2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid (Activator-3) is a potent activator of AMPK

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.

Figure 1

Activator-3, an AMPK mimetic is a potent pan-AMPK activator in cell based and cell free assays. (A) Chemical structures of Activator-3, AMP, ZMP and C2. Bottom panel: Rationale why Activator-3 may likely mimic AMP. (B) pAMPK based dose response curve of Activator-3 in primary rat hepatocytes (left panel) and rat L6 myoblasts (Right Panel). Results are expressed as the increase in activity relative to DMSO control and represent the mean ± SD for three independent experiments. (C) Human recombinant AMPK complexes (α1β1γ1, α2β1γ1, α2β2γ2 and α2β2γ3) expressed in baculo virus were assayed for allosteric activation by Activator-3. The representative four AMPK isozymes used for assays represent different combinations of all known reported isoforms like α1, α2, β1, β2, γ1, γ2 and γ3. EC₅₀ values were calculated by plotting a non-linear curve of log [agonist] vs. response. Results are expressed as the increase in activity relative to DMSO control and represent the mean ± SD for three independent experiments. (D) Enzymes allosterically regulated by AMP (PFK1) or using AMP as a substrate (AK) were assayed in presence of AMP (≤1 mM) or Activator-3 (≤10 µM). Both the enzymes were unaffected by Activator-3. Results are representative of three independent experiments conducted using commercially available kits.

Figure 2

Activator-3 and AMP share common activation mode for AMPK activation and Activator-3 enhances AMPK phosphorylation by upstream kinase LKB1 and protects AMPK complex from PP2C mediated dephosphorylation. (A–D) Recombinant human AMPK α1β1γ1 (A), AMPK α2β1γ1 (B), AMPK α2β2γ2 (C) or AMPK α2β2γ3 (D) were assayed in the presence of AMP (100 µM) alone and AMP (100 µM) and increasing concentration of Activator-3 (0–10 µM). Results are expressed as percentage increase of AMPK activity relative to DMSO control and represent the mean ±SD for three independent experiments. (E) Recombinant human AMPK α1β1γ1 was assayed in the presence of Activator-3 (30 nM; ~EC₅₀ concentration in vitro kinase assay) alone and Activator-3 (30 nM) and increasing concentrations of AMP (0–300 µM). Results are expressed as percentage increase of AMPK activity relative to DMSO control and represent the mean ± SD for three independent experiments. (F) Recombinant human AMPK α1β1γ1 was assayed in the presence of low concentration of ATP (20 µM) and high and physiological concentration of ATP (2 mM) and increasing concentration of Activator-3 (0–1 µM). Results are expressed as percentage change of AMPK activity relative to DMSO control (set as 100%). Results represent the mean ± SD for three independent experiments. (G) Recombinant AMPK complex was purified from HEK-293T cells by transient overexpression of the indicated construct as indicated in methods and assayed in presence of LKB1 alone or in presence of LKB1 and 100 µM AMP or increasing concentration of Activator-3 (10–100 nM) and the representative blot was quantified. (H) The effects of Activator-3 and AMP on dephosphorylation and inactivation of human recombinant AMPK α2β1γ1 by PP2C. Assays were performed either using vehicle (pAMPK alone) or with PP2C or vehicle with PP2C + Mg²⁺ or increasing concentration of Activator-3 (10–100 nM) or 100 µM AMP. The blots were probed with indicated antibodies.

Figure 3

Molecular modelling of AMPK. (A) Complete structure of AMPK heterotrimeric complex with co-crystallized AMP molecules. The missing residues from the incomplete crystallographic structures were homology modeled. α, β and γ subunits are shown in green, blue and gold colors respectively. AMP molecules in the complex are shown in blue sticks. Inset shows a closer view of the three AMP bound sites in the γ subunit; (B) Heatmap showing residue-wise RMSD contribution of AMPK across the trajectory. The color code in the heatmap plot represents the RMSD value for each of the amino acids at a given time-frame. Root mean square fluctuations (RMSF) of the Cα atoms in the AMPK heterotrimeric complex are also shown. The vertical line in the RMSF plot represents the mean RMSF of the complex.

Figure 4

Docking studies of Activator-3 on AMPK. (A–B) AMPK-Activator-3 docked complexes at Site 1 (A) and Site i (B; Center of four CBS domains); considered for the current study. Activator-3 and interacting residues are shown in green and magenta sticks respectively. (C–D) Comparison of binding pose of Activator-3 with C2 (C) and AMP (D). (E) Activator-3 docked at the center of four CBS domains in presence of AMP (pink sticks) at all the three sites known from the crystal structure (PDB ID: 4CFF). In presence of AMP, the Activator-3 (green sticks) is docked at other side of R70 and R152 residues (magenta sticks). CBS domains 1, 2, 3 and 4 are shown in red, blue, cyan and green colors respectively. All the images are shown in stereo view.

Figure 5

R70 and R152 amino acids γ1 subunit of AMPK are required for Activator-3 mediated AMPK activation. (A) The binding energy calculations of Activator-3 bound to wild type and mutant γ subunit of AMPK. (B) pACC based in vitro kinase activities of native human recombinant α1β1γ1 AMPK enzyme and its mutants using 100 µM AMP and 30 nM Activator-3. The activity shown in the above figure was normalized to vehicle control and wild type (WT) control group. Results are the representative of three independent experiments. Statistical analysis was performed using Bonferroni’s Multiple Comparison Test *p < 0.05, **p < 0.01.

Figure 6

Treatment of HSD rats with Activator-3 improves metabolic health. (A–D) OGTT analysis of control and HSD rats on zero day (A and B) and 30 days (C and D). (E–H) Analysis of cholesterol (E), triglyceride (F) free fatty acid (G) and body weight (H) of control and HSD fed rats. Statistical analysis were performed using one way ANOVA followed by tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. (I,J). Western blot analysis of pAMPK, AMPK, pACC, ACC and β-actin of the soleus muscle tissue of normal, HSD fed rats treated with vehicle control or 10 mg/kg /day Activator-3 for 30 days.