Development and optimization of a non-radioactive JNK3 assay.
In light of emerging interest in the relevance of c-Jun NH2-terminal protein kinase 3 (JNK3) as a promising drug target, we describe here an advanced non-radioactive immunosorbent JNK3 activity assay that is applicable for routine screening of small molecule ATP-competitive enzyme inhibitors. We modified and established a JNK3/ATF-2 protocol based on our previously described p38 MAPK method [1] for a substrate-bound non-radioactive procedure that represents a convenient alternative to conventional radioactive protein kinase assays. The objective of the present study was to validate these conditions by using the reference compounds SP600125 and SB203580 to achieve comparable IC(50) results to published data. Furthermore, an IC(50) for staurosporine was determined. The protocol we describe here represents an accessible and robust screening assay for JNK3 inhibitors.
Peifer
Christian
C
Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany. Christian.Peifer@uni-tuebingen.de
Luik
Sabine
S
Thuma
Sabine
S
Herweh
Yvonne
Y
Laufer
Stefan
S
D000255:Adenosine Triphosphate; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007163:Immunosorbent Techniques; D020128:Inhibitory Concentration 50; D048057:Mitogen-Activated Protein Kinase 10 / Q000037:antagonists & inhibitors*; D012015:Reference Standards
D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D048057:Mitogen-Activated Protein Kinase 10