Name | N-(4-Amino-3-chlorobenzoyl)-3-methyl-L-valyl-N-[(2R,3S)-2-ethoxy- 5-oxotetrahydro-3-furanyl]-L-prolinamide |
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Synonyms |
vx765
Belnacasan VX-765 N-(4-Amino-3-chlorobenzoyl)-3-methylvalyl-N-[(2R,3S)-2-ethoxy-5-oxotetrahydro-3-furanyl]-L-prolinamide L-Prolinamide, N-(4-amino-3-chlorobenzoyl)-3-methylvalyl-N-[(2R,3S)-2-ethoxytetrahydro-5-oxo-3-furanyl]- |
Description | Belnacasan (VX-765) is an oral prodrug of VRT-043198, a potent and selective caspase-1 inhibitor with a Ki of 0.8 nM. |
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Related Catalog | |
Target |
Caspase-1 |
In Vitro | Belnacasan inhibits the release of LPS-induced IL-1β and IL-18 by human PBMCs with an IC50 of ~0.7 μM and reduces inflammatory response in murine models of inflammatory disease[1]. VBelnacasan is a potent, specific inhibitor of the caspase-1 subfamily[2]. |
In Vivo | Belnacasan doses 50, 100, and 200 mg/kg significantly (p<0.05) reduces serum IL-1β levels by as much as 60%, whereas 25 mg/kg has a smaller effect (~35% inhibition) that is not statistically significant. It is noteworthy that the effect of Belnacasan on the release of IL-1β induced by LPS reached a plateau at 100 mg/kg. Belnacasan (25, 50, and 100 mg/kg × 2) significantly reduces ear edema. Belnacasan also dose-dependently reduces the concentrations of cytokines, chemokines, and inflammatory mediators in the ear biopsy samples[2]. Belnacasan at doses of 25, 50, and 200 mg/kg significantly delays the time to seizure onset by 1.5- to twofold (p<0.01), reduces the number of seizures by 40% (p<0.01) and the total time spent in EEG seizure activity by 30 to 50% (p<0.01)[3]. |
Kinase Assay | 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]. |
Cell Assay | 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 Belnacasan (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]. |
Animal Admin | Mice[2] Single doses of Belnacasan (10, 21, 43, and 84 mg/kg) in vehicle (25% Cremophor EL in water) are administered via oral gavage. Blood samples (approximately 0.25-0.3 mL) are collected before dose administration and 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 h after dosing via the retroorbital sinus and processed for plasma. A high-performance liquid chromatography/mass spectrometry methodology is used to determine the concentration of Belnacasan and VRT-043198 in plasma samples. Noncompartmental analysis is carried out using WinNonlin Pro, version 4.0.1. Rats[3] Male Sprague-Dawley rats (250-280 g) are used. Belnacasan (25, 50, 200 mg/kg) is dissolved in 20% Cremophor and injected ip in rats once a day for 3 consecutive days. On the fourth day, rats receive Belnacasan, 45 min and 10 min before intrahippocampal injection of kainic acid. Respective controls are similarly injected with vehicle before kainic acid. |
References |
Density | 1.3±0.1 g/cm3 |
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Boiling Point | 779.0±60.0 °C at 760 mmHg |
Molecular Formula | C24H33ClN4O6 |
Molecular Weight | 508.995 |
Flash Point | 424.9±32.9 °C |
Exact Mass | 508.208862 |
PSA | 147.04000 |
LogP | 0.83 |
Vapour Pressure | 0.0±2.7 mmHg at 25°C |
Index of Refraction | 1.589 |
Storage condition | -20℃ |