CY-09 is an NLRP3 inhibitor.

CY-09 在1至10 μM 的剂量范围内，对单钠尿酸（MSU）、尼日尔菌素、ATP诱导的caspase-1激活和IL-1β分泌表现出剂量依赖性抑制作用，且能在LPS预处理的骨髓源性巨噬细胞（BMDMs）中发挥作用。此外，CY-09治疗亦能阻断BMDMs中细胞质LPS引发的非典型NLRP3激活。CY-09与MCC950特异性地抑制NLRP3炎症体激活，对LPS诱导的原效应无影响。CY-09显著抑制尼日尔菌素诱导的ASC寡聚体形成。研究发现，CY-09处理可抑制HEK-293T细胞中Flag-NLRP3与mCherry-NLRP3的相互作用，提示CY-09能够阻止NLRP3寡聚化。

与MCC950相比，CY-09治疗在体内有效抑制了单钠尿酸盐（MSU）注射诱导的IL-1β产生和中性粒细胞浸润。示CY-09能阻断MSU诱导的NLRP3炎症小体激活。CY-09治疗还能在治疗停止后的第25天至第48天内提高NLRP3突变小鼠的存活率。另外，CY-09还抑制了高脂饮食（HFD）处理小鼠脂肪组织中观察到的caspase-1切割。

For ATPase activity assay, purified recombinant human proteins are incubated at 37°C with indicated concentrations of CY-09 for 15 min in the reaction buffer. ATP (25 μM) is then added, and the mixture is further incubated at 37°C for another 40 min. The amount of ATP converted into adenosine diphosphate (ADP) is determined by luminescent ADP detection with ADP-Glo Kinase Assay kit according to the manufacturer's protocol. The results are expressed as percentage of residual enzyme activity to the vehicle-treated enzyme. For ATP binding assay, purified NLRP3 proteins are incubated with ATP binding agarose for 1 h and then different concentrations of CY-09 are added and incubated for 2 h with motion at 4°C. Beads are washed and boiled in loading buffer. Samples are subjected to immunoblotting analysis[1].

To induce NLRP3 inflammasome activation, 5×105/mL BMDMs and 6×106/mL PBMCs are plated in 12-well plates. The following morning, the medium is replaced, and cells are stimulated with 50 ng/mL LPS or 400 ng/mL Pam3CSK4 (for noncanonical inflammasome activation) for 3 h. After that, CY-09 or other inhibitors are added into the culture for another 30 min, and then the cells are stimulated for 4 h with monosodium urate (MSU) (150 μg/mL),?Salmonella typhimurium?(multiplicity of infection) or for 30 min with ATP (2.5 mM) or nigericin (10 μM). Cells are transfected with poly(dA:dT) (0.5 μg/mL) for 4 h or LPS (500 ng/mL) overnight. Cell extracts and precipitated supernatants are analyzed by immunoblot[1].

For the?in vivo?experiments, CY-09 is formulated in a vehicle containing 10% DMSO, 10% Solutol HS 15, and 80% saline,WT or Nlrp3?/? mice at the age of 6 wk, with similar plasma glucose levels and body weights are randomized into different groups. For generation of high-fat diet (HFD)-induced diabetic mice, mice are fed with HFD for 14 wk. The diabetic mice are treated with CY-09 (i.p.) at a dose of 2.5 mg/kg once a day for 6 wk. The mice are maintained with HFD when used for CY-09 treatment and the subsequent experiments[1].