An allosteric activator of AMPK
ZLN024 is an allosteric activator of AMP-activated protein kinase (AMPK) heterotrimers (EC50s = 0.42 and 0.95 ?M for α1β1γ1 and α2β1γ1, respectively).1 Activation of AMPK by ZLN024 requires phosphorylation of AMPK on Thr172, and ZLN024 protects Thr172 from dephosphorylation by protein phosphatase 2Cα.1 ZLN024 stimulates glucose uptake and fatty acid oxidation in L6 myotubes. In primary hepatocytes, ZLN024 decreases fatty acid synthesis and glucose output.1 ZLN024 reduces liver triacylglycerol and total cholesterol content and improves glucose tolerance in db/db mice.1
1.Zhang, L.-N., Xu, L., Zhou, H.-Y., et al.Novel small-molecule AMP-activated protein kinase allosteric activator with beneficial effects in db/db mice.PLoS One8(8)e72092(2013)
Kinase experiment: | Before the scintillation proximity assay (SPA) assay, 200 nM recombinant AMPK protein (α1β1γ1, α2β1γ1, α1β2γ1, α2β2γ1, α1(1-394), α1(1-335), α1(1-312)) is constructed, expressed, purified and fully phosphorylated. The SPA reactions are performed in 96-well plates in a final volume of 50 μL containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 2 μM biotin-SAMS, 2 μM ATP and 7.4×103 Bq/well [γ-33P]ATP. The reactions are initiated by the addition of 50 nM recombinant AMPK protein to the reaction solutions, followed by incubation at 30°C for 2 hr. The reactions are then terminated by the addition of 40 μL of stop solution containing 80 μg Streptavidin-coated SPA beads per well, 50 mM EDTA and 0.1% Triton X-100 in PBS, pH 7.5, followed by incubation for 1 hr. Finally, 160 μL of suspension solution containing 2.4 M CsCl, 50 mM EDTA and 0.1% Triton X-100 in PBS, pH 7.5, is added to the reaction solution to suspend the SPA beads completely. The SPA signals are measured in a Wallac Microbeta plate counter 30 min later[1]. |
Animal experiment: | Mice[1]C57BKS db/db mice are maintained under a 12 hr light-dark cycle with free access to water and food. At 8 weeks of age, male db/db mice are randomly assigned to the various treatment groups by body weight and glucose levels (n=6-8). The treatment groups for the 5-week chronic study are as follows: vehicle (0.5% methylcellulose), ZLN024 (15 mg/kg) and Metformin (250 mg/kg). The treatments are orally administered once daily. The body weights and food intake are measured daily. After 5 weeks of treatment, the mice are killed after a final dose, and the tissues are collected for further analysis. |
参考文献: [1]. Zhang LN, et al. Novel small-molecule AMP-activated protein kinase allosteric activator with beneficial effects in db/db mice. PLoS One. 2013 Aug 20;8(8):e72092. | |
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