Belnacasan (VX-765) is an IL-converting enzyme (ICE)/caspase-1 inhibitor and an orally bioactive prodrug of VRT-043198. Belnacasan modifies the active site of caspase-1 and leads to caspase-1 blockade, which has been associated with tissue protection against inflammatory diseases in animal models.

Caspase-4:<0.6 nM(Ki), Caspase-1:0.8 nM(Ki)

方法：人 PBMCs 和全血用 LPS 和 Belnacasan 处理 18 h，使用 ELISA 检测细胞因子。
结果：Belnacasan 抑制 PBMC 和全血的 IL-1β 释放，IC50 值为 0.67 µM 和 1.9 µM。[1]

方法：为测试体内活性，将 Belnacasan (25-200 mg/kg) 灌胃给药给 CD-1 小鼠，1 h 后静脉注射 LPS (2 mg/kg)。
结果：Belnacasan 在体内抑制 LPS 诱导的 IL-1β 产生。[1]
方法：为检测抗癫痫活性，将 Belnacasan (12.5-200 mg/kg) 腹腔注射给慢性癫痫 C57BL6 小鼠模型，每天两次，持续四天。
结果：Belnacasan 的反复全身给药以剂量依赖的方式显著降低了小鼠的慢性癫痫活动。[2]

Enzyme inhibition is assayed by tracking of the rate of hydrolysis of an appropriate substrate labeled with either p-nitroaniline or aminomethyl coumarin (AMC) as follows: ICE/caspase-1, suc-YVAD-p-nitroanilide; caspase-4, Ac-WEHD-AMC; caspase-6, Ac-VEID-AMC; caspase-3, -7, -8, and -9, Ac-DEVD-AMC; and granzyme B, Ac-IEPD-AMC. Enzymes and substrates are incubated in a reaction buffer [10 mM Tris, pH 7.5, 0.1% (w/v) CHAPS, 1 mM dithiothreitol, and 5% (v/v) DMSO] for 10 min at 37°C. Glycerol is added to the buffer at 8% (v/v) for caspase-3, -6, and -9 and granzyme B to improve stability of enzymes. The rate of substrate hydrolysis is monitored using a fluorometer. Assays for cathepsin B and trypsin are performed[2].

VX-765 is solubilized in DMSO and stored, and then diluted with RPMI 1640 complete medium (DMSO 0.2%) before use[1]. A total of 2×105 cells/well (100 μL cell suspension) is distributed in triplicate in flat-bottom 96-well plates. Either 50 μL of VX-765 (40 μM in RPMI 1640 complete medium containing 0.2% DMSO) or vehicle control is added to appropriate wells. Following a 30-min incubation at 37°C, 50 μL of LPS diluted in RPMI 1640 complete medium is added at final concentrations varying from 0.001 to 10 ng/mL. Cells are returned to a 37°C incubator. At 4 h after LPS addition, 75 μL of supernatant is removed from wells, cleared by centrifugation for 5 min at 1500 rpm, and stored at 4°C until assayed. Cells are returned to a 37°C incubator until 24 h after LPS addition, at which time 100 μL of supernatant is removed, cleared by centrifugation, and stored at 4°C. Supernatants are tested using ELISA kits for IL-1β, IL-6, IL-18, and IL-1α[1].