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Homogeneous and Nonradioactive High-Throughput Screening Platform for the Characterization of Kinase Inhibitors in Cell Lysates

Service of Medical Genetics, CHUV, Lausanne, Switzerland

Nathalie Rouleau

PerkinElmer-Biosignal Inc., Montreal, Canada

Christelle Bielmann

Service of Medical Genetics, CHUV, Lausanne, Switzerland

Julie Bedard

PerkinElmer-Biosignal Inc., Montreal, Canada

Fabienne Maurer

Service of Medical Genetics, CHUV, Lausanne, Switzerland

Nathalie Allaman-Pillet

Service of Medical Genetics, CHUV, Lausanne, Switzerland

Pascal Nicod

Department of Internal Medicine, CHUV, Lausanne, Switzerland

Martina Bielefeld-Sévigny

PerkinElmer-Biosignal Inc., Montreal, Canada

Jacques S. Beckmann

Service of Medical Genetics, Department of Medical Genetics, CHUV, Lausanne, Switzerland

Christophe Bonny

Service of Medical Genetics, CHUV, Lausanne, Switzerland

Roger Bossé

PerkinElmer-Biosignal Inc., Montreal, Canada

Raphaël Roduit

Service of Medical Genetics, Department of Internal Medicine, CHUV, Lausanne, Switzerland, Institut de Recherche en Ophtalmologie, Sion, Switzerland.

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.

Key Words: protein kinases • AlphaScreen • JNK inhibitors

This version was published on December 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 8, 1015-1026 (2006)
DOI: 10.1177/1087057106294697


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