The NLRP3 inflammasome is a multiprotein complex that plays a pivotal role in regulating the innate immune system and inflammatory signaling. Upon activation by PAMPs and DAMPs, NLRP3 oligomerizes and activates caspase-1 which initiates the processing and release of pro-inflammatory cytokines IL-1β and IL-18. NLRP3 is the most extensively studied inflammasome to date due to its array of activators and aberrant activation in several inflammatory diseases. Studies using small molecules and biologics targeting the NLRP3 inflammasome pathway have shown positive outcomes in treating various disease pathologies by blocking chronic inflammation. In this review, we discuss the recent advances in understanding the NLRP3 mechanism, its role in disease pathology, and provide a broad review of therapeutics discovered to target the NLRP3 pathway and their challenges.

NLRP3 inflammasome has recently emerged as an attractive drug target for neurodegenerative disorders. In our continuing studies, a new chemical scaffold was designed as selective inhibitors of NLRP3 inflammasome. Initial characterization of the lead HL16 demonstrated improved however non-selective inhibition on the NLRP3 inflammasome. Structure-activity relationship studies of HL16 identified a new lead, 17 (YQ128), with an IC 50 of 0.30 ± 0.01 μM. Further studies from in vitro and in vivo models confirmed its selective inhibition on the NLRP3 inflammaome and its brain penetration. Furthermore, pharmacokinetic studies in rats at 20 mg/kg indicated extensive systemic clearance and tissue distribution, leading to a half-life of 6.6 hours. However, the oral bioavailability is estimated to be only 10%, which may reflect limited GI permeability and possibly high first-pass effects. Collectively, these findings strongly encourage development of more potent analogs with improved pharmacokinetic properties from this new chemical scaffold.

Background: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, and the most common type of dementia. A growing body of evidence has implicated neuroinflammation as an essential player in the etiology of AD. Inflammasomes are intracellular multiprotein complexes and essential components of innate immunity in response to pathogen- and danger-associated molecular patterns. Among the known inflammasomes, the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome plays a critical role in the pathogenesis of AD. Objective: We recently developed a novel class of small molecule inhibitors that selectively target the NLRP3 inflammasome. One of the lead compounds, JC124, has shown therapeutic efficacy in a transgenic animal model of AD. In this study we will test the preventative efficacy of JC124 in another strain of transgenic AD mice. Methods: In this study, 5-month-old female APP/PS1 and matched wild type mice were treated orally with JC124 for 3 months. After completion of treatment, cognitive functions and AD pathologies, as well as protein expression levels of synaptic proteins, were assessed. Results: We found that inhibition of NLRP3 inflammasome with JC124 significantly decreased multiple AD pathologies in APP/PS1 mice, including amyloid-β (Aβ) load, neuroinflammation, and neuronal cell cycle re-entry, accompanied by preserved synaptic plasticity with higher expression of pre- and post-synaptic proteins, increased hippocampal neurogenesis, and improved cognitive functions. Conclusion: Our study demonstrates the importance of the NLRP3 inflammasome in AD pathological development, and pharmacological inhibition of NLRP3 inflammasome with small molecule inhibitors represents a potential therapy for AD.

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Background NLRP3 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. Method Chemical 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. Result Chemical 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. Conclusion Chemical 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.

Background: NLRP3 inflammasome is a cytosolic and multimeric protein complex that plays essential roles in innate immune responses. Dysregulation of this protein complex has been linked to neuroinflammation and the development of Alzheimer's disease. Therefore, NLRP3 inflammasome represents an attractive target for effective AD treatment development.Method: Small molecule inhibitors based on a recently identified lead NLRP3 inhibitor were designed, synthesized, and biologically characterized using biochemical and cellular assays. In vivo selectivity and target engagement were also tested using mice and 3xTg AD mice.Result: A series of analogs were successfully synthesized and a new chemical scaffold was also designed to provide NLRP3 inhibitors. Studies in murine macrophages demonstrated that the inhibitory potency can be improved by structural modifications and a new lead compound was identified with an IC50 of 0.1 µM. This lead compound also binds to the recombinant NLRP3 protein with a K D of 0.5 µM. Studies in mice also confirmed that this lead compound is a BBB penetrant. Further in vivo studies demonstrated that this lead compound selectively reduces the IL-1ß and engage the NLRP3 inflammasome in 3xTg AD mice. Conclusion:A new lead NLRP3 inhibitor was identified via medicinal chemistry optimization to show selective inhibition on the NLRP3 inflammasome both in vitro and in vivo. The results strongly encourage further testing of this lead inhibitor for its functional activity in AD mice.