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

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

doi:10.1074/jbc.m109.038984

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.

Highlights

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer
The PIP3 generated by activation of PI3K␣ 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 (6, 7); thereby, recruiting it to the plasma membrane
The research presented in this report describes the use of a combination of high content cell-based screening and focused biochemical profiling of selected protein kinases to identify the molecular target of compounds with the desired mechanism of action

Summary

Introduction

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. 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. Often a key limitation of cell-based screening approaches is the identification of the molecular target of the compound For this reason, the cellbased screen was followed by a focused screen of kinases thought to be involved in the regulation of the intracellular localization of FOXO proteins. The cellbased 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 PI3K␣. 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

Methods

Results

Conclusion