In vitro activity: INF39, an acrylate derivative, is an irreversible and nontoxic inhibitor of NLRP3 that is able to decrease interleukin-1β release from macrophages. Pharmacological inhibition of NLRP3 inflammasome activation by INF39 may offer a new approach in the treatment of inflammatory bowel disease. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration. Bioluminescence resonance energy transfer experiments proved that INF39 was able to directly interfere with NLRP3 activation in cells.

Kinase Assay: INF39 at 100 μM concentration in 2% DMSO is added to wells containing immobilized NALP3 protein and preincubated for 55 min at 37°C to mimic normal experimental time (15 min preincubation+40 min incubation with ATP); in the control wells a mixture of buffer and DMSO is added. After the preincubation time the wells areished three times with reaction buffer, and ATP (250 μM) is added for 40 min at 37°C.

Cell Assay: INF39 is able to reduce the steady state (or basal) BRET signal of NLRP3 without affecting the viability of cells, meaning that it can interfere with the basal NLRP3 conformation. INF39 does not block the initial conformational changes suffered by NLRP3 upon sensing the decrease of intracellular K+; however, it affects a second step of NLRP3 conformational change that could be related with the ATPase activity of the receptor and be independent of the decrease of intracellular K+. INF39 reaches the intestinal epithelium without undergoing chemical modifications. After absorption into epithelial cells, it is likely to act locally at the mucosal epithelial level.