CY09 是NLRP3选择性抑制剂。它能够直接与 NLRP3 NACHT 结构域的 ATP 结合基序结合，对 NLRP3 ATP酶的活性产生抑制作用，进而抑制 NLRP3 炎症小体的组装和活化。

CY-09 is an NLRP3 inhibitor.

CY-09 exhibits a dose-dependent inhibitory effect on monosodium urate (MSU), nigericin, ATP-induced caspase-1 activation and IL-1β secretion at the doses of 1 to10 μM in LPS-primed bone marrow-derived macrophages (BMDMs). Cytosolic LPS-induced noncanonical NLRP3 activation in BMDMs can also be blocked by CY-09 treatment. CY-09 and MCC950 specifically inhibit NLRP3 inflammasome activation and have no effect on LPS-induced priming effects. CY-09 treatment remarkably suppresses nigericin-induced ASC oligomerization. It is found that CY-09 treatment inhibits the interaction of Flag-NLRP3 and mCherry-NLRP3 in HEK-293T cells, suggesting that CY-09 blocks NLRP3 oligomerization

Compare with MCC950, CY-09 treatment?in vivo?efficiently suppresses monosodium urate (MSU) injection-induced IL-1β production and neutrophil influx, suggesting that CY-09 can block MSU-induced NLRP3 inflammasome activation?in vivo. CY-09 treatment also increases the survival of?NLRP3mutant mice up to days 30 to 48 even after treatment is stopped at day 25. The caspase-1 cleavage observed in adipose tissue of HFD-treated mice is also suppressed by CY-09

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].

1073612-91-5

C19H12F3NO3S2

423.42

CY 09

DMSO:77 mg/mL(181.8 mM)
Powder: -20°C for 3 years
In solvent: -80°C for 2 years