J774A.1 cells were plated into 96-well plates (1 × 10^5 cells/well) in growth medium overnight. Next day, cells were treated with LPS (1 μg/mL) and various concentrations of compounds for 1 h. Flagellin (1 μg/mL) or poly(dA:dT) (4 μg/mL) in DMEM was added to activate NLRC4 or AIM2 inflammasome, respectively. Flagellin cell-transfection was accomplished utilizing PULSin and allowed to incubate for 6 h.

The NOD-like receptor (NLR) family pyrin-domain-containing 3 (NLRP3) inflammasome, an essential component of the innate immune system, has been emerging as a viable drug target and a potential biomarker for human diseases. In our efforts to develop novel small molecule NLRP3 inhibitors, a 1-(5-chloro-2-methoxybenzyl)-4-phenyl-1H-1,2,3-triazole scaffold was designed via a rational approach based on our previous leads. Structure-activity relationship studies and biophysical studies identified a new lead compound 8 as a potent (IC50: 0.55 ± 0.16 μM), selective, and direct NLRP3 inhibitor. Positron emission tomography (PET) imaging studies of [11C]8 demonstrated its rapid and high brain uptake as well as fast washout in mice and rhesus macaque. Notably, plasma kinetic analysis of this radiotracer from the PET/magnetic resonance imaging studies in rhesus macaque suggested radiometabolic stability. Collectively, our data not only encourage further studies of this lead compound but also warrant further optimization to generate additional novel NLRP3 inhibitors and suitable central nervous system PET radioligands with translational promise.