The chemical structure of DK-139.

Effect of DK-139 on LPS-induced TLR4 activity. HEK-Blue™-hTLR4 cells were treated with different concentrations of LPS (A) or 10 ng/ml LPS in the absence or presence of DK-139 (B). After 12 h, SEAP activity was measured using a microplate reader at 650 nm. The data shown represent the means ± SD of three independent experiments performed in triplicate. ^**P < 0.01.

Inhibitory effect of DK-139 on LPS-induced NF-κB activation. (A) BV2 microglial cells were treated with different concentrations of DK-139 for 30 min, followed by treatment with 0.5 µg/ml LPS for 10 min. Whole-cell lysates were prepared, and Western blotting was performed using phospho-specific antibodies against IκBα (Ser32) and p65/RelA (Ser468), as indicated. GAPDH was used as an internal control to ensure equal protein loading. Each blot is representative of at least three independent experiments. (B) BV2 microglial cells were cultured on coverslips and pretreated with 20 µM DK-139 for 30 min before stimulation with 50 ng/ml LPS. After 30 min, the cells were fixed and incubated with an antibody against phospho-p65/RelA (Ser468) for 2 h, followed by incubation with an Alexa Fluor 488-conjugated (green signal) or Alexa Fluor 555-conjugated (red signal) secondary antibody for 30 min. Nuclear DNA was stained with 1 µg/ml Hoechst 33258 for 10 min (blue signal). Overlay images are shown on the right. Bar, 100 µm. (C) BV2 microglial cells were transfected with 0.1 µg of the 5 × NFκB-Luc plasmid, along with 50 ng of the expression plasmid for Renilla luciferase (pRL-null), to normalize transfection efficiency. At 48 h post-transfection, the cells were either left untreated or treated with different concentrations of DK-139 for 30 min, followed by treatment with 50 ng/ml LPS. After 8 h, firefly luciferase activity was measured and normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. ^*P < 0.05, ^**P < 0.01.

Inhibitory effects of DK-139 on LPS-induced production of pro-inflammatory mediators. (A) BV2 microglial cells were treated with different concentrations of DK-139 for 30 min, followed by treatment with 0.5 µg/ml LPS for 12 h. Total RNA was isolated and the levels of iNOS, COX2, IL-1β, and IL-6 mRNA were determined by RT-PCR; β-actin mRNA was used as an internal control. (B) BV2 microglial cells were treated with different concentrations of DK-139 for 30 min, followed by treatment with 0.5 µg/ml LPS for 12 h. NO production was measured using the Griess reagent. The data represent the mean ± SD of three independent experiments performed in triplicate. ^*P < 0.05, ^**P < 0.01.

Molecular docking simulation of DK-139 into the ATP-binding pocket of Akt. (A) LigPlot analysis. The residues forming hydrogen bonds and hydrophobic interactions with isobavachalcone (left) and DK-139 (right). Red circles denote identical residues interacting with isobavachalcone and with DK139. (B) The 3D structural model of the binding site of isobavachalcone (left) and DK-139 (right) docked to the ATP-binding site of Akt protein, viewed in PyMOL program.

Inhibitory effect of DK-139 on LPS-induced Akt phosphorylation. BV2 microglial cells were treated with different concentrations of DK-139 (A), or AKTi (B) for 30 min, followed by treatment with 0.5 µg/ml LPS for 10 min. Whole-cell lysates were prepared and subjected to Western blotting with phospho-specific antibodies, as indicated. GAPDH was used as an internal control to ensure equal protein loading. (C) BV2 microglial cells were infected with recombinant adenovirus expressing green fluorescence protein (Ad/GFP; Control) at 10 MOI or dominant-active p110-CAAX (Ad5/p110CAAX) at different MOI (5, 10, and 20) for 48 h. Total protein extracts were prepared and subjected to Western blot analysis. GAPDH was used as an internal control to ensure equal protein loading.