Identification of pharmacological inhibitors of the MEK5/ERK5 pathway

https://doi.org/10.1016/j.bbrc.2008.09.087Get rights and content

Abstract

We have identified two novel MEK5 inhibitors, BIX02188 and BIX02189, which inhibited catalytic function of purified, MEK5 enzyme. The MEK5 inhibitors blocked phosphorylation of ERK5, without affecting phosphorylation of ERK1/2 in sorbitol-stimulated HeLa cells. The compounds also inhibited transcriptional activation of MEF2C, a downstream substrate of the MEK5/ERK5 signaling cascade, in a cellular trans-reporter assay system. These inhibitors offer novel pharmacological tools to better characterize the role of the MEK5/ERK5 pathway in various biological systems.

Section snippets

Materials and methods

Cells. HeLa and HEK293T cells were grown in RPMI-1640 with 10% heat inactivated FBS, 2 mM glutamine, and 50 μg/ml gentamycin. All reagents for cell culture were purchased from Invitrogen.

Plasmids. The expression constructs pCNDA-MEK5-CA, pCDNA-ERK5, and pFA-MEF2C were generated by PCR amplification of the respective genes and sub cloning into the expression vectors, pCDNA3.1 (Invitrogen) for MEK5 and ERK5 and pFA (Stratagene) for MEF2C. Constitutively active MEK5 was generated by site directed

Selectivity profile of the MEK5 Inhibitors BIX02188 and BIX02189

High-throughput screening of the Boehringer Ingelheim compound collection was carried out against MEK5. Hits were first screened for selectivity using MEK1 and MEK2. The selective inhibitors were then screened against the panel of kinases listed in Table 1. Optimization of the hits was then carried out to maximize MEK5 potency and selectivity. This led to the identification of two compounds with favorable selectivity profiles. Boehringer Ingelheim. These are the indolinone-6-carboxamides,

Acknowledgments

We thank members of the Protein resources and Biophysics groups at Boehringer Ingelheim Pharmaceuticals, Inc. for production and analysis of purified active enzymes and biomolecular screening group for screening and follow up biochemical assays for this study. We thank Melissa Foerst for her assistance in molecular assay development for MEK5. We thank Elizabeth Mainolfi, Dr. Lore Gruenbaum, Holly Clifford, Steve Fogal, and Dr. Monica Cheng for experimental support and Drs. Christopher

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