To validate the potential use of the compounds in vivo, we first utilized an ex vivo tissue culture model of human prostate tissues [29, Methods S1]. The lead compounds BMH-7, BMH-9, BMH- 15, BMH-21 and BMH-22 clearly induced p53 expression in the epithelial compartment of human prostate tissues indicating tissue permeability (Figure S3). To assess for potential anti-tumor activity in vivo in a mouse model, we used BC3 pleural effusion lymphoma cells that we have previously shown to be highly sensitive to p53 pathway activation [20]. Two compounds (BMH-15, BMH-22) available in larger scale required for the animal studies were tested. Initial flow cytometric analysis of the compound effects in BC3 cells indicated rapid and extensive cell death (BMH-15), or pronounced G2/M phase arrest and cell death (BMH-22) (Figure 3C). We then employed an orthotopic model of B cell lymphoma of BC3luc cells expressing luciferase-reporter [26]. Following BC3luc cell tumor establishment, the mice were treated by i.p. injection (20 mg/kg) three times weekly at which times the tumor bioluminescence was recorded (Figure 3D and 3E). At the given doses, BMH-15 and BMH-22 produced significant (P#0.013) anti-tumor activity. None of the compounds showed any toxic effects in mice as determined by weight curves, well- being, or histology of organs following administration at a similar dose regime for over a three-week period (Figure S4, Methods S1). We conclude that the compounds display significant anti-tumor activity without overt toxicity. The lead molecules act on DNA but diverge in their potential to cause DNA damage Based on the planar heteroaromatic ring structures we addressed the DNA intercalation properties of the compounds. DNA intercalation was assessed by changes in the compound absorbance spectrum by UV-VIS in the presence of DNA. All compounds displayed hypochromic and/or bathochromic shifts befitting DNA binding [30] (Table S4, Methods S1). DNA intercalators lead to lengthening of the double helix and unwinding. We therefore used a DNA unwinding assay, which also distinguishes between a DNA intercalator and a putative TOP1 inhibitor [30,31]. Concordant with the UV-VIS assays, all compounds were found to cause extensive DNA unwinding, but not TOP1 inhibition (Figure S5, Methods S1 and data not shown). Based on these assays, all lead molecules intercalate with DNA. As p53 is highly sensitive to DNA damage [2,32] and as DNA intercalators may cause DNA damage, we assessed whether the compounds cause activation of the DNA damage signaling pathways. The analysis of compound-treated cells for ATM target proteins cH2AX, Ser15 phosphorylated p53 and Ser824 phos- phorylated KAP-1 revealed that DNA damage response was activated by BMH-7 and BMH-15 (Figure 4A). To further estimate the dependency of p53 induction on activated ATM-cascade, we treated the cells with the compounds in the presence or absence of a specific ATM inhibitor KU55933 [33]. p53 stabilization, cH2AX foci formation and KAP-1 phosphorylation by BMH-7 and BMH- 15 were ATM-dependent (Figure 4B and 4C and 4D). BMH-15 induced a prominent cH2AX response also in human prostate tissue (Figure S3B). We conclude that while BMH-7 and BMH-15 elicit an ATM-dependent DNA damage response, more impor- tantly, BMH-9, BMH-21, and BMH-22 do not. BMH-23, analyzed separately, did also not activate ATM-pathway (data not shown). Transcriptional profiling reveals highly similar cellular responses p53, when activated, exerts an extensive transcriptional program affecting hundreds of target genes [34,35]. To validate Novel p53 Pathway Activators the extent to which the lead compounds activate p53 pathway and to gain further information of potential other pathways affected, we used transcriptional profiling. Analysis of the gene expression patterns showed a total of 5926 transcripts undergoing significant up – or down regulation (GSE #12666, Dataset S1). Hierarchical clustering indicated similar transcriptional profiles despite the chemical diversity of the compounds. BMH-9 was closely clustered with BMH-22 and BMH-23 and BMH-21 with BMH-7 and BMH-15 (Figure 5A). Further analysis, as shown by Venn diagrams, indicated extensive mutual sharing of transcriptional targets within the clusters (Figure 5B). All compounds shared a total of 118 targets with a high degree of similarity of the elicited responses (Figure 5C and Dataset S2). To provide indications for the cellular programs activated by the compounds we performed GO assignments of the transcripts using DAVID analysis platform [24,25]. The analysis indicated a significant enrichment of DNA damage checkpoint, response and repair genes, and cell cycle genes (see Table S5 for complete analysis). We further queried KEGG for specific pathways affected. The analysis showed marked enrichment of pathways affecting cell cycle, p53 signaling and ubiquitin mediated proteolysis (Figure 5D and Table S6). To verify the results of the transcriptional profiling, we performed qPCR of a number of transcripts shared by the compounds (8 up- and 10 down regulated transcripts). QPCR analysis closely correlated with the transcriptional profiling (Figure 5E). Genomic signatures of the lead compounds reveal similarities to known drugs To evaluate whether the lead compounds share similarities between drugs with known mechanisms of action, we compared the gene expression profiles elicited by the compounds to those at the Connectivity Map database [36]. The Connectivity Map comprises gene expression profiles of over 2000 experimental and known drugs. Comparison of the query transcriptional profile provides a ranked order of agents triggering similar responses. Clustering analysis of profiles ranking with the lead compounds indicated a high degree of similarity both in terms of positively and negatively ranking hits (Figure 6A). High-ranking scores were obtained for eight topoisomerase inhibitors, eight quinoline derivatives, many of which are used as anti-helminitic drugs, and hycanthone, which was the top-scoring hit with four compounds (Figure 6B and 6C). These findings are fully consistent with the identified high-content screen hits in the Spectrum Collection (Figure 1B). Among the highest scoring hits in the connectivity analysis, 30.5% have previously been implicated in p53 pathway activation (Table S7). Interestingly, the analysis identified frequent hits of drugs in clinical uses other than cancer. These included six phenothiazines, four glycosides, antihistamines and a1-adrenoreceptor antagonists (Figure 6B). However, none of these drugs tested separately affected p53 or caused activation of DNA damage signaling (not shown), suggesting that they may share other parallel activities, which may include anti-tumor properties. Discussion We present here the discovery of novel small-molecule lead compounds and detailed studies of their anti-tumor activities and mechanisms of action. The lead compounds caused death of colon, melanoma, osteosarcoma, and lymphoma tumor cells at sub-low micromolar concentrations while maintaining viability of normal primary cells and human bone marrow. The compounds showed tissue permeability in a human ex vivo prostate tissue model as evidenced by p53 induction, supporting their use in vivo, and two PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12996