Fig 1.
NF-κB induction by TLR7/8 agonists in HEK-TLR cells.
Four TLR7/8 agonists were compared. (A) Structure of R848, (B) Structure of Hybrid-2, (C) Structure of para-amine and (D) Structure of meta-amine. HEK-293 cells transfected with (E) human TLR7 and (F) TLR8 and an NF-κB-driven reporter SEAP gene were stimulated for 18–24 h with TLR agonists. The y-axis shows the level of SEAP activity in the Quanti-blue assay optical density (OD). The x-axis shows the concentration of each compound in mg/ml. Each data point represents the mean ± SD of OD at 650 nm of triplicate culture wells. HEK detection medium alone (negative control) is represented in gray. The TLR7/8 benchmark agonist R848 is represented in black; The TLR 7/8 agonists Para-amine, Meta-amine and Hybrid-2 are represented in blue, green and red respectively. Three stars indicate significance of p < 0.001 (t test) and two green indicate significance of p < 0.01 (t test).
Fig 2.
Hybrid-2 is more potent and effective than R848, para-amine and meta-amine in a whole blood cytokine assay.
Human newborn and adult blood was cultured for 6h with TLR 7/8 agonists R848, para-amine, meta-amine and Hybrid-2 and supernatants collected for TNF or IL-1β ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 6–8. For between-agonist analyses, t test was applied to compare Hybrid-2 to the other compounds. For between age-group analyses, t test was applied to compare Hybrid-2 and R848 in newborns and adults. Statistical significance is denoted as follows: *p<0.05 and **p<0.01, with black star(s) for comparison of Hybrid-2 to R848.
Fig 3.
Hybrid-2 is more potent than R848 in inducing TNF production by human newborn and adult MoDCs.
Stimulation of (A) newborn MoDCs and (B) adult MoDCs for 24 h with Hybrid-2 or R848. Supernatants collected for TNF ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 5. For between-agonist analyses, t test was applied to compare Hybrid-2 to R848. Statistical significance is denoted as follows: *p<0.05 as a black star.
Fig 4.
Crystal structure of Hybrid-2 and R848 with human TLR8.
(A) The C-atoms of Hybrid-2 are shown in green, and the residues on TLR8 are shown in cyan. A water molecule involved in mediating the interaction between the hydroxyl on N1-substituent of Hybrid-2 and Asp545 residue is shown as a red sphere. The hydrogen bonds are represented as dashed lines in magenta, and the bond distances are marked in black. Overlay of crystal structures of Hybrid-2 and R848 with TLR8 is shown in (B). R848 is shown in light red and its TLR8 receptor shown in grey. The electrostatic repulsion between the backbone carbonyl of Gly572 residue and ether oxygen of the C2-ethoxymethyl substituent of R848 is shown in magenta.