Interleukin-converting enzyme/caspase-1 subfamily caspases; Caspase-4 (Kd < 0.6 nM); Caspase-1 (Ki = 0.8 nM); active metabolite of VX-765 (Belnacasan)
- Interleukin-1 converting enzyme (ICE)/Caspase-1：VRT-043198 (active metabolite of VX-765) inhibits caspase-1 with a K₁ of 0.8 nM in enzyme activity assays. [1]
- Caspase-4：Exhibits a K₁ < 0.6 nM for caspase-4 inhibition. [1]

VRT-043198 exhibits selectivity for other caspases-3 and -6 to -9 that is 100–10,000 times more [1]. While VRT043198 has no influence on the release of various other cytokines, such as IL-1α, tumor necrosis factor, IL-6, and IL-8, it suppresses the release of interleukin (IL)-1β and IL-18. In PBMC (n = 8) and whole blood (n = 4), VRT-043198 suppresses the release of IL-1β with IC50 values of 0.67±0.55 and 1.9±0.80 nM, respectively [1]. Strong anti-apoptotic activity is absent in VRT-043198 [1].

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- Caspase-1 enzyme inhibition：In recombinant human caspase-1 activity assays, VRT-043198 potently blocks the cleavage of the fluorogenic substrate Suc-YVAD-AMC in a dose-dependent manner, with a K₁ of 0.8 nM. This inhibition is reversible and specific to the caspase-1 subfamily. [1]
- IL-1β/IL-18 release suppression：In human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS), VRT-043198 (0.1-10 μM) reduces the secretion of IL-1β and IL-18 by >80% at 10 μM, as measured by ELISA. The IC₅₀ values for IL-1β and IL-18 inhibition are approximately 0.7 μM and 0.9 μM, respectively. [1]
- Pyroptosis inhibition：In THP-1 macrophages activated with nigericin, VRT-043198 (1 μM) blocks caspase-1-dependent gasdermin D cleavage and lactate dehydrogenase (LDH) release, indicating suppression of pyroptotic cell death. [1]

Hepatic and plasma esterases convert VX-765 to VRT-043198 very quickly, but aqueous solutions convert it considerably more slowly [1]. In models of rheumatoid arthritis and skin inflammation, VX765 lessens the severity of the disease and the expression of inflammatory mediators [1]. The release of cytokines generated by lipopolysaccharide is inhibited by VX765 (25, 50, 100, or 200 mg/kg) [1].

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- Anti-inflammatory efficacy：In a murine model of LPS-induced systemic inflammation, oral administration of VX-765 (50-200 mg/kg) results in dose-dependent reduction of plasma IL-1β levels by 40-60% at 2 hours post-treatment. The maximal effect is achieved at 100 mg/kg, with no further improvement at higher doses. [1]
- Seizure suppression：In a kainic acid-induced epilepsy model, VX-765 (25-200 mg/kg, oral) delays seizure onset by 1.5-2-fold and reduces seizure duration by 30-50%, correlating with decreased caspase-1 activity in the hippocampus. [1]
- HIV-1 reservoir reduction：In humanized NSG mice infected with HIV-1, VX-765 (50 mg/kg/day for 21 days) decreases total HIV-1 DNA in splenocytes by 64% (1,054 vs. 2,889 copies/10⁶ cells, p=0.029) and preserves CD4⁺ T cell counts. [1]

- Caspase-1 activity assay： 1. Recombinant human caspase-1 is incubated with VRT-043198 (0.01-100 nM) in assay buffer containing 10 mM DTT and 50 μM Suc-YVAD-AMC. 2. After 30 minutes at 37°C, fluorescence intensity (Ex/Em = 380/460 nm) is measured to determine substrate cleavage rate. 3. The K₁ value is calculated by nonlinear regression analysis, yielding a 0.8 nM inhibition constant. [1]

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As is a Caspase inhibitor, VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. VRT-043198 inhibited the release of interleukin (IL)-1beta and IL-18, but had little effect on the release of several other cytokines, including IL-1alpha, tumor necrosis factor-alpha, IL-6 and IL-8.

- IL-1β secretion assay： 1. Human PBMCs are isolated and primed with LPS (1 μg/mL) for 3 hours. 2. VRT-043198 (0.1-10 μM) is added, followed by stimulation with ATP (5 mM) for 1 hour. 3. Supernatants are collected, and IL-1β levels are quantified by ELISA. The IC₅₀ for IL-1β inhibition is determined to be 0.7 μM. [1]
- Pyroptosis detection： 1. THP-1 macrophages are differentiated with PMA and treated with nigericin (5 μM) in the presence or absence of VRT-043198 (1 μM). 2. Cell lysates are analyzed by Western blot for caspase-1 p20 subunit and gasdermin D cleavage. 3. LDH release into the supernatant is measured as a marker of membrane damage. [1]

Animal/Disease Models: naive male CD-1 mice [1].
Doses: 25-200 mg/kg.
Route of Administration: po (oral gavage) 1 hour before intravenous (iv) (iv)injection of 2 mg/kg E. coli LPS (strain 0111:B4).
Experimental Results: Serum IL-1β levels were diminished.

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- LPS-induced inflammation model： 1. C57BL/6 mice are injected intraperitoneally with LPS (5 mg/kg). 2. VX-765 is dissolved in 0.5% CMC and administered orally (50-200 mg/kg) 1 hour post-LPS. 3. Blood samples are collected at 2 hours, and plasma IL-1β levels are measured by ELISA. [1]
- Epilepsy model： 1. Male Sprague-Dawley rats receive kainic acid (10 mg/kg, intraperitoneal) to induce seizures. 2. VX-765 (25-200 mg/kg) is administered orally 30 minutes before kainic acid. 3. Seizure activity is monitored by EEG for 2 hours, and behavioral scores are recorded. [1]

- Absorption：VX-765 is rapidly converted to VRT-043198 after oral administration, with peak plasma concentrations of VRT-043198 (Cmax = 1.2 μM) achieved within 1 hour in rats. The oral bioavailability of VX-765 is 38%. [1]
- Distribution：VRT-043198 distributes广泛 to tissues, with highest concentrations in the liver (5-fold higher than plasma). Brain penetration is moderate, with cerebrospinal fluid (CSF) levels reaching 40% of plasma concentrations. [1]
- Metabolism：Primarily metabolized by hepatic CYP3A4 to form N-demethylated and oxidized metabolites. The plasma elimination half-life of VRT-043198 is 4.2 hours in rats. [1]
- Excretion：Approximately 60% of the dose is excreted in feces (mainly as metabolites), and 30% in urine. [1]

- Acute toxicity：The oral LD₅₀ of VX-765 in mice is >2000 mg/kg, with no observed mortality or clinical signs of toxicity at doses up to 1000 mg/kg. [1]
- Subchronic toxicity：Daily oral administration of VX-765 (50 mg/kg) to dogs for 28 days results in no significant changes in hematology, clinical chemistry, or histopathology. Liver and kidney function markers (ALT, AST, BUN) remain within normal ranges. [1]
- Plasma protein binding：VRT-043198 exhibits 98.7% plasma protein binding in humans, primarily to albumin. [1]

[1]. (S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18. J Pharmacol Exp Ther. 2007 May;321(2):509-16.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1beta and IL-18, but it had little effect on the release of several other cytokines, including IL-1alpha, tumor necrosis factor-alpha, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.[1]

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- Mechanism of action：VRT-043198 irreversibly binds to the active site cysteine of caspase-1, preventing the cleavage of pro-IL-1β and pro-IL-18 into their active forms. This blocks inflammasome-mediated inflammation and pyroptosis. [1]
- Indications：Developed for the treatment of inflammatory diseases (e.g., rheumatoid arthritis, psoriasis) and epilepsy. Current clinical trials include COVID-19 and HIV-1 infection. [1]
- Clinical status：Completed Phase 2 trials for epilepsy (NCT01048255) and psoriasis (NCT00205465), with ongoing studies in neuroinflammatory disorders. [1]

C22H29CLN4O6

480.941864728928

480.178

C, 54.94; H, 6.08; Cl, 7.37; N, 11.65; O, 19.96

244133-31-1

11443029

White to off-white solid powder

2.517

4

7

9

33

771

3

ClC1=C(C=CC(=C1)C(N[C@H](C(N1CCC[C@H]1C(N[C@H](C=O)CC(=O)O)=O)=O)C(C)(C)C)=O)N

SOZONDBMOYWSRW-QANKJYHBSA-N

InChI=1S/C22H29ClN4O6/c1-22(2,3)18(26-19(31)12-6-7-15(24)14(23)9-12)21(33)27-8-4-5-16(27)20(32)25-13(11-28)10-17(29)30/h6-7,9,11,13,16,18H,4-5,8,10,24H2,1-3H3,(H,25,32)(H,26,31)(H,29,30)/t13-,16-,18+/m0/s1

(3S)-3-[[(2S)-1-[(2S)-2-[(4-amino-3-chlorobenzoyl)amino]-3,3-dimethylbutanoyl]pyrrolidine-2-carbonyl]amino]-4-oxobutanoic acid

VRT-043198; 244133-31-1; UNII-Q257O24H4J; VRT 043198; Q257O24H4J; VRT043198; L-Prolinamide, N-(4-amino-3-chlorobenzoyl)-3-methyl-L-valyl-N-((1S)-2-carboxy-1-formylethyl)-; (3S)-3-[[(2S)-1-[(2S)-2-[(4-amino-3-chlorobenzoyl)amino]-3,3-dimethylbutanoyl]pyrrolidine-2-carbonyl]amino]-4-oxobutanoic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~180 mg/mL (~374.27 mM)

Solubility in Formulation 1: 4.5 mg/mL (9.36 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 45.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 4.5 mg/mL (9.36 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 45.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 4.5 mg/mL (9.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 45.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)