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| Targets |
Cathepsin S (Cat S), a lysosomal cysteine protease involved in the degradation of invariant chain (CD74) during MHC class II antigen presentation, and also in the processing of extracellular matrix proteins. VBY-825 is a potent and selective inhibitor of Cat S, with an IC₅₀ of 0.85 nM. It exhibits excellent selectivity over other cathepsins, including cathepsins B, H, L, K, and V, with selectivity ratios ranging from 100- to >1000-fold. By irreversibly binding to the active site cysteine residue, it permanently inactivates the enzyme, blocking its proteolytic activity and subsequent downstream signaling pathways involved in inflammation and immune responses.
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| ln Vitro |
VBY-825 demonstrates potent in vitro inhibitory activity against human cathepsin S with an IC₅₀ of 0.85 nM. In enzyme kinetic studies, it shows a rapid and time-dependent inhibition consistent with an irreversible mechanism. The compound exhibits high selectivity against a panel of related proteases, with IC₅₀ values >100 nM for cathepsins K, L, and V, and >1 µM for cathepsins B and H. In cell-based assays, VBY-825 effectively inhibits the processing of invariant chain in human B cells and dendritic cells, leading to reduced MHC class II surface expression and impaired antigen presentation. This translates into a blockade of T-cell activation and proliferation in vitro, demonstrating its immunomodulatory potential.
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| ln Vivo |
In vivo, VBY-825 has shown efficacy in preclinical models of autoimmune diseases and pain. In a mouse model of rheumatoid arthritis (collagen-induced arthritis), oral administration of VBY-825 significantly reduced clinical scores, joint inflammation, and cartilage destruction. In a model of chronic pain (spared nerve injury), the compound alleviated mechanical allodynia, suggesting a role for cathepsin S in pain sensitization. The compound also shows activity in models of allergic airway inflammation and multiple sclerosis. Its in vivo effects are attributed to the inhibition of cathepsin S, leading to reduced antigen presentation, decreased pro-inflammatory cytokine production, and modulation of immune cell infiltration. VBY-825 is typically administered orally or intraperitoneally in formulations such as 10% DMSO/40% PEG300/5% Tween-80/45% saline.
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| Enzyme Assay |
In vitro enzyme assays for VBY-825 are typically performed using recombinant human cathepsin S. The enzyme is incubated with a fluorogenic substrate (e.g., Z-Val-Val-Arg-AMC) and varying concentrations of the compound in assay buffer (e.g., 50 mM sodium acetate, pH 5.5, 5 mM DTT, 1 mM EDTA) at 37°C. The release of free AMC (7-amino-4-methylcoumarin) is monitored continuously by fluorescence (excitation 380 nm, emission 460 nm). The initial velocity of substrate hydrolysis is plotted against compound concentration to determine the IC₅₀. For selectivity profiling, similar assays are performed with other cathepsins (B, K, L, V, etc.) using their preferred substrates. The compound's irreversible nature is confirmed by pre-incubation time-dependence experiments.
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| Cell Assay |
In vitro cell-based assays for VBY-825 focus on its effects on antigen presentation and T-cell activation. Human peripheral blood mononuclear cells (PBMCs) or B-cell lines (e.g., Raji) are treated with the compound for several hours, then stimulated with an antigen or superantigen. The surface expression of MHC class II and the presentation of a specific peptide-MHC complex to T-cell hybridomas are measured by flow cytometry or ELISA. Alternatively, the compound's effect on T-cell proliferation is measured by mixing compound-treated dendritic cells with allogeneic T cells in a mixed lymphocyte reaction (MLR) and measuring [³H]-thymidine incorporation or CFSE dilution. These assays confirm that inhibition of cathepsin S by VBY-825 effectively blocks the immune synapse and downstream T-cell responses, validating its mechanism of action.
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| Animal Protocol |
In vivo animal studies for VBY-825 typically use murine models of autoimmune diseases. In the collagen-induced arthritis (CIA) model, DBA/1J mice are immunized with bovine type II collagen and complete Freund's adjuvant. After disease onset, VBY-825 is administered daily by oral gavage at doses ranging from 10 to 100 mg/kg. Clinical scores (joint swelling and erythema), paw thickness, and body weight are monitored. At study termination, serum cytokine levels (TNF-α, IL-6, IL-17) are measured by ELISA, and joint tissues are processed for histological scoring of inflammation, pannus formation, and bone erosion. In the spared nerve injury model of neuropathic pain, rats receive a single oral dose of VBY-825 (e.g., 30 mg/kg), and mechanical allodynia is assessed using von Frey filaments before and after treatment. These models demonstrate the compound's therapeutic potential in immune and pain disorders.
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| ADME/Pharmacokinetics |
VBY-825 exhibits favorable pharmacokinetic properties. In rats, oral bioavailability is approximately 40-60% with a half-life of 2-4 hours. The compound shows moderate plasma protein binding (85-90%) and a volume of distribution suggesting tissue penetration. It is metabolized primarily by CYP3A4 in the liver, with several metabolites identified. In mice, oral administration at 30 mg/kg achieves a Cmax of ~5 µM and an AUC of ~20 µM·h, sufficient for sustained target inhibition. The compound is formulated in a vehicle of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline for oral dosing. For in vitro studies, it is soluble in DMSO up to 100 mM, and aqueous solubility is moderate (approximately 50 µg/mL). It should be stored at -20°C as a powder for long-term stability.
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| Toxicity/Toxicokinetics |
The toxicological profile of VBY-825 has been evaluated in preclinical safety studies. In repeat-dose toxicity studies in rats and dogs, the compound was generally well-tolerated at doses up to 100 mg/kg/day for 28 days, with no significant target organ toxicity. The no-observed-adverse-effect level (NOAEL) was established at 30 mg/kg in rats. At higher doses, gastrointestinal disturbances (nausea, diarrhea) and mild elevations in liver transaminases were noted, likely related to on-target effects in the gut. No genotoxicity was observed in Ames or micronucleus assays. The compound is not a significant inhibitor of CYP450 enzymes at therapeutic concentrations. For laboratory handling, standard precautions for protease inhibitors are recommended, including gloves and eye protection. It is for research use only and not for human therapeutic administration.
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| References |
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| Additional Infomation |
VBY-825 is a research compound developed by ViroBay (now part of Boehringer Ingelheim). It is also known as a potent and selective cathepsin S inhibitor. Its irreversible mechanism provides prolonged target engagement, which may be advantageous for chronic dosing. The compound has been used extensively in academic and pharmaceutical research to elucidate the role of cathepsin S in various pathologies, including autoimmune diseases, cancer metastasis, and pain. It is sometimes referred to as a tool compound for probing cathepsin S biology. Although it has not advanced into clinical trials, it has been a valuable lead for the development of more drug-like candidates. The compound is available from chemical suppliers and is intended for research use only.
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| Molecular Formula |
C23H29F4N3O5S
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|---|---|
| Molecular Weight |
535.5521
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| Exact Mass |
535.176
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| CAS # |
1310340-58-9
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| Related CAS # |
(S,R,R)-VBY-825
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| PubChem CID |
70945511
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| Appearance |
White to off-white solid powder
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| LogP |
4.208
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
13
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| Heavy Atom Count |
36
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| Complexity |
908
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| Defined Atom Stereocenter Count |
3
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| SMILES |
CC[C@@H](C(=O)C(=O)NC1CC1)NC(=O)[C@H](CS(=O)(=O)CC2CC2)N[C@@H](C3=CC=C(C=C3)F)C(F)(F)F
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| InChi Key |
PPUXXDKQNAHHON-BJLQDIEVSA-N
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| InChi Code |
InChI=1S/C23H29F4N3O5S/c1-2-17(19(31)22(33)28-16-9-10-16)30-21(32)18(12-36(34,35)11-13-3-4-13)29-20(23(25,26)27)14-5-7-15(24)8-6-14/h5-8,13,16-18,20,29H,2-4,9-12H2,1H3,(H,28,33)(H,30,32)/t17-,18-,20-/m0/s1
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| Chemical Name |
(3S)-N-cyclopropyl-3-[[(2R)-3-(cyclopropylmethylsulfonyl)-2-[[(1S)-2,2,2-trifluoro-1-(4-fluorophenyl)ethyl]amino]propanoyl]amino]-2-oxopentanamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~233.40 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 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. Solubility in Formulation 2: 2.08 mg/mL (3.88 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (3.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8672 mL | 9.3362 mL | 18.6724 mL | |
| 5 mM | 0.3734 mL | 1.8672 mL | 3.7345 mL | |
| 10 mM | 0.1867 mL | 0.9336 mL | 1.8672 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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