Inhibition of NLRP3 by a CNS-penetrating indazole scaffold

Abstract Low-grade inflammation is a hallmark of ageing and a key cause of age-related impairments and diseases 1 . The NOD-like receptor NLRP3 senses a variety of danger signals and environmental insults, resulting in pro-inflammatory response, inflammasome formation and pyroptosis 2,3 . Its aberrant activation has been linked to many acute and chronic diseases ranging from atherosclerosis to Alzheimer’s disease and cancer, making NLRP3 an attractive therapeutic target 4,5 . Here we report the discovery, characterization, and structure of an indazole-based NLRP3 antagonist, BAL-1516, which potently inhibits inflammasome formation in monocytes and microglia. The cryo-electron microscopy structure of BAL-1516 bound to NLRP3 reveals a previously undescribed compound binding site at a surface groove of the nucleotide-binding domain with contacts to the FISNA and WHD subdomains. The characteristic feature of BAL compound binding is the formation of three hydrogen bonds to the peripheral β-strand of the triple-ATPase; two from the indazole’s nitrogen atoms and a third from the compounds’ linker region. Additional phenyl and thiazole moieties render the compound hydrophobic, allowing excellent blood-brain barrier penetration. The compound binding site is highly specific for NOD-like receptors, and the optimized compound BAL-1516 is able to directly bind mouse NLRP3 despite two conservative residue changes in the binding interface. The BAL compounds represent a first-in-class family of NLRP3 inhibitors, providing a broad design space, including covalent and degradative properties, for the development of NLRP3-directed therapeutics. The innate immune system contains cytosolic proteins that sense cellular stress caused by bacterial, viral and fungal infections or sterile inflammation, to control cellular integrity 2 . NLRP3 is a well-studied member of the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) that is involved in the activation of the inflammasome, a multiprotein complex that mediates inflammation 6 . Upon detection of stress or pathogen-associated signals, NLRP3 triggers the activation of caspase-1, which leads to the production of pro-inflammatory cytokines such as IL-1β and IL-18, driving inflammatory responses and ultimately pyroptotic cell death. In the context of neuroinflammation, NLRP3 plays a significant role in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis 7 . Research into targeting NLRP3 signalling with CNS-penetrating molecules holds potential for developing therapeutic strategies to alleviate neuroinflammatory conditions and to slow the progression of neurodegenerative diseases.