Novel p53 Pathway Activators activated and stabilized p53, of which four are for the first time described here (Figure 1B and Table S1). Of the eleven, five drugs intercalate with DNA, and four are topoisomerase 1 (TOP1) or topoisomerase 2 (TOP2) inhibitors. The results indicated that the p53 high-content imaging screen can be applied for testing of both novel and known drugs for their anti-tumor activities. We then proceeded to screen synthetic small-molecule chemical compound libraries (40,000 compounds) at a relatively low compound concentration (3 mM) in order to increase the likelihood to select for potent p53 activating molecules. Nine small-molecule compounds that consistently activated the p53 reporter at low micromolar concentrations were discovered. The chemical structures of six compounds amenable for larger scale syntheses are presented in Figure 1C and were used in subsequent studies. Searches for previously reported p53 activating small-molecule compounds indicated that the lead compounds identified here, BMH-7, BMH-9, BMH-15, BMH- 21, BMH-22, and BMH-23 were novel. Furthermore, among the identified leads, there were two sets of compounds (BMH-7 and BMH-15, BMH-22 and BMH-23) that were structurally highly related to each other (Figure 1C). The compound chemical characteristics indicated excellent predictions for the compound solubility, permeability and oral bioavailability (Lipinsky’s rule of five) [27] (Table S2). We then assessed the ability of the compounds to activate the p53 pathway. Dose-titration experiments of the leads indicated that all were highly potent inducers of the p53DsRed activation (30–65% positive cells) as compared to nutlin-3 (15% positive cells) and were active at nanomolar (BMH-21) to low micromolar range (BMH-7, BMH-9, BMH-15, BMH-22, BMH-23) (Figure 2A). Notably, high compound concentrations ($10 mM) caused extensive cell death eventually quenching the reporter signal. All compounds markedly stabilized p53 and increased the levels of its transcriptional targets, Hdm2 and p21Cip1 (Figure 2B), whereas p53 mRNA levels were in fact suppressed by four out of six compounds (Figure S1). Figure 2. Lead compounds activate p53 pathway. A) p53DsRed reporter activation. A375 cells stably expressing p53DsRed reporter were incubated with the lead compounds and nutlin-3 for 24 hours, followed by high-content image analysis. The percentage of cells expressing p53DsRed reporter is shown. The experiment was performed in triplicate and represents $1700 cells per datapoint. Error bars represent SD. B) p53 stabilization and target protein regulation. A375 cells were treated with BMH-7, -9, -15, -22, nutlin-3 (5 mM), and BMH-21 (0.5 mM) for 24 hours followed by analyses for p53, Hdm2 and p21 protein levels. Gapdh was used as a loading control. C) p53-dependent gene regulation. Isogenic HCT116 p53+/+ and p532/2 cells were treated with the compounds followed by qPCR for p53 target genes. Fold induction of the relative levels of the transcripts in the p53+/+ and p532/2 cells are shown. Data represent duplicate biological experiments and duplicate qPCR reactions. Error bars represent SD. doi:10.1371/journal.pone.0012996.g002 PLoS ONE | www.plosone.org 4 September 2010 | Volume 5 | Issue 9 | e12996