Abstract
AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation. Activator-3 enhanced AMPK phosphorylation by upstream kinase LKB1 and protected AMPK complex against dephosphorylation by PP2C. Molecular modeling analyses followed by in vitro mutant AMPK enzyme assays demonstrate that Activator-3 interacts with R70 and R152 of the CBS1 domain on AMPK γ subunit near AMP binding site. Activator-3 and C2, a recently described AMPK mimetic, bind differently in the γ subunit of AMPK. Activator-3 unlike C2 does not show cooperativity of AMPK activity in the presence of physiological concentration of ATP (2 mM). Activator-3 displays good pharmacokinetic profile in rat blood plasma with minimal brain penetration property. Oral treatment of High Sucrose Diet (HSD) fed diabetic rats with 10 mg/kg dose of Activator-3 once in a day for 30 days significantly enhanced glucose utilization, improved lipid profiles and reduced body weight, demonstrating that Activator-3 is a potent AMPK activator that can alleviate the negative metabolic impact of high sucrose diet in rat model.
Highlights
Diabetes is a progressive disease of multiple metabolic disorders
The importance of AMP binding for AMPK activation is further demonstrated by mutations that localize to the CBS domains of the γ subunit in AMPK in Wolff-Parkinson-White Syndrome46–49. 5-aminoimidazole-4-carboxamide riboside (AICAR)-derived ZMP is an AMP analog and a potent AMPK activator that binds to the CBS domain of γ subunit[6]
Similar EC50 were observed for pAMPK in HepG2 cells (Fig. S1A) and pACC in HepG2, primary rat hepatocytes and L6 cells as well (Supplementary Fig. S1B–D)
Summary
Diabetes is a progressive disease of multiple metabolic disorders. The main cause is the absolute or relative deficiency of insulin. AMPK-mediated phosphorylation of ACC1 inhibits synthesis of free fatty acids through the modulation of intracellular concentration of Malonyl-CoA, a clinically validated lipid biomarker. AMP is a natural activator of AMPK and binds to the allosteric site of Cystein-β synthase (CBS) domains of different γ subunits (regulatory subunit) and indirectly promotes activity at the catalytic domain of α subunit[6]. 5-aminoimidazole-4-carboxamide riboside (AICAR)-derived ZMP is an AMP analog and a potent AMPK activator that binds to the CBS domain of γ subunit[6]. We report in vitro and in vivo characterization of Activator-3, a novel AMP mimetic small molecule and its probable binding sites in the CBS domain of the γ subunit of AMPK and its mechanism of action of AMPK activation
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