Together the PRRs activate the innate immune system in response to conserved pathogen-derived molecules such as nucleic acids and cell wall components. NOD2 is one of the best characterized cytoplasmic PRRs and is expressed primarily in antigen-presenting cells and certain mucosal epithelia. NOD2 is thought to undergo a conformational change upon binding the bacterial cell wall component muramyl dipeptide to enable ATP binding, oligomerization and recruitment of the A 484954 serine/threonine kinase RIP2. This in turn leads to recruitment of additional effector kinases including TAK1/TAB1 and the formation of a polyubiquitinated signaling complex that stimulates canonical NF-kB and MAPK pathways leading to increased synthesis of pro-inflammatory cytokines and chemokines. Such co-activation has been shown to be necessary for an optimal adaptive immune response in T and B lymphocytes. Given the central role of NOD2 in regulating innate immune signaling it is perhaps not surprising that mutations in this PRR are associated with chronic inflammatory and autoimmune diseases. Although the associations are less robust, variants in NOD2 and the closely related NOD1 have also been linked to a variety of other inflammatory conditions including adult-onset sarcoidosis and atopic diseases. Whether abnormal NOD2 signaling contributes to the pathogenesis of inflammatory diseases in which there is no mutation in NOD2 remains to be established. The role of NOD2 in initiating innate immune responses, and its genetic association with inflammatory diseases, identifies NOD2 as a potential target for therapeutic intervention. To our knowledge the only compounds demonstrated to inhibit NOD2 signaling are the plant-derived polyphenol curcumin, and arene-chromium diterpenes based on the anti-inflammatory pseudopterosins from a sea coral. Moreover, although a A 943931 dihydrochloride cell-based high-throughput screen for small molecule NOD2 inhibitors has been conducted no selective compounds were identified. Screening large compound collections for NOD2 inhibitors using a traditional biochemical/receptor binding approach has not been feasible due to the difficulty in expressing and purifying large quantities of functional NOD2 protein. Consequently, we employed a cellbased screening approach to search for inhibitors of MDPstimulated cytokines coupled with extensive selectivity profiling in NOD2-independent assays. We describe here the cellular activity and structure-activity relationship for a benzimidazole diamide series exhibiting highly selective inhibition of NOD2 signaling pathways.