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Many human tumors have activated the protein kinase Akt either by activation of the oncogene PI3Kα or by inactivation of the tumor suppressor PTEN.When activated, Akt phosphorylates and thereby inactivates FOXO transcription factors, key effector molecules of the P I3 K/Akt pathway.In order to identify potential molecular targets or compounds with therapeutic potential we developed a high-throughput cellular imaging assay that follows the intraceltular location of the FoxO3a protein in tumor cells.Using this assay we screened 21000 shRNAs and a collection of approximately 35,000 small molecules.We discovered previously unrecognized FOXO-repressor functions of several human genes.We will present data that show the essential role of one of these genes in the maintenance of the oncogenic properties of malignant melanoma suggesting it as a potential therapeutic target for cancer treatment.Furthermore, we identified chemical agents capable of inducing the translocation of a FOXO reporter protein from the cytoplasm to the nucleus.242 compounds were found that selectively induced the nuclear translocation of GFP-FOXO yet did not affect general nuclear export, 54 were found to inhibit PI3Kα directly.Using the results of the nuclear translocation assay a structure activity relationship (SAR) was developed for pyrazolopyrimidines derivatives.Computational analysis of the data indicated that morpholine and meta-phenol were the preferred substituents in R1 and R4 positions on the pyrazolopyrimidine scaffold, respectiyely.This was confirmed by virtual docking experiments.This lead to the synthesis of ETP-45658, a pyrazolo pyrimidine that inhibits PI3Kα with an IC50 value of 22 nanomolar.ETP-45658 is selective against a panel of 72 non related protein kinases.It inhibits several PI3K pathway effectors in treated tumor cell lines and efficiently inhibits growth of cancer cell lines.Transcriptional profiling of treated cells indicated that ETP-45658 did not induce significant transcriptional alterations of genes related to classical toxicity.In mice treated with a single administration of ETP-45658 inhibited AKT and eI41G phosphorylation in mammary ductal ceils that constitutively express myristylated p110α confirming mechanism of action in vivo.