Mechanistic Insights of Sulfonamide‐Based NLRP3 Inhibitors for the Treatment of Neurodegenerative Diseases

Neuroinflammation has proven to play an important role in the pathogenesis of neurodegenerative diseases by encouraging the expression and maturation of pro-inflammatory regulators. Studies have shown the upregulation of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), traumatic brain injury (TBI), and many more. The NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome is the key regulator in the maturation and release of these inflammatory cytokines and is therefore an attractive target for suppressing inflammation. Upon assembly and activation of the multimeric NLRP3 inflammasome complex comprised of NLRP3, ASC protein, and pro-caspase-1, autoproteolytic cleavage of pro-caspase-1 occurs. Activated caspase-1 is then able to cleave pro-IL-1β and pro-IL-18 and release their biologically active forms, IL-1β and IL-18, consequently leading to an inflammatory cell death known as pyroptosis (Figure 1). Our recent medicinal chemistry campaign has led to a series of novel small molecule inhibitors that are suggested to selectively target NLRP3 and show both in vitro and in vivo activity to inhibit the release of IL-1β and IL-18 with sub-micromolar potencies. Through various assays and photoaffinity labeling experiments using a chemical probe, we have revealed and validated the mechanisms and binding of our lead compound, YQ128, to NLRP3. YQ128 is able to inhibit caspase-1 and GSDMD cleavage as well as ASC speck formation in mouse macrophages; however, it has no effect on the NLRP3 expression, suggesting its target is upstream of ASC speck and caspase-1 but downstream of priming. Through target identification studies using a photoaffinity labeling (PAL) technique, we found that YQ128 specifically interacts with NLRP3, but not other components of the NLRP3 inflammasome complex. Competition studies with the parent compound and other known NLRP3 inhibitors suggested the specific and unique binding of YQ128 to NLRP3. The results from other biological assays proposed its binding to the NACHT domain of NLRP3. Using our chemical probe in a pull-down assay, we also discovered other potential targets within the inflammasome pathway, including a protein that was recently termed "undruggable". These results suggest a novel mechanism of inhibition by our compounds and further studies are currently underway to improve these inhibitors as potential therapeutics for neuroinflammation in neurodegenerative diseases.