(5Z)-7-Oxozeaenol

Kinase experiment:

For screening TAK1 inhibitors, insect expression vectors for TAK1 and TAB1 are co-infected into Sf9 cells. After 2 days of incubation, cell lysates are immunoprecipitated with anti-TAK1 antibody (M-17). The immunoprecipitates are incubated with various compounds (5Z-7-Oxozeaenol, etc.) and subsequently incubated with 2 μg of myelin basic protein and 10 μCi of [γ-32P]ATP (3,000 Ci/mmol) in 10 μL of the kinase buffer containing 10 mM HEPES (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2 at 30°C for 5 min. Samples are separated by 10% SDS-PAGE, and 32P incorporated into myelin basic protein is quantified with a bioimage analyzer. The catalytic activity of MEK1 is determined by activation of ERK2 to phosphorylate dure. The catalytic activity of MEKK1 is measured with 2 μg of myelin basic protein as a substrate in the kinase buffer. For subsequent kinase assays, immunoprecipitates are incubated with 5 μCi of [γ-32P]ATP (3,000 Ci/mmol) and 1 μg of bacterially expressed MKK6 or GST-IκBα-(1-72) in 10 μL of the kinase buffer at 25°C for 2 min. Samples are separated by 10% SDS-PAGE and visualized by autoradiography[1].

References:

[1]. Ninomiya-Tsuji J, et al. A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase. J Biol Chem. 2003 May 16;278(20):18485-90.
[2]. Dakas PY, et al. Modular synthesis of radicicol A and related resorcylic acid lactones, potent kinase inhibitors. Angew Chem Int Ed Engl. 2007;46(36):6899-902.
[3]. Takehana K, et al. A radicicol-related macrocyclic nonaketide compound, antibiotic LL-Z1640-2, inhibits the JNK/p38 pathways in signal-specific manner. Biochem Biophys Res Commun. 1999 Apr 2;257(1):19-23.