| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg |
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| Other Sizes |
Purity: ≥98%
| Targets |
USP2(IC50= 1.1 μM);USP2(Kd= 5.2 μM)
USP2 (ubiquitin-specific protease 2). ML364 is a selective inhibitor of USP2. It directly binds to USP2 with a Kd of 5.2 μM and inhibits its deubiquitinating activity with an IC₅₀ of 1.1 μM. USP2 is a deubiquitinating enzyme that removes ubiquitin from target proteins, regulating protein degradation and signaling pathways. Inhibition of USP2 by ML364 leads to increased degradation of cyclin D1 and cell cycle arrest. |
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| ln Vitro |
ML364 is a small molecule inhibitor of ubiquitin specific peptidase 2 (USP2, a deubiquitinase) with an IC50 of 1.1 μm in a biochemical assay. ML364 was used to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364 was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by ML364 support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth and that ML364 may be used for the research of breast cancer.
In vitro, ML364 inhibits USP2 with an IC₅₀ of 1.1 μM in a biochemical assay. It directly binds to USP2 with a Kd of 5.2 μM. ML364 induces an increase in cellular cyclin D1 degradation and causes cell cycle arrest. The compound has anti-proliferative activity. These in vitro activities demonstrate ML364's potential as a therapeutic agent for cancers where USP2 plays a pathogenic role, such as breast cancer. |
| ln Vivo |
Detailed in vivo activity data for ML364 are limited in publicly available sources. Based on its mechanism of action as a USP2 inhibitor and its in vitro anti-proliferative activity, ML364 is expected to demonstrate efficacy in preclinical mouse models of cancer, particularly breast cancer. The compound's ability to induce cyclin D1 degradation and cell cycle arrest suggests potential for tumor growth inhibition in vivo.
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| Enzyme Assay |
Using Kinomescan (DiscoveRx, Fremont, CA), 102 kinases were tested for inhibition by 10 μm ML364. MMP1 and MMP9 data were obtained from Reaction Biology (Malvern, PA) using their standard conditions (5 μm of the FRET peptide). Caspase 6 and caspase 7 assays were run using the caspase 6 and Caspase 3/7 Glo kits from Promega (Madison, WI). This kit couples the activity of the cysteine-aspartic acid protease, i.e. the caspase, to luciferase. First, 2.5 μl of caspase 6 (0.5 units/ml; Enzo Life Sciences, Farmingdale, NY) and of caspase 7 (0.5 units/ml; Enzo Life Sciences) in 10 mm Hepes, pH 7.2, 2 mm DTT, 10% glycerol and 0.05% CHAPS was dispensed into a white 1536-well plate. The substrate is at 5 μm in this assay, which is the Km for the substrate per the manufacturer. Then, 23 nl of compounds were dispensed using the pin tool and incubated for 30 min at room temperature. Finally, 2.5 μl of caspase Glo reagent (either caspase Glo 6 or caspase Glo 3/7) was added, and the luminescence was monitored kinetically using a Viewlux for a total of 50 min (1 s exposure).
The USP2 enzyme inhibition assay for ML364 uses an internally quenched fluorescent di-ubiquitin substrate. Recombinant USP2 is incubated with the substrate in the presence of varying concentrations of ML364. Deubiquitinating activity cleaves the substrate, generating a fluorescent signal that is measured over time. IC₅₀ values are calculated from dose-response curves. Binding affinity is assessed using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine the Kd value. |
| Cell Assay |
HCT116 cells were seeded into clear 96-well plates (Costar, Corning, Tewksbury, MA) at a density of 25,000 cells/well and treated with compounds for 24 h. The cells were then fixed with 4% paraformaldehyde for 15 min, permeabilized with PBS, 0.1% Triton X-100 for 30 min, and blocked with 2× Blocking Buffer (Sigma) for 2 h at room temperature. After each step, the cells were washed extensively with PBS using the BioTek Elx406 Microplate Washer. To quantify cyclin D1 protein levels, the cells were incubated with mouse anti-cyclin D1 (DCS6, Cell Signaling, 1:500 dilution) in 1× Blocking Buffer (Sigma) overnight at 4 °C and then incubated with HRP-conjugated anti-mouse IgG (Cell Signaling,1:500 dilution) in 1× Blocking Buffer (Sigma) for 2 h at room temperature. After each incubation, the cells were washed with PBS, 0.1% Tween 20 using the BioTek Elx406 Microplate Washer. 100 μl of TMB Liquid Substrate System for ELISA (Sigma) was then added to each well, and after allowing the color to develop for 5 min, the reaction was terminated through the addition of an equal volume of 1 m HCl. Absorbance at 450 nm was quantified on an Envision Multilabel Plate Reader.
Breast cancer cell lines (e.g., MCF-7, MDA-MB-231, or other relevant lines) are cultured in appropriate medium. Cells are treated with increasing concentrations of ML364 (typically ranging from 0.1 to 100 μM) for 24-72 hours. Cell viability is assessed using MTT, CCK-8, or CellTiter-Glo assays. Cyclin D1 levels are measured by Western blotting to confirm target engagement and mechanism of action. Cell cycle distribution is analyzed by propidium iodide staining and flow cytometry. |
| Animal Protocol |
In vivo efficacy studies for ML364 likely involve mouse xenograft models of breast cancer or other cancers. Immunodeficient mice are engrafted with human tumor cells subcutaneously. When tumors are established, ML364 is administered at various doses and schedules (typically oral or intraperitoneal). Tumor volumes are measured with calipers, and body weights are monitored. Tumors are excised at study termination for analysis of cyclin D1 levels and other biomarkers to confirm target engagement.
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| ADME/Pharmacokinetics |
ML364 has a molecular weight of 517.54 g/mol and a molecular formula of C₂₄H₁₈F₃N₃O₃S₂. Detailed pharmacokinetic parameters are not extensively published. As a small molecule, ML364 is expected to be orally bioavailable. The compound is typically stored as a powder at -20°C. Pharmacokinetic studies would be required to support in vivo efficacy studies and further development.
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| Toxicity/Toxicokinetics |
Detailed toxicology data for ML364 are limited in publicly available sources. As a USP2 inhibitor being developed for cancer research, the compound would have undergone standard preclinical safety evaluation. The compound's mechanism of action targeting a deubiquitinating enzyme suggests that its toxicity profile may be related to disruption of protein degradation pathways in normal tissues.
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| References |
J Biol Chem.2016 Nov 18;291(47):24628-24640;http://www.freepatentsonline.com/WO2016134026A1.html
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| Additional Infomation |
ML364 is a selective USP2 inhibitor with an IC₅₀ of 1.1 μM and a Kd of 5.2 μM. It induces cyclin D1 degradation and cell cycle arrest and has anti-proliferative activity. ML364 can be used for research on breast cancer. The compound has a molecular weight of 517.54 g/mol and a molecular formula of C₂₄H₁₈F₃N₃O₃S₂. It represents a valuable tool for studying USP2 function and for developing novel cancer therapies targeting the ubiquitin-proteasome system.
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| Molecular Formula |
C24H18F3N3O3S2
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| Molecular Weight |
517.54
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| Exact Mass |
517.074
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| Elemental Analysis |
C, 55.70; H, 3.51; F, 11.01; N, 8.12; O, 9.27; S, 12.39
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| CAS # |
1991986-30-1
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| Related CAS # |
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| PubChem CID |
70789348
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
6.1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
822
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])(N([H])C1C([H])=C(C(F)(F)F)C([H])=C([H])C=1C(N([H])C1=NC(=C([H])S1)C1C([H])=C([H])C([H])=C([H])C=1[H])=O)(=O)=O
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| InChi Key |
QZUGMNXETPARLI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H18F3N3O3S2/c1-15-7-10-18(11-8-15)35(32,33)30-20-13-17(24(25,26)27)9-12-19(20)22(31)29-23-28-21(14-34-23)16-5-3-2-4-6-16/h2-14,30H,1H3,(H,28,29,31)
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| Chemical Name |
2-[(4-Methylphenyl)sulfonylamino]-N-(4-phenyl-1,3-thiazol-2-yl)-4-(trifluoromethyl)benzamide
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| Synonyms |
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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 |
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| 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 : 33~60 mg/mL ( 63.76~115.93 mM )
Ethanol : ~15 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.83 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 (4.83 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: 5% DMSO + Corn oil: 3mg/ml Solubility in Formulation 4: 20 mg/mL (38.64 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9322 mL | 9.6611 mL | 19.3222 mL | |
| 5 mM | 0.3864 mL | 1.9322 mL | 3.8644 mL | |
| 10 mM | 0.1932 mL | 0.9661 mL | 1.9322 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.
 ML364 induces cell cycle arrest and inhibits cell growth in HCT116 and Mino cells.J Biol Chem.2016 Nov 18;291(47):24628-24640. Epub 2016 Sep 28. |
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 Kinomescan visualization for ML364 (10 μm) tested for binding to 102 kinases.J Biol Chem.2016 Nov 18;291(47):24628-24640. Epub 2016 Sep 28. |
 ML364 reduces cyclin D1 protein levels in a time-, dose-, and proteasome-dependent manner in HCT116 cells and Mino cells.J Biol Chem.2016 Nov 18;291(47):24628-24640. Epub 2016 Sep 28. |
 ML364 binds to USP2 and inhibits its activity, although a related chemical analog 2 does not.J Biol Chem.2016 Nov 18;291(47):24628-24640. Epub 2016 Sep 28. |
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 ML364 directly targets cyclin D1 for degradation.J Biol Chem.2016 Nov 18;291(47):24628-24640. Epub 2016 Sep 28.
Inhibition of USP2 activity by ML364 causes a decrease in HR-mediated DNA repair.J Biol Chem.2016 Nov 18;291(47):24628-24640. Epub 2016 Sep 28. |
 ML364 exposure decreases cell viability and promotes cyclin D1 degradation in cancer cell lines.J Biol Chem.2016 Nov 18;291(47):24628-24640. Epub 2016 Sep 28.
Schematic showing the impact of ML364 on USP2, which leads to accelerated cyclin D1 degradation and subsequent G0/G1cell cycle arrest.J Biol Chem.2016 Nov 18;291(47): 24628-24640. Epub 2016 Sep 28. |
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