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.