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. 2009 Oct 9;284(41):28392-28400.
doi: 10.1074/jbc.M109.038984. Epub 2009 Aug 18.

Chemical interrogation of FOXO3a nuclear translocation identifies potent and selective inhibitors of phosphoinositide 3-kinases

Wolfgang Link  1 , Julen Oyarzabal  1 , Beatriz G Serelde  1 , Maria Isabel Albarran  1 , Obdulia Rabal  1 , Antonio Cebriá  1 , Patricia Alfonso  1 , Jesus Fominaya  1 , Oliver Renner  1 , Sandra Peregrina  1 , David Soilán  1 , Plácido A Ceballos  1 , Ana-Isabel Hernández  1 , Milagros Lorenzo  1 , Paolo Pevarello  1 , Teresa G Granda  1 , Guido Kurz  1 , Amancio Carnero  1 , James R Bischoff  2
Affiliations

Chemical interrogation of FOXO3a nuclear translocation identifies potent and selective inhibitors of phosphoinositide 3-kinases

Wolfgang Link et al. J Biol Chem. .

Abstract

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3Kalpha. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.

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Figures

FIGURE 1.
FIGURE 1.
Image-based GFP-FOXO relocation assay identified inhibitors of PI3K/Akt signaling. A, screening strategy to identify targeted kinase inhibitors of the PI3K/Akt signaling pathway. B, sample images of FOXO translocation induced by confirmed positives from the primary screen for FOXO relocators. Upper panels, U2foxRELOC cells were treated with 1% DMSO or ETP-7701 (10 μm) for 1 h. Middle panels, U2nesRELOC assay to exclude compounds that are general inhibitors of nuclear export. Middle left, the fluorescent signal (green) and DAPI stain of nuclei (blue) of U2nesRELOC cells treated with 1% DMSO for 1 h. Middle right, the fluorescent signal (green) and DAPI stain of nuclei (blue) of U2nesRELOC cells treated with leptomycin B (4 nm). Lower left, the fluorescent signal (green) and DAPI stain of nuclei (blue) of U2nesRELOC cells treated with 10 μm ETP-7701. Lower right, the fluorescent signal (green) and DAPI stain of nuclei (blue) of U2nesRELOC cells treated with 10 μm ETP-6459.
FIGURE 2.
FIGURE 2.
Identification of pyrazolopyrimidines as PI3Kα inhibitors. A, chemical structures, IC50 for inhibition of PI3Kα, fluorescent images, and the EC50 to induce GFP-FOXO nuclear translocation in cells and of the pyrazolopyrimidine compounds identified from the primary screen. Results are given as the mean ± S.E. of three independent experiments performed in triplicate. B, U2OS cells were treated with the compound indicated at 10 μm or as a positive control LY294002 (20 μm) for 4 h; then total protein was analyzed by immunoblot with the antibody indicated. One representative experiment is shown for three performed. C, ETP-7382 inhibits PI3K/Akt signaling in treated cells. U2OS cells were treated with ETP-7382 (10 μm) or as a positive control LY294002 (20 μm) for 4 h; then total protein was analyzed by immunoblot. One representative experiment is shown of three performed. Phosphorylation sites, P-Akt (serine 473), P-FOXO3a (threonine 32), and P-p70/S6K (threonine 389), were identified using specific phosphoantibodies purchased from Cell Signaling.
FIGURE 3.
FIGURE 3.
A, schematic representation of interactions of compounds bound to PI3Kγ. The two-dimensional representations were carried out with the MOE package (Chemical Computing Group, Inc. Molecular Operating Environment, MOE 2007.09 (2007). Montreal, Quebec, Canada). Upper left quadrant, pyrazolopyrimidine scaffold docked into the PI3Kγ structure. Upper right quadrant, reference compound, LY294002 docked into the PI3Kγ structure. Lower left quadrant, reference compound, PI-103 docked into the PI3Kγ structure. Lower right quadrant, ETP-456658 docked into the PI3Kγ structure. B, IC50 values of ETP-45658 for various phosphoinositide 3-kinases. IC50 values were determined as described under supplemental information.
FIGURE 4.
FIGURE 4.
A, ETP-45658 is a potent inhibitor of PI3K/Akt signaling in vitro and in vivo. ETP-45658 induces a dose-dependent increase in GFP-FOXO nuclear translocation. U2foxRELOC cells were treated with ETP-45658 at the concentrations indicated in the figure for 1 h. B, ETP-45658 inhibits PI3K/Akt signaling in cells. U2OS cells were treated with 10 μm ETP-45658 or 20 μm LY294002 for 4 h. Total protein extracts were analyzed by immunoblot. One representative blot of three independent experiments is shown. The phosphorylation sites, P-Akt (serine 473), P-FOXO3a (threonine 32), P-p70/S6K (threonine 389), P-GSK3-b (serine 9), p-p44MAPK (threonine 202/tyrosine 204), and p-p42MAPK (threonine 202/tyrosine 204) were detected using phosphospecific antibodies purchased from Cell Signaling. C, ETP-45658 inhibits PI3K signaling in vivo. Photomicrographs (×200 magnification) of mammary glands from virgin female mice of the transgenic line MMTV-myrp110α are depicted. Mammary glands no. 9 (right hind leg) and no. 10 (left hind leg) of 3 mice were injected with ETP-45658 (22.7 mg/kg), PI-103 (22.7 mg/kg), or vehicle. The images shown are representative mammary duct staining of mice treated with vehicle (left) or ETP-45658 (right). The inset in the left lower corner of each panel shows a representative region of the epithelial layer of a duct in higher magnification (×400). All immunohistochemistry staining was performed in parallel. The phosphorylation sites detected are as follows: P-Akt (serine 473), P-eIF4G (serine 1108), and P-p70/S6K (threonine 389). The epithelial-specific signal was quantified, and values of intensity of the immunohistochemical images of compound-treated mammary glands were compared with the values of the respective vehicle control images. Legend: ***, p < 0.001.
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