Mechanistic insights of novel NLRP3 inhibitors as potential AD therapeutics

Abstract

AbstractBackgroundNLRP3 inflammasome is an essential component of innate immunity and its activation leads to the production of interleukin (IL)‐1β and IL‐18, and promotes inflammatory cell death. Recent studies have indicated a critical role for the NLRP3 inflammasome in the pathogenesis of Alzheimer’s disease (AD), where neuroinflammation has been recognized as an essential player. Thus, novel NLRP3 inhibitors represent a novel approach to develop AD therapeutics.MethodChemical probes based on a novel lead NLRP3 inhibitor that exhibits promising neuroprotective activities in AD models were synthesized to understand mode of action. Recombinant human NLRP3 protein was produced with his‐tag attached on both N‐ and C‐terminus. Photo‐affinity labeling and pull‐down assays were conducted using cell lysates of J774A.1 cells, and analyzed by silver staining and western blotting. Binding studies were performed using the recombinant human NLRP3 protein by microscale thermophoresis (MST) technique. ATPase activity of NLRP3 protein was analyzed by ADP glow assay. Drug affinity responsive target stability (DARTS) studies were performed using J774A.1 cell lysates.ResultChemical probes exhibited comparable potency as the lead compound to inhibit the release of IL‐1β from J774A.1 cells. Protein labeling and pull‐down studies clearly indicated the selective recognition of NLRP3 protein from J774A.1 cell lysates by the chemical probe. Further competition studies revealed that our lead inhibitor bins to NLRP3 differently than MCC950, the known NLRP3 inhibitor. Binding studies also confirmed the direct interaction of our inhibitor with the NLRP3 protein. DARTS studies also showed that upon incubation with the lead inhibitor, the stability of NLRP3 by pronase digestion was increased. We also confirmed that our NLRP3 inhibitor does not inhibit the ATPase activity of NLRP3.ConclusionChemical biology studies employing molecular biology, biochemical and biophysical techniques established that small molecule NLRP3 inhibitors derived from a novel chemical scaffold directly bind to the NLRP3 protein without interfering its ATPase activity, thus representing a novel mode of action. The results also indicated a distinct mode of action compared to the known NLRP3 inhibitor MCC950. Collectively, the results strongly encourage developing new analogs based on this novel chemical scaffold as novel NLRP3 inhibitors and potential AD therapeutics.