2. Apoptosis
  3. Caspase
  4. Belnacasan

Belnacasan (VX-765) is an orally bioactive proagent of VRT-043198, which is a potent and selective inhibitor of IL-converting enzyme (ICE)/caspase-1 with Kis of 0.8 nM and less than 0.6 nM for caspase-1 and caspase-4, respectively. Belnacasan (VX-765) inhibits the release of LPS-induced IL-1β and IL-18 by human PBMCs with an IC50 of ~0.7 μM.

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Belnacasan Chemical Structure

Belnacasan Chemical Structure

CAS No. : 273404-37-8

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10 mM * 1 mL in DMSO
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Customer Review

Based on 111 publication(s) in Google Scholar

Belnacasan Related Antibodies

Top Publications Citing Use of Products

107 Publications Citing Use of MCE Belnacasan

WB

    Belnacasan purchased from MedChemExpress. Usage Cited in: J Leukoc Biol. 2019 Feb;105(2):401-410.  [Abstract]

    PMA-differentiated CASP4/5-deficient THP-1 cells are primed with 100 ng/mL LPS for 4 h, and are then left uninfected or are infected with S. Typhimurium (MOI 25, log-phase), in the presence of 25 μM VX765 to suppress cell death and cellular release of the ASC speck.

    Belnacasan purchased from MedChemExpress. Usage Cited in: Toxicology. 2018 Dec 1;410:26-40.  [Abstract]

    HUVECs are primed with 1 μg/mL LPS for 2 h, and then pretreated with or without 5 μM VX-765 for 1 h; following by treatment with Acrolein (50 μM) for 24 h. Western blot is performed to determine the expression of cleaved caspase-1, IL-1β and IL-18.

    Belnacasan purchased from MedChemExpress. Usage Cited in: Brain Behav Immun. 2017 Oct;65:99-110.  [Abstract]

    VX765 treatment successfully inhibits the caspase 1 activity and reduces nuclear TDP-43 and cleaved caspase-3 protein levels in the hippocampus of BDE-47-treated mice.

    Belnacasan purchased from MedChemExpress. Usage Cited in: Brain Behav Immun. 2016 Aug;56:175-86.  [Abstract]

    The effects of NLRP3 inflammasome inhibitor VX-765 on NF-κB and CD11b levels in Sham and OVX groups. Sham operation or bilateral ovariectomy is performed and then the mice are subcutaneously administrated with the chemicals as indicated for four weeks. (A) p-p65 protein level with representative protein bands presented on top of histogram; (B) CD11b protein level with representative protein bands presented on top of histogram.

    View All Caspase Isoform Specific Products:

    View All Isoforms
    Caspase 1 Caspase 2 Caspase 3 Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10 Caspase 12 Caspase Caspase 11 Caspase-13

    • Biological Activity

    • Protocol

    • Purity & Documentation

    • References

    • Customer Review

    Description

    Belnacasan (VX-765) is an orally bioactive proagent of VRT-043198, which is a potent and selective inhibitor of IL-converting enzyme (ICE)/caspase-1 with Kis of 0.8 nM and less than 0.6 nM for caspase-1 and caspase-4, respectively. Belnacasan (VX-765) inhibits the release of LPS-induced IL-1β and IL-18 by human PBMCs with an IC50 of ~0.7 μM[1][2].

    IC50 & Target[1]

    Caspase-1

     

    In Vivo

    Belnacasan reduces inflammatory response in murine models of inflammatory disease[1].
    Belnacasan (50-200 mg/kg) significantly reduces serum IL-1β levels by as much as 60%. 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 (25-100 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 (25-200 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].

    MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
    Clinical Trial
    Molecular Weight

    509.00

    Formula

    C24H33ClN4O6
    CAS No.
    Appearance

    Solid

    Color

    White to off-white

    SMILES

    ClC1=C(N)C=CC(C(N[C@@H](C(C)(C)C)C(N2[C@H](C(N[C@H]3CC(O[C@H]3OCC)=O)=O)CCC2)=O)=O)=C1
    Shipping

    Room temperature in continental US; may vary elsewhere.
    Storage
    Powder -20°C 3 years
    In solvent -80°C 2 years
    -20°C 1 year
    Solvent & Solubility
    In Vitro: 

    DMSO : 100 mg/mL (196.46 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

    H2O : 1 mg/mL (1.96 mM; ultrasonic and warming and heat to 60°C)

    Preparing
    Stock Solutions
    Concentration Solvent Mass 1 mg 5 mg 10 mg
    1 mM 1.9646 mL 9.8232 mL 19.6464 mL
    5 mM 0.3929 mL 1.9646 mL 3.9293 mL
    View the Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

    * Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

    • Molarity Calculator

    • Dilution Calculator

    Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

    Mass
    =
    Concentration
    ×
    Volume
    ×
    Molecular Weight *

    Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

    This equation is commonly abbreviated as: C1V1 = C2V2

    Concentration (start)

    C1

    ×
    Volume (start)

    V1

    =
    Concentration (final)

    C2

    ×
    Volume (final)

    V2

    In Vivo:

    Select the appropriate dissolution method based on your experimental animal and administration route.

    For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
    To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
    The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

    • Protocol 1

      Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

      Solubility: ≥ 2.5 mg/mL (4.91 mM); Clear solution

      This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

      Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
    • Protocol 2

      Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

      Solubility: ≥ 2.5 mg/mL (4.91 mM); Clear solution

      This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

      Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.

    For the following dissolution methods, please prepare the working solution directly. It is recommended to prepare fresh solutions and use them promptly within a short period of time.
    The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

    • Protocol 1

      Add each solvent one by one:  50% PEG300    50% Saline

      Solubility: 5 mg/mL (9.82 mM); Clear solution; Need ultrasonic

    • Protocol 2

      Add each solvent one by one:  15% Cremophor EL    85% Saline

      Solubility: 3.33 mg/mL (6.54 mM); Clear solution; Need ultrasonic

    In Vivo Dissolution Calculator
    Please enter the basic information of animal experiments:

    Dosage

    mg/kg

    Animal weight
    (per animal)

    g

    Dosing volume
    (per animal)

    μL

    Number of animals

    Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
    Please enter your animal formula composition:
    %
    DMSO +
    % 
    +
    %
    Tween-80 +
    %
    Saline
    Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
    The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
    Calculation results:
    Working solution concentration: mg/mL
    Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).
    The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
    Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
     If the continuous dosing period exceeds half a month, please choose this protocol carefully.
    Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
    Purity & Documentation

    Purity: 99.99%

    COA (250 KB) HNMR (228 KB) LCMS (339 KB) Elemental Analysis Report (107 KB)

    Handling Instructions (2659 KB)
    References
    Kinase Assay
    [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].

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.
    Cell Assay
    [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 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].

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.
    Animal Administration
    [2][3]

    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.

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.
    References

    Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

    Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
    H2O / DMSO 1 mM 1.9646 mL 9.8232 mL 19.6464 mL 49.1159 mL
    DMSO 5 mM 0.3929 mL 1.9646 mL 3.9293 mL 9.8232 mL
    10 mM 0.1965 mL 0.9823 mL 1.9646 mL 4.9116 mL
    15 mM 0.1310 mL 0.6549 mL 1.3098 mL 3.2744 mL
    20 mM 0.0982 mL 0.4912 mL 0.9823 mL 2.4558 mL
    25 mM 0.0786 mL 0.3929 mL 0.7859 mL 1.9646 mL
    30 mM 0.0655 mL 0.3274 mL 0.6549 mL 1.6372 mL
    40 mM 0.0491 mL 0.2456 mL 0.4912 mL 1.2279 mL
    50 mM 0.0393 mL 0.1965 mL 0.3929 mL 0.9823 mL
    60 mM 0.0327 mL 0.1637 mL 0.3274 mL 0.8186 mL
    80 mM 0.0246 mL 0.1228 mL 0.2456 mL 0.6139 mL
    100 mM 0.0196 mL 0.0982 mL 0.1965 mL 0.4912 mL

    * Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

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    Help & FAQs
    • Do most proteins show cross-species activity?

      Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

    Keywords:

    Belnacasan273404-37-8VX-765VX765VX 765CaspaseInhibitorinhibitorinhibit

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