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Purity: ≥98%
ML323 (ML-323; ML 323) is a novel, potent and highly selective USP1-UAF1 inhibitor with potential antitumor activity. It inhibits USP1-UAF1 with an IC50 of 76 nM. ML323 inhibits the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. ML323 links deubiquitination to DNA damage responses. ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. USP1-UAF1 is a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.
| Targets |
USP1-UAF1, in Ub-Rho assay(IC50=76 nM);USP1-UAF1(Ki= 68 nM)
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| ln Vitro |
A highly effective inhibitor of the USP1-UAF1 deubiquitinase complex, ML-323 (ML323) exhibits excellent selectivity against unrelated proteases, deSUMOylase, deneddylase, and human DUBs. With an IC50 of 76 nM in a ubiquitin-rhodamine (Ub-Rho) assay and 174 nM and 820 nM in orthogonal gel-based assays employing K63-linked diubiquitin (di-Ub) and monoubiquitinated PCNA (Ub-PCNA) as substrates, respectively, ML-323 is a strong inhibitor of USP1-UAF1. ML-323 most likely uses an allosteric mechanism to produce its inhibitory effect. ML-323's measured inhibition constants are 68 nM and 183 nM for the free enzyme (Ki) and the enzyme-substrate complex (K'i). Additionally, ML-323 increases the cytotoxicity of cisplatin in osteosarcoma and non-small cell lung cancer cells.[1].ML-323 (ML323) is a probe molecule with excellent selectivity toward USP1/UAF1 and reversible, nanomolar inhibitory activity. Furthermore, ML-323 raises endogenous monoubiquitination levels of PCNA and FANCD2, two known cellular targets of USP1/UAF1 and amplifies the cytotoxicity of cisplatin[2].
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| ln Vivo |
ML323 promotes viral replication in vivo.[3]
Mice were pretreated with ML323 before i.p. injection of LPS. ML323 treatment reduced serum level of IFN-β but did not considerably decrease the amount of TNF-α (Fig. 5, G and H), indicating that ML323 is active in vivo. We then investigated the regulatory effects of ML323 on IFN-β expression and viral replication in the context of VSV infection in vivo. ML323 treatment inhibited VSV-induced IFN-β production in peritoneal lavage (Fig. 5 I) and suppressed TBK1 protein expression in peritoneal exudate cells (Fig. 5 J). Furthermore, VSV infection induced IFN-β secretion was much less in sera of ML323-treated mice than in that of control mice (Fig. 5 K). Accordingly, IFN-β mRNA expression in the liver, lung, and spleen of ML323-treated mice was lower than that in controls (Fig. 5 L). In accordance with reduced IFN-β expression, VSV replication in the liver, lung, and spleen of ML323-treated mice was higher than that in controls (Fig. 5 M). Severe infiltration of immune cells was observed in the lungs of ML323-treated mice, compared with that of control mice after VSV infection (Fig. 5 N). Collectively, these data indicate that ML323 attenuates VSV-induced IFN-β expression and thus promotes VSV replication, both in vitro and in vivo. |
| Enzyme Assay |
ML-323 is tested in duplicate at a single dose of 10 μM for DUB profiling. As a substrate, Ub-7-amido-4-methylcoumarin (AMC) is used to track the DUB activities. The initial linear portion of the slope (signal/min) is the only part of the fluorescence signal increase from free AMC that is analyzed over time. An enzyme's activity of 100% is applied when it contains no compounds. ML-323 is subjected to threefold serial dilutions against 70 proteases, starting at 20 μM, in order to perform protease profiling. Before adding the proper enzyme substrates, the compound is pre-incubated with proteases for five to fifteen minutes. By analyzing the fluorescent signal from peptides that have been fluorescently labeled, the enzyme activities are determined [1].
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| Cell Assay |
Using CCK-8 solution, a cell counting kit (CCK) assay determines the viability of the cells. In six-well plates, cells are seeded at a density of 300–500 cells per well for the colony-forming assay, and they are grown there for an entire night. Then, at the indicated concentrations, cells are treated with ML-323 alone, Cisplatin alone, or a combination of Cisplatin and ML-323 (1:1 or 1:4). As a control, cells that were treated with the same volume of DMSO and saline are employed. To enable colony formation, fresh growth medium is added after 48 hours of treatment, and cells are then incubated for a further 5–10 days.The cells are treated with ML-323 at the recommended concentrations or an equivalent volume of DMSO for UV combination treatment. The medium is removed after 48 hours, and the prescribed dosage of radiation is applied to the cells at 254 nm. To enable colony formation, fresh growth medium is added, and the cells are incubated for a further five to ten days. As controls, the cells that are not exposed to UV light but are treated with either ML-323 or an equivalent volume of DMSO are denoted as 100%. Cells are fixed with methanol and stained with 0.5% crystal violet after the colonies have formed. Colonies with more than 50 cells are given a score. Plates in triplicate are used to calculate the number of colonies.With GraphPad Prism, the dose-response curves are created, and CalcuSyn is used for analysis to determine the combination index, which is based on the percentage of affected cells following the addition of fixed ratios of cisplatin and the USP1-UAF1 inhibitor[1].
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| Animal Protocol |
In vivo LPS challenge[3]
C57BL/6J mice (females, 6–8 wk old) were i.p. injected with 10 mg/kg ML323 for 6 h and then i.p. injected with 10 mg/kg LPS for 2 h. Serum levels of IFN-β and TNF-α were measured by ELISA. Viral pathogenesis in mice[3] C57BL/6J mice (females, 8 wk old) were i.p. infected with VSV (5 × 107 PFU/mouse). 10 mg/kg ML323 was i.p. administered 6 h before VSV infection. Lungs from control or virus-infected mice were dissected, fixed in 10% (vol/vol) phosphate-buffered formalin, embedded into paraffin, sectioned, stained with hematoxylin and eosin (H&E) solution, and examined by light microscopy for histological changes. |
| References |
[3]. J Exp Med. 2017 Dec 4; 214(12): 3553–3563.
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| Additional Infomation |
Optimal activation of TANK-binding kinase 1 (TBK1) is crucial for initiation of innate antiviral immunity and maintenance of immune homeostasis. Although several E3 ubiquitin ligases have been reported to regulate TBK1 activation by mediating its polyubiquitination, the functions of deubiquitinase on TBK1 activity remain largely unclear. Here, we identified a deubiquitinase complex, which is formed by ubiquitin specific peptidase 1 (USP1) and USP1-associated factor 1 (UAF1), as a viral infection-induced physiological enhancer of TBK1 expression. USP1-UAF1 complex enhanced TLR3/4 and RIG-I-induced IFN regulatory factor 3 (IRF3) activation and subsequent IFN-β secretion. Mechanistically, USP1 and UAF1 bound to TBK1, removed its K48-linked polyubiquitination, and then reversed the degradation process of TBK1. Furthermore, we found that ML323, a specific USP1-UAF1 inhibitor, attenuated IFN-β expression and enhanced viral replication both in vitro and in vivo. Therefore, our results outline a novel mechanism for the control of TBK1 activity and suggest USP1-UAF1 complex as a potential target for the prevention of viral diseases.[3]
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| Molecular Formula |
C23H24N6
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| Molecular Weight |
384.480
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| Exact Mass |
384.206
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| Elemental Analysis |
C, 71.85; H, 6.29; N, 21.86
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| CAS # |
1572414-83-5
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| Related CAS # |
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| PubChem CID |
60167849
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| Appearance |
White solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
515.4±60.0 °C at 760 mmHg
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| Flash Point |
265.5±32.9 °C
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| Vapour Pressure |
0.0±1.3 mmHg at 25°C
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| Index of Refraction |
1.651
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| LogP |
4.61
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
29
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| Complexity |
492
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N([H])(C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])=C([H])N=N1)C1C(C([H])([H])[H])=C([H])N=C(C2=C([H])C([H])=C([H])C([H])=C2C([H])(C([H])([H])[H])C([H])([H])[H])N=1
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| InChi Key |
VUIRVWPJNKZOSS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H24N6/c1-16(2)20-6-4-5-7-21(20)23-24-14-17(3)22(27-23)25-15-18-8-10-19(11-9-18)29-13-12-26-28-29/h4-14,16H,15H2,1-3H3,(H,24,25,27)
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| Chemical Name |
N-(4-(1H-1,2,3-triazol-1-yl)benzyl)-2-(2-isopropylphenyl)-5-methylpyrimidin-4-amine
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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 : 49~76 mg/mL ( 127.44~197.66 mM )
Ethanol : ~38 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.50 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 (6.50 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 (6.50 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 | 2.6009 mL | 13.0046 mL | 26.0092 mL | |
| 5 mM | 0.5202 mL | 2.6009 mL | 5.2018 mL | |
| 10 mM | 0.2601 mL | 1.3005 mL | 2.6009 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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