DEVELOPMENT OF A NONRADIOACTIVE, TIME-RESOLVED FLUORESCENCE ASSAY FORTHE MEASUREMENT OF JUN N-TERMINAL KINASE-ACTIVITY

Activated transcription factor AP-1 is composed of c-Jun homodimers or c-Jun/c-Fos heterodimers and mediates expression of several gene prod ucts 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 phosph orylation of c-Jun may lead to a novel class of therapeutics. A nonrad ioactive, high-throughput, time-resolved fluorescence assay was develo ped to measure and identify inhibitors of JNK activity. This assay uti lized a lanthanide (europium)-labeled antibody that was specific for N -terminally phosphorylated c-Jun. The optimized europium-based assay w as approximately 15-fold more sensitive compared to a similar P-32-bas ed JNK assay. Compounds that were identified as inhibitors of JNK usin g the europium-based assay also inhibited JNK activity in the P-32-bas ed assay with similar IC50 values. The europium-based JNK assay elimin ates the contamination problems associated with the use of radioactivi ty. The sensitivity and safety of the europium-based assay make it ame nable to robotics that will significantly increase screening throughpu t.