ML-323
Based on 17 publication(s) in Google Scholar
ML-323 is a reversible, potent USP1-UAF1 inhibitor with IC50 of 76 nM in a Ub-Rho assay. The measured inhibition constants of ML-323 for the free enzyme (Ki) is 68 nM.
For research use only. We do not sell to patients.
- Purity: 99.51%
- CAS No.: 1572414-83-5
- Formula: C23H24N6
- Molecular Weight:384.48
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Storage:Powder -20°C, 3 years , 4°C, 2 years ; In solvent -80°C, 1 year , -20°C, 6 months
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Biological Activity
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Chemical Information
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Solvent & Solubility
- Protocol
- Purity & Documentation
- References
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Help & FAQs
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Publications Citing Use of MedChemExpress (MCE) ML-323
More- Nat Commun. 2026 Feb 12;17(1):1214. [Abstract]
- Nat Commun. 2025 May 16;16(1):4564. [Abstract]
- Nat Commun. 2022 Mar 31;13(1):1700. [Abstract]
- Nucleic Acids Res. 2024 Mar 21;52(5):2340-2354. [Abstract]
- Redox Biol. 2026 May:92:104086. [Abstract]
- J Immunother Cancer. 2026 Jan 14;14(1):e013498. [Abstract]
- Int J Biol Sci. 2022 May 1;18(8):3122-3136. [Abstract]
- Cell Death Dis. 2022 Nov 10;13(11):951. [Abstract]
- EMBO J. 2015 May 12;34(10):1385-98. [Abstract]
- Cell Chem Biol. 2021 Jun 17;28(6):855-865.e9. [Abstract]
- J Med Chem. 2022 Oct 27;65(20):13645-13659. [Abstract]
- Cell Prolif. 2020 Oct;53(10):e12908. [Abstract]
- J Neurosci. 2023 Mar 1;43(9):1456-1474. [Abstract]
- Sci Rep. 2025 Feb 18;15(1):5876. [Abstract]
- Viruses. 2023 Feb 28;15(3):655. [Abstract]
- Anim Cells Syst. 2024 May 11;28(1):237-250. [Abstract]
- Hum Cell. 2025 Apr 18;38(3):91. [Abstract]
Customer Validation & Images
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Bio/Physico-chemical Assay
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Bio/Physico-chemical Assay
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Bio/Physico-chemical Assay
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WB
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Cell Proliferation/Viability Assay
Biological Activity
IC50: 76 nM (USP1-UAF1, in Ub-Rho assay)[1]
Ki: 68 nM (USP1-UAF1)[1]
ML-323 (ML323) is a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. ML-323 is a potent USP1-UAF1 inhibitor with IC50 values of 76 nM in a ubiquitin-rhodamine (Ub-Rho) assay and 174 nM and 820 nM in orthogonal gel-based assays using K63-linked diubiquitin (di-Ub) and monoubiquitinated PCNA (Ub-PCNA) as substrates, respectively. ML-323 probably exerts its inhibitory effect through an allosteric mechanism. The measured inhibition constants of ML-323 for the free enzyme (Ki) and the enzyme-substrate complex (K’i) are 68 nM and 183 nM. Besides, ML-323 potentiates Cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells[1]. ML-323 (ML323), a probe molecule that displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1. In addition, ML-323 potentiates the cytotoxicity of Cisplatin and increases endogenous monoubiquitination levels of both PCNA and FANCD2, two known cellular targets of USP1/UAF1[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Chemical Information
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CAS No. 1572414-83-5
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Appearance Solid
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Molecular Weight 384.48
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Formula C23H24N6
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Color White to off-white
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SMILES
CC1=CN=C(C2=C(C(C)C)C=CC=C2)N=C1NCC3=CC=C(N4N=NC=C4)C=C3
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Shipping
Room temperature in continental US; may vary elsewhere.
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Storage
Powder -20°C 3 years 4°C 2 years In solvent -80°C 1 year -20°C 6 months
Publications (17)
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Journal Impact Factor
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Most Recent
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Nat Commun
Human iPSC-based Modeling of Pulmonary Fibrosis Reveals p300/CBP Inhibition Suppresses Alveolar Transitional Cell State. [Abstract]2026 Feb 12;17(1):1214. PMID: 41680175 -
Nat Commun
2025 May 16;16(1):4564. PMID: 40379682 -
Nat Commun
USP8 inhibition reshapes an inflamed tumor microenvironment that potentiates the immunotherapy. [Abstract]2022 Mar 31;13(1):1700. PMID: 35361799 -
Nucleic Acids Res
USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress. [Abstract]2024 Mar 21;52(5):2340-2354. PMID: 38180818
ML-323 purchased from MedChemExpress. Usage Cited in: Nucleic Acids Res. 2024 Mar 21;52(5):2340-2354. [Abstract]
ML-323 (20 μM; 2.5 h) suppressesed the binding of MRE11 and EXO1 to nascent DNA in HeLa PRIMPOL-overexpressing cells.
ML-323 purchased from MedChemExpress. Usage Cited in: Nucleic Acids Res. 2024 Mar 21;52(5):2340-2354. [Abstract]
ML-323 (10-30 μM; 3.5 h) suppressesed the accumulation of replication-associated ssDNA gaps induced by exposure to 150μM cisplatin or 0.4mM HU in U2OS and HeLa PRIMPOL-overexpressing cells.
ML-323 purchased from MedChemExpress. Usage Cited in: Nucleic Acids Res. 2024 Mar 21;52(5):2340-2354. [Abstract]
ML-323 (20 μM; 4.5 h) suppressesed the accumulation of replication-associated ssDNA gaps induced by exposure to 0.4 mM HU in HeLa PRIMPOL-overexpressing cells.
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Redox Biol
USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers. [Abstract]2026 May:92:104086. PMID: 41844497 -
J Immunother Cancer
CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy. [Abstract]2026 Jan 14;14(1):e013498. PMID: 41534899 -
Int J Biol Sci
2022 May 1;18(8):3122-3136. PMID: 35637948
ML-323 purchased from MedChemExpress. Usage Cited in: Int J Biol Sci. 2022 May 1;18(8):3122-3136. [Abstract]
ML-323 (8-32 μM; 48 h) reduced the expression of TAZ and downstream genes in Hippo signaling pathway in 143B and HOS cells.
ML-323 purchased from MedChemExpress. Usage Cited in: Int J Biol Sci. 2022 May 1;18(8):3122-3136. [Abstract]
ML-323 (8-32 μM; 7-14 d) weakened the ability of OS cells in forming colonies.
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Cell Death Dis
Inhibition of USP1 activates ER stress through Ubi-protein aggregation to induce autophagy and apoptosis in HCC. [Abstract]2022 Nov 10;13(11):951. PMID: 36357365 -
EMBO J
Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway. [Abstract]2015 May 12;34(10):1385-98. PMID: 25862789 -
Cell Chem Biol
Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2. [Abstract]2021 Jun 17;28(6):855-865.e9. PMID: 33979649 -
J Med Chem
Discovery of Potent OTUB1/USP8 Dual Inhibitors Targeting Proteostasis in Non-Small-Cell Lung Cancer. [Abstract]2022 Oct 27;65(20):13645-13659. PMID: 36221183 -
Cell Prolif
Comprehensive analysis of ubiquitin-specific protease 1 reveals its importance in hepatocellular carcinoma. [Abstract]2020 Oct;53(10):e12908. PMID: 32951278
ML-323 purchased from MedChemExpress. Usage Cited in: Cell Prolif. 2020 Oct;53(10):e12908. [Abstract]
ML-323 (50 μM; 24 hours) treatment down-regulates USP1 and WDR48 in MHCC97H and SK-Hep-1 cells. And also decreases the protein expression of PCNA, cyclin D1 and cyclin E1.
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J Neurosci
USP1/UAF1-stabilized METTL3 promotes reactive astrogliosis and improves functional recovery after spinal cord injury through m6A modification of YAP1 mRNA. [Abstract]2023 Mar 1;43(9):1456-1474. PMID: 36653190 -
Sci Rep
N6-methyladenosine (m6A)-forming enzyme METTL3 controls UAF1 stability to promote inflammation in a model of colitis by stimulating NLRP3. [Abstract]2025 Feb 18;15(1):5876. PMID: 39966502 -
Viruses
Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities against New World Alphaviruses. [Abstract]2023 Feb 28;15(3):655. PMID: 36992362 -
Anim Cells Syst
Blocking SLC7A11 attenuates the proliferation of esophageal squamous cell carcinoma cells. [Abstract]2024 May 11;28(1):237-250. PMID: 38741950 -
Hum Cell
LncRNA OLMALINC promotes osteosarcoma progression through USP1-mediated autophagy suppression. [Abstract]2025 Apr 18;38(3):91. PMID: 40249458
Solvent & Solubility
DMSO : ≥ 49 mg/mL (127.44 mM; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
* "≥" means soluble, but saturation unknown.
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, 1 year; -20°C, 6 months. When stored at -80°C, please use it within 1 year. When stored at -20°C, please use it within 6 months.
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, 1 year; -20°C, 6 months. When stored at -80°C, please use it within 1 year. When stored at -20°C, please use it within 6 months.
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
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.
Add each solvent one by one: 10% DMSO 40% PEG300 5% Tween-80 45% Saline
Solubility: ≥ 2.5 mg/mL (6.50 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.
Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: ≥ 2.5 mg/mL (6.50 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.
Please enter the basic information of animal experiments:
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Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
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%DMSO +
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
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%+
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+%Tween-80 + +
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%Saline +
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).
Working solution concentration: 0.22 mg/mL
Method for preparing stock solution: mg drug dissolved in μL DMSO. Stock solution concentration: mg/mL.
1. Take μL DMSO stock solution;
2. Add μL .
μL , mix evenly;
3. Then add μL Tween 80, mix evenly;
4. Then add μL
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.
Protocol
For DUB profiling, ML-323 is tested at a single-dose of 10 μM in duplicate. The DUB activities are monitored using Ub-7-amido-4-methylcoumarin (AMC) as a substrate. The increase in fluorescent signal from free AMC is monitored over time, although only the initial linear portion of slope (signal/min) is used for analysis. The activity of enzyme with no compound is treated as 100%. For protease profiling, ML-323 is tested using threefold serial dilutions starting at 20 μM against 70 proteases. Proteases are pre-incubated with the compound for 5-15 min before the addition of the appropriate enzyme substrates. The enzyme activities are measured by reading the fluorescent signal from fluorescently labeled peptides[1].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Cell viability is measured by a cell counting kit (CCK) assay using CCK-8 solution. For the colony-forming assay, cells are seeded at a density of 300-500 cells per well in six-well plates and grown overnight. Cells are then treated with ML-323 alone, Cisplatin alone or a combination of Cisplatin and ML-323 (1:1 or 1:4) at the indicated concentrations. Cells treated with an equal volume of DMSO and saline are used as a control. After 48 h of treatment, fresh growth medium is added, and cells are incubated for an additional 5-10 d to allow for colony formation. For UV combination treatment, the cells are treated with ML-323 at the indicated concentrations or an equal volume of DMSO. After 48 h, the medium is removed, and cells are irradiated at 254 nm at the indicated dosage. Fresh growth medium is added, and the cells are incubated for an additional 5-10 d to allow for colony formation. The cells without UV irradiation but treated with ML-323 or an equal volume of DMSO are used as controls and designated as 100%. After the formation of the colonies, cells are fixed with methanol and stained with 0.5% crystal violet. Colonies consisting of >50 cells are scored. The number of colonies is determined from triplicate plates. The dose-response curves are generated using GraphPad Prism and analyzed by using CalcuSyn to calculate the combination index, which is determined for the fraction of cells affected after the addition of fixed ratios of cisplatin and the USP1-UAF1 inhibitor[1].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Purity & Documentation
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Data Sheet (284 KB)
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SDS (393 KB)
- English - EN (393 KB)
- Français - FR (393 KB)
- Deutsch - DE (393 KB)
- Norwegian - NO (393 KB)
- Español - ES (393 KB)
- Swedish - SV (393 KB)
- Italian - IT (393 KB)
- Portuguese - PT (393 KB)
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Handling Instructions (2659 KB)
References
[1]. Liang Q, et al. A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses. Nat Chem Biol. 2014 Apr;10(4):298-304. [Content Brief]
[2]. Dexheimer TS, et al. Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex. National Center for Biotechnology Information; 2010-2012 Oct 23. [Content Brief]
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, 1 year; -20°C, 6 months. When stored at -80°C, please use it within 1 year. When stored at -20°C, please use it within 6 months.
| Optional Solvent | Concentration Solvent Mass | 1 mg | 5 mg | 10 mg | 25 mg |
|---|---|---|---|---|---|
| DMSO | 1 mM | 2.6009 mL | 13.0046 mL | 26.0092 mL | 65.0229 mL |
| 5 mM | 0.5202 mL | 2.6009 mL | 5.2018 mL | 13.0046 mL | |
| 10 mM | 0.2601 mL | 1.3005 mL | 2.6009 mL | 6.5023 mL | |
| 15 mM | 0.1734 mL | 0.8670 mL | 1.7339 mL | 4.3349 mL | |
| 20 mM | 0.1300 mL | 0.6502 mL | 1.3005 mL | 3.2511 mL | |
| 25 mM | 0.1040 mL | 0.5202 mL | 1.0404 mL | 2.6009 mL | |
| 30 mM | 0.0867 mL | 0.4335 mL | 0.8670 mL | 2.1674 mL | |
| 40 mM | 0.0650 mL | 0.3251 mL | 0.6502 mL | 1.6256 mL | |
| 50 mM | 0.0520 mL | 0.2601 mL | 0.5202 mL | 1.3005 mL | |
| 60 mM | 0.0433 mL | 0.2167 mL | 0.4335 mL | 1.0837 mL | |
| 80 mM | 0.0325 mL | 0.1626 mL | 0.3251 mL | 0.8128 mL | |
| 100 mM | 0.0260 mL | 0.1300 mL | 0.2601 mL | 0.6502 mL |
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