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 targets caspase-1 (interleukin-converting enzyme, ICE) and caspase-4. It is a selective inhibitor of the caspase-1 subfamily, displaying Ki values of 0.8 nM for caspase-1 and 0.6 nM for caspase-4. It inhibits the release of interleukin (IL)-1β and IL-18, while showing minimal effect on the release of other cytokines such as IL-1α, TNF-α, IL-6, and IL-8.

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]

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In vitro, VRT-043198 is a potent and selective inhibitor of caspase-1 and caspase-4. It has Ki values of 0.8 nM for caspase-1 and 0.6 nM for caspase-4. It specifically inhibits the release of IL-1β and IL-18.

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]

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In vivo activity data for VRT-043198 are limited, as it is the active metabolite of VX-765. VX-765 has been studied for its anti-inflammatory effects in various disease models. As a blood-brain barrier permeable caspase-1 inhibitor, VRT-043198 has potential for studying neuroinflammation.

- 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.

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In vitro enzyme inhibition assays for VRT-043198 are performed using purified caspase-1 and caspase-4 enzymes. Enzyme activity is measured using fluorogenic peptide substrates that are cleaved by caspases. The compound is incubated with the enzyme and substrate, and the inhibition of substrate cleavage is quantified. Ki values (0.8 nM for caspase-1, 0.6 nM for caspase-4) are calculated from inhibition curves.

- 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]

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In vitro cellular assays for VRT-043198 are performed using immune cells or cell lines. Cells are stimulated to induce IL-1β and IL-18 production (e.g., with LPS) and treated with the compound. Cytokine levels in the culture medium are measured by ELISA. The compound's ability to inhibit IL-1β and IL-18 release is quantified.

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]

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In vivo animal experiments for VRT-043198 are conducted using the prodrug VX-765, which is metabolized to VRT-043198. A typical protocol involves administration of VX-765 via oral or intraperitoneal routes to animal models of inflammatory diseases. Efficacy is assessed by measuring IL-1β and IL-18 levels, inflammation markers, and disease severity.

Absorption: VX-765 is rapidly converted to VRT-043198 after oral administration, and the peak plasma concentration (Cmax = 1.2 μM) of VRT-043198 in rats is reached within 1 hour. The oral bioavailability of VX-765 is 38%. [1]
- Distribution: VRT-043198 is widely distributed in tissues, with the highest concentration in the liver (5 times higher than the plasma concentration). It has moderate brain permeability, and the concentration in cerebrospinal fluid (CSF) can reach 40% of the plasma concentration. [1]
- Metabolism: It is mainly metabolized by CYP3A4 in the liver, producing N-demethylated and oxidative metabolites. The plasma elimination half-life of VRT-043198 in rats is 4.2 hours. [1]
- Excretion: Approximately 60% of the dose is excreted in feces (mainly in the form of metabolites), and 30% is excreted in urine. [1]

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VRT-043198 has a molecular weight of 496.94 and molecular formula C₂₂H₂₉ClN₄O₆. It is a blood-brain barrier permeable compound. It is the active metabolite of VX-765 (Belnacasan). Further PK details are available from the manufacturer's data sheets.

Acute toxicity: The oral LD₅₀ of VX-765 in mice was >2000 mg/kg, and no death or clinical signs of toxicity were observed at doses up to 1000 mg/kg. [1]
- Subchronic toxicity: No significant changes in hematology, clinical chemistry or histopathology were observed after dogs were given oral administration of VX-765 (50 mg/kg) daily for 28 consecutive days. Liver and kidney function indicators (ALT, AST, BUN) were all within the normal range. [1]
- Plasma protein binding rate: The plasma protein binding rate of VRT-043198 in humans was 98.7%, mainly bound to albumin. [1]

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Toxicological data for VRT-043198 are not extensively reported. As a research-use compound, its safety profile has not been formally evaluated in comprehensive preclinical toxicology studies. The compound is intended for research purposes only and is not approved for human therapeutic use. Standard laboratory safety precautions should be followed when handling this compound.

[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-chlorophenyl)-formyl]-amino}-3,3-dimethyl-butyryl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydrofuran-3-yl)-amide (VX-765) is an oral prodrug for the absorption of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-formyl]-amino}-3,3-dimethyl-butyryl)-pyrrolidine-2-yl]-formyl}-amino)-4-oxo-butyric acid (VRT-043198), which is a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits selectivity of up to 100 to 10,000-fold for other caspases -3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effect of VRT-043198 on the release of cytokines from monocytes in vitro and the in vivo effects of orally administered VX-765 in various animal models. In peripheral blood monocytes and whole blood cultures from healthy subjects stimulated with the bacterial product, VRT-043198 inhibited the release of interleukin (IL)-1β and IL-18, but had little effect on the release of several other cytokines, including IL-1α, tumor necrosis factor-α, IL-6, and IL-8. Conversely, VRT-043198 showed little or no observable activity in apoptosis models and did not affect the proliferation of activated primary T cells or T cell lines. VX-765 is efficiently converted to VRT-043198 after oral administration and inhibits lipopolysaccharide-induced cytokine secretion. In addition, VX-765 can reduce disease severity and expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor that can be used to treat inflammatory diseases. [1]

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- Mechanism of action: VRT-043198 binds irreversibly to the cysteine residue at the active site of caspase-1, preventing pro-IL-1β and pro-IL-18 from cleaving into their active forms. This blocks inflammasome-mediated inflammation and pyroptosis. [1]
- Indications: It has been developed for the treatment of inflammatory diseases such as rheumatoid arthritis, psoriasis and epilepsy. Ongoing clinical trials include COVID-19 and HIV-1 infection. [1]
- Clinical status: Phase II clinical trials for epilepsy (NCT01048255) and psoriasis (NCT00205465) have been completed, and studies on neuroinflammatory diseases are currently underway. [1]

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VRT-043198 is the active metabolite of VX-765 (Belnacasan). It is a potent, selective, and blood-brain barrier permeable inhibitor of caspase-1 and caspase-4 with Ki values of 0.8 nM and 0.6 nM, respectively. It inhibits IL-1β and IL-18 release. This product is for research use only.

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.)