Chemical Interrogation of FOXO3a Nuclear Translocation Identifies Potent and Selective Inhibitors of Phosphoinositide 3-Kinases

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 PI3Kα. 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. 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 PI3Kα. 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. The phosphoinositide 3-kinase (PI3K) 4The abbreviations used are: PI3Kphosphoinositide 3-kinaseGFPgreen fluorescent proteinDAPI4′,6-diamidino-2-phenylindolePTENphosphatase and tensin homologFOXOForkhead box OSARstructure-activity relationshipmTORmammalian target of rapamycin. /Akt pathway is activated in a variety of solid and non-solid tumors (1Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5150) Google Scholar) and therefore is considered as a potential intervention point for anticancer therapeutics. Activation of the pathway is frequently caused by mutations in PI3Kα that enhance its catalytic activity, leading to the generation of phosphatidyl 3,4,5-trisphosphate (PIP3) (2Zhao L. Vogt P.K. Oncogene. 2008; 27: 5486-5496Crossref PubMed Scopus (467) Google Scholar) or by mutations or deletions in the tumor suppressor PTEN (phosphatase and tensin homolog) that result in its loss of function. PTEN antagonizes the activity of PI3Kα through the dephosphorylation PIP3 (3Myers M.P. Pass I. Batty I.H. Van der Kaay J. Stolarov J.P. Hemmings B.A. Wigler M.H. Downes C.P. Tonks N.K. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13513-13518Crossref PubMed Scopus (1008) Google Scholar). In addition, PI3Kα can be activated by mutations in certain receptor-tyrosine kinases as well as by mutations in the oncogene KRAS (4Downward J. Semin. Cell Dev. Biol. 2004; 15: 177-182Crossref PubMed Scopus (678) Google Scholar, 5Carnero A. Blanco-Aparicio C. Renner O. Link W. Leal J.F. Curr. Cancer Drug. Targets. 2008; 8: 187-198Crossref PubMed Scopus (646) Google Scholar). phosphoinositide 3-kinase green fluorescent protein 4′,6-diamidino-2-phenylindole phosphatase and tensin homolog Forkhead box O structure-activity relationship mammalian target of rapamycin. The PIP3 generated by activation of PI3Kα or sustained by the inactivation of PTEN binds to a subset of lipid-binding domains in downstream targets such as the pleckstrin homology (PH) domain of the oncogene Akt (6Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2110) Google Scholar, 7Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1048) Google Scholar); thereby, recruiting it to the plasma membrane. Once at the plasma membrane, Akt can be activated (8Kennedy S.G. Wagner A.J. Conzen S.D. Jordán J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (980) Google Scholar, 9Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (914) Google Scholar). When active, Akt phosphorylates several effector molecules including the Forkhead box O (FOXO) transcription factors (10Calnan D.R. Brunet A. Oncogene. 2008; 27: 2276-2288Crossref PubMed Scopus (923) Google Scholar, 11Burgering B.M. Oncogene. 2008; 27: 2258-2262Crossref PubMed Scopus (167) Google Scholar). FOXO proteins are a family of conserved polypeptides that bind to DNA as a monomer and activate the transcription of genes that are involved in numerous biologically relevant processes such as metabolism, differentiation, proliferation, longevity, and apoptosis (12Accili D. Arden K.C. Cell. 2004; 117: 421-426Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar, 13Ho K.K. Myatt S.S. Lam E.W. Oncogene. 2008; 27: 2300-2311Crossref PubMed Scopus (150) Google Scholar). Akt phosphorylates FOXO proteins at three conserved consensus sites, which leads to conformational changes that facilitate CRM-1-mediated nuclear export (14Turner J.G. Sullivan D.M. Curr. Med. Chem. 2008; 15: 2648-2655Crossref PubMed Scopus (127) Google Scholar, 15Van Der Heide L.P. Hoekman M.F. Smidt M.P. Biochem. J. 2004; 380: 297-309Crossref PubMed Scopus (554) Google Scholar). Nuclear FOXO proteins function as regulators of transcription, whereas cytoplasmic FOXO proteins are considered inactive. It is well established that FOXO is negatively regulated by various proliferative and anti-apoptotic signaling pathways that activate the PI3K/Akt signaling cascade (11Burgering B.M. Oncogene. 2008; 27: 2258-2262Crossref PubMed Scopus (167) Google Scholar). Therefore, we chose to employ a high content imaging approach to monitor the nucleocytoplasmic translocation of a GFP-FOXO3a fusion protein in U2OS cells (U2foxRELOC) (16Zanella F. Rosado A. García B. Carnero A. Link W. Chembiochem. 2008; 9: 2229-2237Crossref PubMed Scopus (69) Google Scholar, 17Zanella F. Rosado A. Garcia B. Carnero A. Link W. BMC Cell Biol. 2009; 10: 14Crossref PubMed Scopus (36) Google Scholar) as the readout for biological inhibition of PI3K/Akt signaling. The rapid kinetics of the assay allowed us to reduce the incubation time and minimize possible toxic effects that might interfere with the analysis. Furthermore, this image-based high-throughput strategy provides a filter for adequate solubility, permeability, and stability in a cellular context and enables compounds that produce artifacts or cytotoxicity to be identified on a single cell basis. Often a key limitation of cell-based screening approaches is the identification of the molecular target of the compound. For this reason, the cell-based screen was followed by a focused screen of kinases thought to be involved in the regulation of the intracellular localization of FOXO proteins. This screen identified pyrazolopyrimidine derivatives as inhibitors of PI3Kα. Finally, a combination of computational and synthetic medicinal chemistry was used to optimize the chemical features required for activity. Here we report the discovery of a novel series of PI3K inhibitors discovered by cellular high content screening that are potent, selective, and demonstrate mechanism of action in vivo. Compounds were purchased from ChemDiv (San Diego, CA), BioFocus (Cambridge, UK), and Life Chemicals (Burlington, Canada). LY294002 was from Calbiochem. Leptomycin B was purchased from LC Laboratories (Woburn, MA). Different but complementary criteria were considered for compound selection and purchase: (a) chemistry perspective: drug-like compounds and (b) biological point of view: structures focused on kinases (kinases libraries) as well as a diverse set of compounds exploring different chemical space with a potential biological activity. Compounds were purchased from ChemDiv and Life Chemicals. Their structure and purity (>90%) have been validated by NMR and/or LCMS. The compounds that have been synthesized in-house are at least 95% pure by LCMS and NMR (see supplemental information for details). The GFP-FOXO3a fusion protein was kindly provided by T. Finkel. The U2nesRELOC assay uses the reporter construct pRevMAPKKnesGFP, which has been described earlier (18Henderson B.R. Eleftheriou A. Exp. Cell Res. 2000; 256: 213-224Crossref PubMed Scopus (351) Google Scholar, 19Zanella F. Rosado A. Blanco F. Henderson B.R. Carnero A. Link W. Assay Drug. Dev. Technol. 2007; 5: 333-341Crossref PubMed Scopus (37) Google Scholar). pRevMAPKKnesGFP carries the nuclear export signal from MAPKK cloned between the BamHI and AgeI sites of pRev(1.4)-GFP, sandwiched between the Rev and the GFP-coding sequences. Cell lines were obtained from the American Type Culture Collection (ATTC). U2OS (human osteosarcoma) was cultured in Dulbecco's modified Eagle's medium. PC3 (human prostate carcinoma), MCF7 (human breast carcinoma), HCT116 (human colon carcinoma), 768-0 (human renal carcinoma), U251 (human glioblastoma) were grown in RPMI. All media were supplemented with 10% fetal bovine serum (Sigma) and antibiotics-antimycotics. Cells were maintained in a humidified incubator at 37 °C with 5% CO2 and passaged when confluent using trypsin/EDTA. The U2nesRELOC assay and the U2foxRELOC assay have been described previously (16Zanella F. Rosado A. García B. Carnero A. Link W. Chembiochem. 2008; 9: 2229-2237Crossref PubMed Scopus (69) Google Scholar, 19Zanella F. Rosado A. Blanco F. Henderson B.R. Carnero A. Link W. Assay Drug. Dev. Technol. 2007; 5: 333-341Crossref PubMed Scopus (37) Google Scholar). Briefly, cells were seeded at a density of 1.0 × 105 cells/ml into black wall clear bottom 96-well microplates (BD Biosciences) after 12 h of incubation at 37 °C with 5% CO2, 2 μl of each test compound were transferred from the mother plates to the assay plates. Cells were incubated in the presence of the compounds for 1 h. Then cells were fixed, and the nucleus stained with DAPI (Invitrogen). Finally the plates were washed with 1× phosphate-buffered saline twice and stored at 4 °C before analysis. Detailed methods are described in supplemental information. The Gold 3.1 program (21Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar) was used to carry out docking of LY294002, PI-103, and ETP-45658 5During the preparation of this report, a compound with the identical structure as ETP-45658 was described as an inhibitor of PI3K in an international application published under the patent cooperation treaty (WO2008/115974A2) by Wyeth Pharmaceuticals. to with the structure of with M.E. O. Stephens L. Hawkins P.T. M.P. Williams R.L. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The was using the the docking used was a the of The function was used to docking and protein for to of were on the each the structures out of genetic were For we the for LY294002 and to with the structure of for LY294002 M.E. O. Stephens L. Hawkins P.T. M.P. Williams R.L. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and the for as described by (21Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar). and key is as a criteria for the docking between LY294002 and the are and when the for LY294002 is For PI-103, docking using a to the one described by (21Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar) in which the was to a to and the is into the by the and we that the of is in into the by the of in the obtained with have that this of the of the docking of in on the was as the protein structure for docking of The strategy used to biologically inhibitors of PI3K/Akt signaling is in the we used a cellular imaging assay that the intracellular of FOXO proteins (16Zanella F. Rosado A. García B. Carnero A. Link W. Chembiochem. 2008; 9: 2229-2237Crossref PubMed Scopus (69) Google Scholar). U2OS cells that a green fluorescent protein of were seeded in 96-well assay plates and treated with compounds at a of for 1 h. A of 33,992 compounds were for to the translocation the GFP-FOXO3a reporter protein from the cytoplasm into the The nuclear of by the LY294002, was as activity and used as a were as compounds that have an activity compounds were as positive a of A of an compound is in compounds that be inhibitors of the nuclear export selective of FOXO nuclear the of the compounds to the localization of a nuclear export signal fluorescent reporter protein F. Rosado A. Blanco F. Henderson B.R. Carnero A. Link W. Assay Drug. Dev. Technol. 2007; 5: 333-341Crossref PubMed Scopus (37) Google Scholar) was Briefly, the nuclear export assay uses U2OS cells that a Rev protein, which a The fluorescent signal of cells to the with a nuclear export inhibitor such as the reporter protein in the cell nucleus of the the nuclear localization of Rev protein and were as inhibitors as inhibitors of the nuclear an of a positive compound is in the of The positive in the FOXO translocation assay and that were in the nuclear export assay were as FOXO The FOXO relocators were protein kinases that are known to at sites involved in the regulation of its intracellular localization such as or by such as PI3Kα and (10Calnan D.R. Brunet A. Oncogene. 2008; 27: 2276-2288Crossref PubMed Scopus (923) Google Scholar, H. J. Cell Sci. 2007; PubMed Scopus Google Scholar). inhibitors of or were compounds chemical of which or were identified as biochemical inhibitors of PI3Kα. the FOXO relocators that were inhibitors of were an of protein kinases that have been in signaling that FOXO proteins compounds several receptor-tyrosine kinases whereas activity was In a of the FOXO relocators were inhibitors of nuclear were inhibitors of were inhibitors of and were as inhibitors of an Pyrazolopyrimidine derivatives were the most potent biochemical inhibitors of PI3Kα identified from the screen The for inhibition of PI3Kα from to 12 In clear nuclear FOXO translocation of between and the compounds Akt at in treated cells The was the most potent biochemical inhibitor of PI3Kα and the most potent of FOXO nuclear translocation obtained from the screen Therefore, this compound was for mechanism of action of signaling downstream of Akt was in cells treated with the of on and of on was the mechanism of action of as an inhibitor of signaling. on the were for into the features of required to the PI3Kα and cellular activity, we a combination of virtual screening and synthetic medicinal In the a and was on of structures of the and using the obtained from the FOXO screen and from PI3Kα biochemical a virtual screen compound of compounds was using the This was followed by using as the by the with PI3Kα activity. to the of of compounds were as of FOXO and of were biochemical inhibitors of PI3Kα a of of indicated that an an as or docking the that a key in the or the The structure of O. C. Stephens L. Williams R.L. PubMed Scopus Google Scholar) in with the compounds LY294002 M.E. O. Stephens L. Hawkins P.T. M.P. Williams R.L. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and (21Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar) that the to as a to a key with in the (21Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar). Therefore, the synthetic chemistry used as one of the key substituents for the and as described in The in 1 that an with in the were and in as by the to the PI3K inhibitor This structure-activity relationship was by from A.J. K. P. A. A. L. P. J. A. N. L. M.H. C. P. A. J. Med. Chem. 2008; PubMed Scopus Google chemistry and of the pyrazolopyrimidine chemistry and of the pyrazolopyrimidine ETP-45658, which a and was found to be a potent inhibitor of PI3Kα with an of The compound was to with in a to LY294002 and the for in the are with and The PI3Kα and which are found in human ETP-45658 as the PI3Kα protein ETP-45658 was for its to and ETP-45658 was and potent it activity with an of ETP-45658 but activity the and DNA with of and ETP-45658 was a of protein kinases of kinases and were at a of it that ETP-45658 is selective phosphoinositide ETP-45658 was a potent of nuclear translocation with an of and supplemental The compound a of Akt on after a of U2OS cells The of ETP-45658 to the of the Akt and was with the in Akt ETP-45658 caused a 95% in the of at and a on at Akt It is clear the inhibition of the of on was of on with LY294002 and the of the on of is and therefore incubation with the inhibitor be required to In addition, the of the was this was to the inhibition of PI3K by ETP-45658 or by ETP-45658 activity was be ETP-45658 of cells the of kinases of cellular for the PI3K ETP-45658 the of after 1 h of with this the cell of PC3 cells treated for h with ETP-45658 a clear with of apoptosis as indicated by the of a of cells that ETP-45658 PI3K signaling in treated tumor The activity of ETP-45658 was using a small of human cell lines with mutations in PI3Kα or loss of function of the of The cellular for inhibition of were found to be between and was in cell lines with mutations in PI3K or mutations in is to the of PI3K/Akt pathway inhibitors as a the activity of ETP-45658 in vivo was ETP-45658 was in lines under of the which leads to PI3Kα activation in the of O. Blanco-Aparicio C. Leal J.F. Carnero A. Cancer Res. 2008; PubMed Scopus Google Scholar). The were with ETP-45658 or into a positive a of was with the compound after the were and were for The signal was and of of the of were with the of the in the of Akt on was in the of with ETP-45658 as with the mice. The of Akt was to of the The of on and on were In a clear of the of was with ETP-45658 a in the in the that ETP-45658 PI3K signaling in vivo. of the high of genetic in the PI3K/Akt signaling cascade in of human is to inhibitors of this pathway to as a The in this report the of a combination of high content cell-based screening and focused biochemical of protein kinases to the molecular target of compounds with the mechanism of This was followed by molecular modeling and medicinal chemistry that led to the discovery of ETP-45658 a potent and selective inhibitor of The screen of 33,992 compounds followed the intracellular of the Akt in tumor cells (16Zanella F. Rosado A. García B. Carnero A. Link W. Chembiochem. 2008; 9: 2229-2237Crossref PubMed Scopus (69) Google Scholar). Compounds found that the nuclear translocation of nuclear export were a small of protein kinases involved in the regulation of FOXO nuclear This identified pyrazolopyrimidine derivatives as inhibitors of PI3Kα. we inhibitors of Akt or This is to an of compound collection the family of protein kinases or the that the inhibitors of kinases that are in compound collection were to the cell and therefore be as in the cell-based we have biochemical using compound we be which is A of FOXO relocators were identified that activity protein kinases that phosphorylates FOXO or kinases that that compounds target proteins of the PI3K/Akt compounds were found to be inhibitors of several receptor-tyrosine which for activity in the FOXO F. C.L. J. Cancer Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) in a screen for inhibitors of nuclear compounds were obtained between the three chemical were found of which were PI3Kα. 1 were which have been as for H. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). The is on and has been as inhibitors of A J. Chem. Sci. 1998; PubMed Scopus Google Scholar). has been as protein inhibitors J. C. J.L. L. T. P. S.D. A. B. F. J.L. D. D. A. D. H. L. J. Med. Chem. 2006; PubMed Scopus Google Scholar). The most frequent was in of the FOXO relocators key chemical and features were FOXO relocators were found to have the were different and a cell-based assay to screen for of the signaling J. A. Chem. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) identified as an inhibitor of which found to be a inhibitor of PI3Kα. a of was identified in the image-based screen used in the and PI3Kα A was for the using the FOXO nuclear localization with the PI3Kα biochemical The that and with are the preferred substituents in and of the pyrazolopyrimidine scaffold. The most ETP-45658, in the and in the of the pyrazolopyrimidine scaffold. It is an inhibitor of PI3Kα with an of It is potent and as well as of PI3Kα that are found in human The compound and DNA at docking that the compound LY294002, ETP-45658 a key between the in the and the of in which the by the of M.E. O. Stephens L. Hawkins P.T. M.P. Williams R.L. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar); in a report A.J. K. P. A. A. L. P. J. A. N. L. M.H. C. P. A. J. Med. Chem. 2008; PubMed Scopus Google Scholar) identical between and its key ETP-45658, (21Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (968) Google and D. T. Cancer Sci. 2007; PubMed Scopus Google Scholar) the LY294002 structure and are potent potent PI3K the with through the by the that as D. T. Cancer Sci. 2007; PubMed Scopus Google Scholar). I. H. T. H. K. T. J. Natl. Cancer 2006; PubMed Scopus Google Scholar). PI3Kα as well as A. B. A. F. L. L. de A. H. T. T. N. P. P. Cancer Res. 2007; PubMed Scopus Google Scholar). ETP-45658 is a PI3K inhibitor with activity. ETP-45658 the mechanism of action in and in is a in the of Akt at of FOXO at and of at in cells after with the compound. The for of the nuclear translocation of FOXO of with the of obtained for biochemical inhibition of PI3Kα. ETP-45658 a with in treated The compound has activity in a variety of cell and this activity to be of PI3Kα and PTEN we obtained in cells with PI3Kα and PTEN or or cells with PI3Kα and PTEN or have been for PI3K inhibitors C. Oncogene. 2008; 27: PubMed Scopus Google Scholar). ETP-45658 is in vivo. The of that a PI3K in cells with the compound caused the changes in molecular of pathway including and to optimize the to a compound for with

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