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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
ONX-0914 TFA specifically targets the LMP7 (beta5i) subunit of the immunoproteasome. The immunoproteasome (composed of beta1i, beta2i, and beta5i/LMP7) is responsible for generating peptides for presentation by MHC class I molecules. Its activity is crucial for the production of pro-inflammatory cytokines, including IL-23, TNF-alpha, and IL-6, and for the activation and proliferation of Th17 cells. By irreversibly binding to the active site of LMP7, ONX-0914 inhibits its chymotrypsin-like proteolytic activity. This leads to reduced processing of antigenic peptides, decreased inflammatory signaling, and suppression of autoimmune responses. The compound is selective for LMP7 over the constitutive proteasome subunits beta1, beta2, and beta5.
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| ln Vitro |
ONX-0914 suppresses the presentation of antigen specific to LMP7. ONX-0914 inhibits T cell development and the generation of cytokines by mice splenocytes[1].
In vitro, ONX-0914 TFA blocks the production of pro-inflammatory cytokines from immune cells. In mouse splenocytes stimulated with anti-CD3/CD28 antibodies, ONX-0914 (1-10 microM) inhibits the production of IL-23, IL-6, and TNF-alpha in a dose-dependent manner. The compound also suppresses the differentiation of naive CD4+ T cells into Th17 cells, as measured by reduced IL-17A production in the culture supernatant. The IC50 for inhibition of LMP7 enzymatic activity in cell lysates is in the sub-micromolar to low micromolar range. In human peripheral blood mononuclear cells (PBMCs), ONX-0914 (5-20 microM) reduces cytokine secretion following stimulation with LPS or T cell activators. It exhibits low off-target activity against other proteasome subunits. |
| ln Vivo |
OnX-0914 (2-10 mg/kg; intraperitoneally; on days 4, 6, and 8) ameliorates mouse arthritis[1]. The T and B cell–dependent CIA (collagen-induced arthritis) model likewise experienced a quick therapeutic response when treated with ONX-0914 (2, 6 and 10 mg per kg body weight on days 25, 27, 29, 31, and 33; iv)[1].
In vivo, ONX-0914 TFA has demonstrated efficacy in preclinical models of autoimmune and inflammatory diseases. In the collagen-induced arthritis (CIA) model in mice, oral or intraperitoneal administration of ONX-0914 (10-30 mg/kg/day) attenuates the progression of experimental arthritis, reducing joint swelling, histological damage (synovitis, cartilage erosion, bone destruction), and the incidence of disease. The compound reduces serum levels of pro-inflammatory cytokines and autoantibodies. In models of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), ONX-0914 treatment delays disease onset and reduces clinical severity. Inflammatory bowel disease (IBD) models also show benefit. The compound is noncompetitive and irreversible, leading to sustained target inhibition. |
| Enzyme Assay |
Cell-free assays are used to measure LMP7 enzymatic activity. Purified immunoproteasome complexes (containing LMP7, LMP2, and MECL-1) or recombinant LMP7 are incubated with a fluorogenic peptide substrate specific for the chymotrypsin-like activity (e.g., Suc-LLVY-AMC, 50 microM) in assay buffer (25 mM HEPES, pH 7.5, 0.5 mM EDTA, 0.01% SDS, and 0.01% sodium azide). Varying concentrations of ONX-0914 TFA (0.001-100 microM) are added to the reaction mixture and pre-incubated with the enzyme for 15-30 minutes at 37degC. The reaction is initiated by adding the substrate and monitored continuously for 60-90 minutes using a fluorescence plate reader (ex/em 380/460 nm). The IC50 is calculated from the dose-response curve. For the mycobacterial proteasome, the Ki is 5.2 microM. To confirm irreversibility, a dilution or dialysis experiment can be performed.
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| Cell Assay |
A cell-based assay uses mouse splenocytes or human PBMCs. For Th17 differentiation, naive CD4+ T cells are isolated from mouse spleens using magnetic bead separation. Cells are seeded in 96-well plates (1×10⁵ cells/well) coated with anti-CD3 (10 microg/mL) and soluble anti-CD28 (2 microg/mL) in the presence of TGF-beta (5 ng/mL), IL-6 (20 ng/mL), and anti-IFN-gamma (10 microg/mL) to promote Th17 differentiation. ONX-0914 TFA (0.1-10 microM) is added at the time of culture initiation. After 72-96 hours, IL-17A levels in the culture supernatant are measured by ELISA. For cytokine secretion assays, splenocytes (2×10⁵ cells/well) are stimulated with anti-CD3/CD28 for 48 hours with or without ONX-0914, and IL-23, IL-6, and TNF-alpha are measured by ELISA. For cell viability, an MTT assay is performed to exclude direct cytotoxicity.
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| Animal Protocol |
Animal/Disease Models: Collagen antibody-induced arthritis (CAIA, Arthritis was induced in balb/c (Bagg ALBino) mouse with antibodies specific for type II collagen (mAb) and endotoxin)[1]
Doses: 2, 6 or 10 mg per kg body weight Route of Administration: Iv; treated on days 4, 6 and 8 Experimental Results: Blocked disease progression in a dose-dependent manner and completely ameliorated visible signs of disease at the highest dose. The collagen-induced arthritis (CIA) model is used in male DBA/1J mice (8-10 weeks). Mice are immunized intradermally at the base of the tail with 100 microg of bovine type II collagen (CII) emulsified 1:1 in Complete Freund‘s Adjuvant (CFA). On day 21, a booster immunization with 100 microg CII in Incomplete Freund‘s Adjuvant (IFA) is administered. ONX-0914 TFA (10-30 mg/kg) is formulated in vehicle (e.g., 10% DMSO, 5% Tween-80, 85% saline) and administered by oral gavage or intraperitoneal injection daily from day 21 to day 42 (or until study end). Clinical scores of arthritis are assessed three times weekly: 0 = normal; 1 = mild swelling and erythema of digit or ankle; 2 = moderate swelling and erythema of entire paw; 3 = severe swelling and erythema of entire paw; 4 = ankylosis with deformity. Paw thickness is measured with calipers. Histopathology of hind paws (after euthanasia) is scored for inflammation, pannus formation, cartilage erosion, and bone resorption. Serum levels of anti-CII antibodies and cytokines (IL-6, TNF-alpha, IL-17) are measured by ELISA. The EAE model in C57BL/6 mice immunized with MOG35-55 peptide is also used. ONX-0914 reduces clinical severity of EAE. |
| ADME/Pharmacokinetics |
The TFA salt form has a molecular weight of 571.66 g/mol (free base). The lyophilized powder should be stored at -20degC in a sealed container, protected from light and moisture, where it is stable for up to 3 years. For solution storage, prepare 10-50 mM stocks in DMSO and store at -80degC for up to 6 months and at -20degC for up to 1 month. The compound is soluble in DMSO (10-20 mg/mL). For in vivo oral administration, it can be formulated in 10% DMSO + 5% Tween-80 + 85% saline, 0.5% methylcellulose, or other suitable vehicles. For intraperitoneal injection, the same vehicle can be used. The TFA counterion enhances solubility. The compound is a selective LMP7 inhibitor.
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| Toxicity/Toxicokinetics |
This product is for research use only and is not for human therapeutic use. No specific toxicity data is available for ONX-0914 TFA. In preclinical studies of collagen-induced arthritis, mice treated with ONX-0914 at doses up to 30 mg/kg/day for 3-4 weeks showed no significant weight loss, mortality, or overt signs of toxicity (changes in activity, fur, or behavior). The immunoproteasome is primarily expressed in immune cells, and its inhibition is expected to be well-tolerated because constitutive proteasome function remains intact. However, long-term inhibition may impair immune function, including increased susceptibility to infections. Standard laboratory safety practices (gloves, lab coat, safety glasses) should be followed. Avoid inhalation of powder. The compound is not an FDA-approved drug.
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| References |
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| Additional Infomation |
The immunoproteasome is a distinct isoform of the proteasome that is induced by inflammatory cytokines. It plays an important role in the generation of MHC class I epitopes, immune cell activation, and autoimmune diseases. LMP7 (beta5i) is one of three catalytic subunits of the immunoproteasome. ONX-0914 is a selective LMP7 inhibitor that has been used extensively as a chemical probe to dissect the role of the immunoproteasome in inflammation and autoimmunity. The compound has shown activity in models of arthritis, multiple sclerosis, inflammatory bowel disease, and lupus. The noncompetitive, irreversible mode of action ensures sustained target engagement. The parent non-deuterated compound is ONX-0914 (PR-957). This compound is not a clinically approved drug. Supplier information must not be included.
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| Molecular Formula |
C33H41F3N4O9
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| Molecular Weight |
694.70
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| Related CAS # |
ONX-0914;960374-59-8
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| Appearance |
White to off-white solid powder
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 :~100 mg/mL (~143.95 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.60 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (3.60 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.60 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 | 1.4395 mL | 7.1974 mL | 14.3947 mL | |
| 5 mM | 0.2879 mL | 1.4395 mL | 2.8789 mL | |
| 10 mM | 0.1439 mL | 0.7197 mL | 1.4395 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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