Identification and characterization of potent, selective small molecule RIP2 kinase inhibitors (INM6P.422)

NOD1 and NOD2 are cytoplasmic PRRs, which bind peptides derived from bacterial peptidoglycans. Signaling via these receptors involves recruitment and activation of RIP2 kinase, which results in NF-κB- and MAPK-dependent pro-inflammatory cytokine production. To enable a chemical biology approach to evaluate the role of RIP2 in inflammation we screened an in-house compound collection. Optimization of an aminoquinoline template led to the discovery of GSK’214 and related analogs. GSK’214 potently and reversibly inhibited human, rat and mouse RIP2 enzymatic activity (IC50 3-10 nM). Selectivity was demonstrated against a panel of 273 commercially-available kinases. NOD2-mediated signaling induced by muramyl dipeptide (MDP) was used to characterize functional activity, in vitro and in vivo. GSK’214 inhibited cytokine (TNFα, IL1β, IL-6 and IL-8) production (IC50 20 nM) by human monocytes stimulated by MDP, but not by ligands specific for TLR2, TLR4, TLR7, TLR9, IL1R and TNFR. Using an acute mouse peritonitis assay, orally administered GSK’214 inhibited neutrophil influx and cytokine release induced by MDP, but not LPS. Next, we used these inhibitors to probe the role of RIP2 in a murine TNBS-colitis model of intestinal inflammation. Selective inhibition of RIP2 kinase activity reduced disease severity to a degree comparable to prednisolone. These results demonstrate the potential of these inhibitors to elucidate the contribution of RIP2 kinase-dependent signaling in inflammation.