Identification of Selective Small Molecule Inhibitors of the Nucleotide-Binding Oligomerization Domain 1 (NOD1) Signaling Pathway
Figure 10
Suppression of human monocyte IL-8 secretion by selective NOD1 inhibitors.
(A) Comparison of activity between original hit compounds and more potent analogues 16 and 22. Monocytes freshly isolated from human whole blood were pre-treated with NOD1 compounds (0.05, 0.5 and 5 µM) prior to stimulation with 25 µg/mL Tri-DAP. An inhibitor of RIP2 kinase was included as a positive control. The amount of IL-8 present in the medium after 24-hour incubation was determined by HTRF assay. (B) The more potent quinazolininone compound 22 selectively inhibits Tri-DAP but not MDP stimulated IL-8 secretion. Monocytes were pre-treated with compound at the indicated concentrations for 1 hour prior to stimulation with either 25 µg/mL Tri-DAP or 0.1 µg/mL MDP for 24 hours. IL-8 release was determined by HTRF assay. The RIP2 inhibitor blocked responses to both NOD agonists, as expected, and was included as a positive control. Results are expressed as percent inhibition of stimulated IL-8 secretion. The data shown in (A) is the mean inhibition (± SD) observed across multiple experiments (n = 2 or 4) for each compound and data in (B) is the mean ± SD of triplicate treatment wells from an individual experiment repeated once with similar results.