In vitro activity: Boceprevir (SCH 503034) has an average Ki value of 14 nM over a large number of runs in the HCV NS3 protease continuous assay. In the 72-h bicistronic subgenomic cell-based replicon assay in HuH-7 cells, the EC50 and EC90 values are determined to be 0.20 μM and 0.35 μM, respectively. Boceprevir is also found to be a very weak inhibitor of HNE (Ki=26 μM) representing a selectivity of 2200.

Kinase Assay: Boceprevir is a novel, potent, highly selective, orally bioavailable HCV NS3 protease inhibitor with Ki of 14 nM in both enzyme assay and EC90 of 350 nM in cell-based replicon assay.

Cell Assay: J774A.1 cells (a murine macrophage cell line) are plated at 5×104 cells/well in a 96 multiwell plate for 24 hours in RPMI medium supplemented with 10% of fetal bovine serum (FBS). The cells are primed with Escherichia coli 0111:B4 LPS(1 μg/ml) for 4 hours and then ATP (5 mM) for 30 minutes to induce the NLRP3 inflammasome formation. The supernatants are collected and levels of IL-1β are measured with a mouse IL-1β ELISA kit. To test the inhibitory effects of 16673-34-0 on NLRP3 inflammasome activation, cells are co-treated with 16673-34-0 (400μM) or Glyburide (400μM) at the time of ATP for 30 minutes, and IL-1β levels are used as read-out.