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| Targets |
β1i (LMP2) immunoproteasome subunit (equipped with the reversible boronic acid warhead).
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| ln Vitro |
In splenocytes from wt or LMP7-deficient mice, treatment with ML604440 (300 nM; overnight) had an impact on the surface expression of H-2Kb [2]. ML604440 treatment (300 nM; 24 hours) did not substantially stain human PBMC or mice splenocytes. ML604440 (300 nM; 3 days) had no influence on the overall quantity of CD4+ T cells that produce IL-17A [2].
ML604440 (at 300 nM), alone, did not inhibit IL-6 secretion from LPS-stimulated mouse splenocytes or human PBMCs. When ML604440 (300 nM) was combined with the LMP7-selective inhibitor PRN1126 (300 nM), IL-6 secretion from LPS-stimulated mouse splenocytes and human PBMCs was significantly reduced to a level similar to that achieved with the dual inhibitor ONX 0914 (300 nM). This indicates that co-inhibition of LMP2 and LMP7 is required for suppressing cytokine production. ML604440 (300 nM), alone, did not affect the differentiation of naïve mouse CD4+ T cells into IL-17A-producing Th17 cells under polarizing conditions. When ML604440 (300 nM) was combined with PRN1126 (300 nM), Th17 cell differentiation was significantly suppressed, similar to the effect of ONX 0914 (300 nM). ML604440 (300 nM), alone, did not reduce the cell surface expression of the MHC-I molecule H-2Kb on wild-type mouse splenocytes. When ML604440 (300 nM) was combined with PRN1126 (300 nM), H-2Kb surface expression was significantly reduced, similar to the effect of ONX 0914 (300 nM). This reduction was not observed on LMP7-deficient splenocytes, confirming the specificity of the combined inhibition.[2] |
| ln Vivo |
LMP2 is stained in vivo by ML604440 treatment (ip; 10 mg/kg; once daily; 7 days) without causing appreciable alterations to the projection behind it [3].
In a dextran sulfate sodium (DSS)-induced colitis model, daily treatment with ML604440 (15 mg/kg, subcutaneously) alone did not protect mice from weight loss compared to vehicle-treated mice. However, co-administration of ML604440 (15 mg/kg) with PRN1126 (40 mg/kg) significantly reduced weight loss and colon shortening. In an experimental autoimmune encephalomyelitis (EAE) model, intermittent treatment (three times per week) with ML604440 (15 mg/kg, subcutaneously) alone, starting on the immunization day, did not delay disease onset or reduce severity compared to the vehicle group. In contrast, the combination of ML604440 (15 mg/kg) with PRN1126 (40 mg/kg) significantly delayed disease onset and reduced disease incidence.[2] |
| Cell Assay |
IL-6 Secretion Assay: Mouse splenocytes or human PBMCs were incubated with inhibitors (e.g., 300 nM ML604440, PRN1126, or their combination) or DMSO control and stimulated with LPS (3 µg/ml) overnight. After 20 hours, the concentration of IL-6 in the supernatant was determined by ELISA according to the manufacturer's protocol.[2]
Th17 Differentiation Assay: Magnetically purified murine CD4+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of Th17-polarizing cytokines (TGF-β and IL-6) and neutralizing antibodies against IL-4 and IFN-γ. Inhibitors (e.g., 300 nM ML604440, PRN1126, or their combination) or DMSO were added to the culture. After 3 days, cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours. Intracellular IL-17A expression in CD4+ cells was then analyzed by flow cytometry.[2] MHC-I Surface Expression Assay: Splenocytes from wild-type or LMP7-deficient mice were incubated overnight with inhibitors (e.g., 300 nM ML604440, PRN1126, or their combination) or DMSO. Cells were then stained with an anti-mouse H-2Kb-PE antibody after blocking Fc receptors. Stained cells were analyzed by flow cytometry to measure MHC-I surface expression levels.[2] |
| Animal Protocol |
Animal/Disease Models: Male C57BL/6J mice (6-8 weeks old) were injected with anti-platelet monoclonal antibodies [3]
Doses: 10 mg/kg Route of Administration: intraperitoneal (ip) injection; 10 mg/kg; one time/day; 7-day Experimental Results: LMP2 is inhibited in vivo. There was no significant improvement in platelet counts after immunizing mice with a monoclonal rat anti-mouse CD41 platelet antibody. For in vivo studies, ML604440 was dissolved in a vehicle containing 5% ethanol, 10% PEG300 in an aqueous solution of 20% (w/v) sulfobutylether-β-cyclodextrin and 10 mM sodium citrate (pH 6). It was administered subcutaneously (s.c.) at a dose of 15 mg/kg. DSS-Induced Colitis Model: Colitis was induced in 8-10-week-old mice by adding 3% DSS to drinking water for 5-7 days. Mice were treated daily with vehicle, ML604440 (15 mg/kg, s.c.), PRN1126 (40 mg/kg, s.c.), or a combination of both. Body weight was recorded daily. Colon length was measured at the endpoint. Experimental Autoimmune Encephalomyelitis (EAE) Model: C57BL/6 mice were immunized subcutaneously with MOG35-55 peptide in CFA and received pertussis toxin intraperitoneally on day 0 and intravenously on day 2. Mice were treated intermittently (three times per week) with vehicle, ML604440 (15 mg/kg, s.c.), PRN1126 (40 mg/kg, s.c.), or a combination of both, starting on the day of immunization. Clinical disease scores were assessed daily.[2] |
| Toxicity/Toxicokinetics |
The study indicated that after overnight incubation of spleen cells with LMP2 and LMP7 inhibitors (including ML604440), cell death (propidium iodide-positive cells) was not increased compared to the control group, suggesting that acute cytotoxicity did not exist at the tested concentration (300 nM) in this specific assay. [2]
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| References |
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| Additional Infomation |
ML604440 is a dipeptide borate inhibitor described as a selective LMP2 inhibitor. Unlike the epoxyketone inhibitor LU-001i, ML604440 does not cause the immunoproteasome subunit band to shift toward higher apparent molecular weights in Western blot analysis, possibly due to its reversible binding mechanism. This study showed that selective inhibition of LMP2 by ML604440 alone was insufficient to reduce MHC-I surface expression, inhibit IL-6 secretion, inhibit Th17 differentiation, or improve disease symptoms in mouse colitis and EAE models. Only when ML604440 was used in combination with the selective LMP7 inhibitor (PRN1126) did a synergistic effect, including significant therapeutic effects, occur, highlighting the need for simultaneous inhibition of both LMP2 and LMP7 subunits to exert broad anti-inflammatory activity in autoimmune diseases. [2]
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| Molecular Formula |
C17H24BF3N2O4
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|---|---|
| Molecular Weight |
388.189675331116
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| Exact Mass |
388.178
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| CAS # |
1140517-08-3
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| PubChem CID |
57655545
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| Appearance |
White to yellow solid powder
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
27
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| Complexity |
527
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| Defined Atom Stereocenter Count |
1
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| SMILES |
FC(C1C=CC=CC=1C(NC(C)(C)C(N[C@H](B(O)O)CC(C)C)=O)=O)(F)F
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| InChi Key |
IRZZVDHGNHGNAI-ZDUSSCGKSA-N
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| InChi Code |
InChI=1S/C17H24BF3N2O4/c1-10(2)9-13(18(26)27)22-15(25)16(3,4)23-14(24)11-7-5-6-8-12(11)17(19,20)21/h5-8,10,13,26-27H,9H2,1-4H3,(H,22,25)(H,23,24)/t13-/m0/s1
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| Chemical Name |
[(1R)-3-methyl-1-[[2-methyl-2-[[2-(trifluoromethyl)benzoyl]amino]propanoyl]amino]butyl]boronic acid
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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 : ~100 mg/mL (~257.61 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.44 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 (6.44 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 (6.44 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 | 2.5761 mL | 12.8803 mL | 25.7606 mL | |
| 5 mM | 0.5152 mL | 2.5761 mL | 5.1521 mL | |
| 10 mM | 0.2576 mL | 1.2880 mL | 2.5761 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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