Identification of novel inflammasome inhibitors via cellular NLRP3 target engagement assays

The NLRP3 (NOD-like receptor family, pyrin domain-containing protein 3) inflammasome, a multiprotein complex, plays a crucial role in triggering the release of pro-inflammatory cytokines like interleukin-1 beta. Abnormal activation of NLRP3 can mediate an aberrant immune response to viral infections and is associated with inflammatory diseases. In this study, the goal was to identify bioactive, potent, and specific inhibitors of NLRP3 that could modulate the inflammasome pathway and assess their potential therapeutic relevance. An innovative workflow was assembled by setting up a robust cellular-based high throughput screening (HTS) target engagement (TE) tool to identify potent NLRP3 inhibitors and validate their functional effect on the inflammasome downstream signaling cascade. A subset of 2,500 compounds from the European Chemical Biology Library (ECBL) was screened and validated inhibitors were subjected to a similarity study by state-of-the-art computational tools to comprehend their specific impact on inflammasomal signaling nodes upstream of NLRP3 and to propose feasible anti-inflammatory drugs. Ultimately, ten compounds were selected and validated in functional checkpoints of the NLRP3 inflammasome pathway, such as caspase-1 activity and IL-1β release, proving the validity of this HTS TE method for identifying NLRP3 inhibitors. Subsequent similarity-based clustering suggested the organization of the active compounds into three primary groups, linked to NF-κB signaling, ROS-induced NLRP3 activation, and NLRP3 induction pathway in response to microbial and related insults. Overall, these findings demonstrate the robustness and efficiency of the target-engagement methodology to capture bioactive inflammasome inhibitors with diverse mechanisms of action. Seven of the identified NLRP3 inhibitors were characterized as novel inflammasome inhibitors with therapeutic potential.