Figure 1.
Cell-based screening scheme used to identify selective NOD2 inhibitors.
The compound collection was screened for inhibitors of MDP-induced IL-8 secretion in 293/hNOD2 stable cells. Active inhibitors were then tested for selectivity against IL-8 induced by alternative pathways including NOD1, TNFR1 and TLR2 as well as for direct inhibitors of RIP2 kinase. The activity of selective NOD2 inhibitors was then confirmed in cells expressing NOD2 endogenously and against agonist-independent NOD2 signaling.
Figure 2.
GSK669 inhibits NOD2 but not NOD1 mediated responses.
(A) Structure of GSK669, the original hit identified from the NOD2 HTS screening cascade. (B) Concentration response curves showing GSK669 selectively inhibits MDP-stimulated IL-8. IL-8 secretion in either MDP treated 293/hNOD2 or iE-DAP treated 293/hNOD1 stable cell lines was measured in the presence of increasing concentrations of GSK669. Data are mean ± SD from 6 independent assays. (C) Selective inhibition of NOD2-mediated MAPK phosphorylation by GSK669. Serum-starved 293/hNOD1 or NOD2 stable cells were pre-incubated with compound at the concentrations indicated and then stimulated for 1 hour with either Tri-DAP or MDP, respectively. Phospho-p38, JNK and ERK1/2 were identified in cell lysates by western blotting. A RIP2 inhibitor compound was used as a positive control. Similar results were obtained in 5 (293/hNOD2) and 2 (293/hNOD1) separate experiments.
Table 1.
Activity of NOD2 selective compounds and inhibitors of IKK and RIP2 in cell-based assays used for hit identification and triage.
Figure 3.
GSK669 exhibits NOD2 selectivity in cells that express endogenous NOD1 and NOD2.
Concentration response curves of GSK669 and its close analogue GSK400 for the inhibition of MDP (A) but not Tri-DAP (B) stimulated IL-8 secretion in HCT116 cells which express functionally active NOD1 and NOD2. Cells were pre-incubated with compounds for 1 hour prior to addition of NOD agonists. IL-8 secreted into medium was assayed after 24 hours. The same RIP2 inhibitor used in Figure 2C was included as a positive control. Data are the average percent inhibition obtained from 1 (RIP2), 2 (GSK400) or 4 (GSK669) separate experiments.
Figure 4.
Structure-activity relationship for GSK669 analogs in the MDP-stimulated IL-8 production assay in 293/hNOD2 cells.
Figure 5.
Structure-activity relationship for N-methyl analogs in the MDP-stimulated IL-8 production assay in 293/hNOD2 cells.
Figure 6.
The activity of GSK669 is specific for inhibition of MDP-stimulated NOD2 responses.
(A and B) Over-expression of NOD2 by viral transduction in HEK293T cells induces secretion of IL-8. Cells were infected (MOI = 50) with NOD1 or NOD2 bacmam virus alone for either 24 hours (A) or for 6 hours followed by removal of virus and stimulation with iE-DAP (300 ng/mL) or MDP (30 ng/mL) for an additional 24 hours (B). GSK669 (5 μM) failed to block IL-8 secretion induced by NOD1/2 virus alone but selectively blocked the MDP response in NOD2 transduced cells. The IKK inhibitor (0.5 μM) suppressed IL-8 release under all conditions. Data in (A and B) are the mean ± SD and are representative of two experiments. (C and D) Liposome-mediated transfection of ssRNA dose-dependently induces IFNβ gene expression in 293/hNOD2 but not 293/hNOD1 stable cells and the response was unaffected by GSK669 (0.16–16 μM) and a RIP2 inhibitor (0.1 μM). Cells were pre-incubated with compound for 30 minutes prior to transfection with ssRNA. IFNβ mRNA levels were measured after 6 hours by real-time RT-PCR. Data are presented as fold increase over mock transfected cells (mean ± SD) and are representative of two independent experiments.
Figure 7.
GSK669 and its analog GSK717 suppress cytokine secretion by primary human monocytes.
NOD2 inhibitors suppress cytokine secretion in primary human monocytes activated via NOD2 alone as well as via synergistic NOD2-TLR2 co-activation. (A) Monocytes isolated from whole blood were immediately pre-incubated with NOD2 inhibitor compounds (5 μM) for 1 hour and then stimulated with MDP (0.1 μg/mL) for 24 hours. The concentration of IL-1β, IL-6, IL-8 and TNFα in the conditioned medium was determined. Data are mean ± SD of triplicate treatments from a representative experiment repeated once with similar results. (B) GSK717 blocks synergy between NOD2 and TLR2. Primary human monocytes were pre-incubated for 1 hour with either RIP2 inhibitor alone (0.05–1.6 μM), GSK717 alone (0.5–16 μM) or the combination of GSK717 (0.5–16 μM) and RIP2 inhibitor (at a fixed concentration of 1.6 μM) and then stimulated with MDP (0.1 μg/mL), Pam2CSK4 (100 pg/mL) or both agonists. IL-8 secreted into the medium was determined after 24 hours. The dotted line indicates the level of IL-8 released by co-stimulated cells treated with the highest concentration of RIP2 inhibitor. Results are representative of three independent experiments.