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Development of a Nonradioactive, Time-Resolved Fluorescence Assay for the Measurement of Jun N-Terminal Kinase Activity

Department of Pharmacology, Signal Pharmaceuticals, Inc, 5555 Oberlin Drive, San Diego, CA 92121, Department of Molecular Pharmacology, Isis Pharmaceuticals, Inc., 2280 Faraday Ave., Carlsbad, CA 92008

Tony Hunter

Molecular Biology & Virology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037

Helen Brady

Department of Pharmacology, Signal Pharmaceuticals, Inc, 5555 Oberlin Drive, San Diego, CA 92121

Brion W. Murray

Department of Pharmacology, Signal Pharmaceuticals, Inc, 5555 Oberlin Drive, San Diego, CA 92121

Mark E. Goldman

Department of Pharmacology, Signal Pharmaceuticals, Inc, 5555 Oberlin Drive, San Diego, CA 92121, Axiom Biotechnologies, Inc., 3550 General Atomics Court, San Diego, CA 92121

Activated transcription factor AP-1 is composed of c-Jun homodimers or c-Jun/c-Fos heterodimers and mediates expression of several gene products that have been implicated in disease pathogenesis. Activation of AP-1 is dependent on phosphorylation of c-Jun by Jun N-terminal kinase (JNK). Therefore, identification of inhibitors of JNK-mediated phosphorylation of c-Jun may lead to a novel class of therapeutics. A nonradioactive, high-through-put, time-resolved fluorescence assay was developed to measure and identify inhibitors of JNK activity. This assay utilized a lanthanide (europium)-labeled antibody that was specific for N-terminally phosphorylated c-Jun. The optimized europium-based assay was approximately 15-fold more sensitive compared to a similar ³²P-based JNK assay. Compounds that were identified as inhibitors of JNK using the europium-based assay also inhibited JNK activity in the ³²P-based assay with similar IC₅₀ values. The europium-based JNK assay eliminates the contamination problems associated with the use of radioactivity. The sensitivity and safety of the europium-based assay make it amenable to robotics that will significantly increase screening throughput.

Journal of Biomolecular Screening, Vol. 2, No. 4, 213-223 (1997)
DOI: 10.1177/108705719700200406


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