Squaramides enhance NLRP3 inflammasome activation by lowering intracellular potassium.

The NLRP3 inflammasome is a component of the inflammatory response to infection and injury, orchestrating the maturation and release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-18, and triggering pyroptotic cell death. Appropriate levels of NLRP3 activation are needed to avoid excessive tissue damage while ensuring host protection. Here we report a role for symmetrical diarylsquaramides as selective K+ efflux-dependent NLRP3 inflammasome enhancers. Treatment of macrophages with squaramides potentiated IL-1ß secretion and ASC speck formation in response to K+ efflux-dependent NLRP3 inflammasome activators without affecting priming, endosome cargo trafficking, or activation of other inflammasomes. The squaramides lowered intracellular K+ concentration which enabled cells to respond to a below-threshold dose of the inflammasome activator nigericin. Taken together these data further highlight the role of ion flux in inflammasome activation and squaramides as an interesting platform for therapeutic development in conditions where enhanced NLRP3 activity could be beneficial.

Selective inhibition of the K+ efflux sensitive NLRP3 pathway by Cl- channel modulation.

The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18, and contributes to inflammation exacerbating disease. Fenamate non-steroidal anti-inflammatory drugs (NSAIDs) were recently described as NLRP3 inflammasome inhibitors via chloride channel inhibition. Fenamate NSAIDs inhibit cyclooxygenase (COX) enzymes, limiting their potential as therapeutics for NLRP3-associated diseases due to established side effects. The aim here was to develop properties of the fenamates that inhibit NLRP3, and at the same time to reduce COX inhibition. We synthesised a library of analogues, with feedback from in silico COX docking potential, and IL-1ß release inhibitory activity. Through iterative screening and rational chemical design, we established a collection of chloride channel inhibiting active lead molecules with potent activity at the canonical NLRP3 inflammasome and no activity at COX enzymes, but only in response to stimuli that activated NLRP3 by a K+ efflux-dependent mechanism. This study identifies a model for the isolation and removal of unwanted off-target effects, with the enhancement of desired activity, and establishes a new chemical motif for the further development of NLRP3 inflammasome inhibitors.

Is Targeting the Inflammasome a Way Forward for Neuroscience Drug Discovery?

Neuroinflammation is becoming increasingly recognized as a critical factor in the pathology of both acute and chronic neurological conditions. Inflammasomes such as the one formed by NACHT, LRR, and PYD domains containing protein 3 (NLRP3) are key regulators of inflammation due to their ability to induce the processing and secretion of interleukin 1ß (IL-1ß). IL-1ß has previously been identified as a potential therapeutic target in a variety of conditions due to its ability to promote neuronal damage under conditions of injury. Thus, inflammasome inhibition has the potential to curtail inflammatory signaling, which could prove beneficial in certain diseases. In this review, we discuss the evidence for inflammasome contributions to the pathology of neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease, epilepsy, and acute degeneration following brain trauma or stroke. In addition, we review the current landscape of drug development targeting the NLRP3 inflammasome.

Design, Synthesis and Evaluation of Oxazaborine Inhibitors of the NLRP3 Inflammasome.

The NLRP3 inflammasome is an important regulator of the sterile inflammatory response, and its activation by host-derived sterile molecules leads to the intracellular activation of caspase-1, processing of the pro-inflammatory cytokines interleukin-1ß (IL-1ß)/IL-18, and pyroptotic cell death. Inappropriate activation of NLRP3 drives a chronic inflammatory response and is implicated in several non-communicable diseases, including gout, atherosclerosis, type II diabetes and Alzheimer's disease. In this study, we report the design, synthesis and biological evaluation of novel boron compounds (NBCs) as NLRP3 inflammasome inhibitors. Structure-activity relationships (SAR) show that 4-fluoro substituents on the phenyl rings retain NLRP3 inhibitory activity, whereas more steric and lipophilic substituents diminish activity. Loss of inhibitory activity is also observed if the CCl3 group on the oxazaborine ring is replaced by a CF3 group. These findings provide additional understanding of the NBC series and will aid in the development of these NLRP3 inhibitors as tool compounds or therapeutic candidates for sterile inflammatory diseases.