Mechanistic Characterization of a Small Molecule as a Direct NLRP3 Inhibitor via Binding to the NACHT Domain

The NLRP3 inflammasome has recently emerged as a viable drug target, and small-molecule inhibitors of this protein complex are being actively tested in preclinical disease models. However, the mechanisms of action for the majority of these compounds remain unclear. Our recent medicinal chemistry campaign led to the discovery of several potent lead NLRP3 inhibitors from a distinct chemical scaffold. Herein, further characterization using biophysical, biochemical, chemical biology, and molecular biology approaches of one of the lead inhibitors revealed direct binding to the NACHT domain of the NLRP3 protein. The studies also suggested potentially multiple binding sites within the NACHT domain that can be targeted by different small-molecule inhibitors. Furthermore, an activity-based probe with high labeling efficacy was developed and can serve as a valuable tool to contribute to ongoing efforts in understanding NLRP3 biology and in developing NLRP3-targeted therapies for various diseases.