The association of NOD2 mutations with particular autoimmune and inflammatory diseases highlights its importance in innate immune regulation and infers that inhibition of NOD2 may represent a viable anti-inflammatory therapeutic strategy. However, few known components of the NOD2 A 331440 dihydrochloride signaling pathway represent attractive opportunities for pharmacological intervention. Consequently, to our knowledge, selective NOD2 inhibitors have yet to be developed. The identification of the benzimidazole diamide series by a cell-based screening approach as described here, therefore demonstrates that the NOD2 pathway can indeed be selectively inhibited. The initial hit compound and its related analogues, GSK400 and GSK717, possess sub-micromolar in vitro activity and appear to exhibit significant selectivity for inhibition of NOD2-mediated responses since they do not block any of the other IL-8/NF-kB inducing pathways investigated including NOD1, TNFR1 and TLR2. 5-TAMRA SE Although the primary HTS utilized HEK293 cells stably expressing either exogenous NOD1 or NOD2, compound selectivity for inhibition of NOD2 signaling was maintained in immortalized and primary cells possessing endogenous functional NOD1 and NOD2 proteins. Although the compounds are selective inhibitors of NOD2 signaling, the identity of their target protein and mechanism of action are unknown. These compounds did not inhibit the activity of RIP2 kinase or greater than 300 additional kinases. Moreover, the compounds lacked a typical activation loop/hinge-binding motif lending additional support to the conclusion that they are unlikely to be acting directly as kinase inhibitors. The data demonstrating lack of effect of GSK669 on cellular MDP uptake as well as on ����ligand���� independent activation in NOD2 overexpression systems suggest interference by the compound somewhere between intracellular processing of MDP and NOD2 oligomerization. Interestingly, these compounds blocked MDPinduced responses, but did not inhibit cytokine production induced by either NOD2 over-expression or single-stranded RNA. An alternative explanation of this finding, however, is that NOD2 activation by each process incurs subtle mechanistic differences. Thus, MDP, ssRNA, or expression-induced NOD2 oligomerization may each induce distinct NOD2 conformations, oligomeric structures, requirements for nucleotide binding, or complexes with other proteins, with the inhibitors possessing specificity for the MDP-induced mechanism since this was how they were originally selected in the primary screen. Our data suggesting a competitive interaction between MDP and the inhibitor GSK717 also implies engagement of NOD2. Lastly, other proteins identified as components of the NOD2 signaling complex, such as GRIM19 and TRIM27, may represent potential targets for GSK669 activity.