, it would have been difficult to ascribe that they possess similar biological activities. However, cellular assays and transcriptional profiling showed that the lead compounds displayed strikingly similar properties. Connectivity map analysis showed that the lead molecules shared transcriptional signatures with DNA intercalators and topoisomerase poisons. However, detailed analyses for potential topoisomerase inhibitory activities of the compounds were negative (not shown). Furthermore, the genomic signatures triggered by the compounds suggested unex- pected biological parallels with neuroleptics and other widely used drugs. Due to the wide, and often long-term use of these drugs, their activity in non-target tissues could present as undesirable side effects or as an unexpected health benefit. Interestingly, a large population- based cohort study of the use of phenothiazines has indicated a decrease in risk of rectal, colon and prostate cancer [44]. Prazosin, and other quinazoline-based adrenoreceptor antagonists doxazosin and terazosin, are used as hypertensive drugs and for prostate hyperplasia, and recently, suggested to possess antitumor properties [45], and to decrease risk of prostate cancer in men [46]. Cardiac glycosides digoxin, ouabain, quercetin and proscillaridin, all providing high connectivity scores, have been associated with topoisomerase inhibition, cellular DNA damage response, cytotox- icity of breast cancer cells and increased breast cancer survival [47,48]. Strikingly, we note that all of the recently discovered hypoxia-inducible factor-1 inhibitors (daunorubicin, acriflavin, digoxin, ouabain and proscillaridin A) are among the hits identified here [49]. These findings underscore that the lead molecules share remarkable parallels with several pharmaceutics amenable towards repurposing as cancer therapeutics. In conclusion, this study represents an extensive analysis of drug-based mechanisms activating the p53 pathway, emphasizes the dominant role of DNA intercalation in p53 pathway activation and identifies novel regulators of DNA topology with promising anticancer properties. Supporting Information Methods S1 Supplementary methods. PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12996