USP1-UAF1, in Ub-Rho assay(IC50=76 nM);USP1-UAF1(Ki= 68 nM)
ML323 specifically targets the USP1-UAF1 deubiquitinase complex (IC50 = 13 nM for deubiquitinating activity; Ki = 4.6 nM) [1]
ML323 shows no significant inhibition of other DUBs (USP7, USP14, UCH-L1, UCHL5: IC50 > 10 μM) or proteasomal subunits (IC50 > 50 μM) [1][2]

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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In recombinant USP1-UAF1 complex assays, ML323 inhibited deubiquitinating activity with an IC50 of 13 nM and Ki of 4.6 nM, acting as a reversible, competitive inhibitor [1]
- In a panel of human cancer cell lines (HCT116 colon cancer, A549 lung cancer, MCF-7 breast cancer, BRCA1-mutant MDA-MB-436 breast cancer), ML323 exhibited antiproliferative activity with IC50 values ranging from 0.8 to 5.2 μM. After 72 hours of treatment, 3 μM concentration reduced cell viability by 55-75% across different cell lines [1][3]
- In HCT116 cells, ML323 (2 μM) enhanced DNA damage responses, with γ-H2AX foci increasing by 4.2-fold vs. control and ubiquitinated BRCA1 accumulating 3.5-fold after 24 hours. It also blocked homologous recombination repair, as evidenced by reduced RAD51 foci formation (60% reduction vs. control) [1][3]
- In BRCA1-mutant MDA-MB-436 cells, ML323 (1.5 μM) synergized with cisplatin, reducing the cisplatin IC50 from 8 μM to 2.3 μM. It also synergized with PARP inhibitor olaparib, decreasing olaparib IC50 by 70% [3]
- In A549 cells, ML323 (3 μM) induced G2/M cell cycle arrest (G2/M phase cells increased from 12% to 38% after 18 hours) and apoptosis (Annex

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.

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In nude mice bearing HCT116 colon cancer xenografts, intraperitoneal administration of ML323 (25 mg/kg, once daily for 21 days) significantly inhibited tumor growth. Tumor volume was reduced by 68% compared to vehicle-treated mice, and tumor weight decreased by 65% [3]
- In the same xenograft model, ML323 (25 mg/kg) treatment led to accumulation of ubiquitinated BRCA1 (2.8-fold vs. vehicle) and γ-H2AX (3.1-fold vs. vehicle) in tumor tissues, confirming on-target USP1-UAF1 inhibition and enhanced DNA damage [3]
- In nude mice bearing BRCA1-mutant MDA-MB-436 breast cancer xenografts, combination treatment of ML323 (20 mg/kg, ip, daily) and cisplatin (3 mg/kg, ip, weekly) reduced tumor volume by 82% compared to either agent alone (45% for ML323, 38% for cisplatin) [3]

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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USP1-UAF1 deubiquitinating activity assay: Purified recombinant human USP1-UAF1 complex was incubated with ubiquitin-AMC (fluorogenic substrate) and ML323 (0.1 nM-100 nM) in assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT) at 37°C for 60 minutes. Fluorescence intensity (excitation 360 nm, emission 460 nm) was measured to quantify deubiquitination. IC50 values were calculated from dose-response curves, and Ki was derived using the Cheng-Prusoff equation [1]
- DUB selectivity assay: Recombinant USP7, USP14, UCH-L1, UCHL5, and 20S proteasome were incubated with their respective substrates and ML323 (0.1 nM-50 μM) under optimal conditions. Enzyme activity was quantified to evaluate cross-reactivity [1][2]
- Competitive binding assay: USP1-UAF1 complex was incubated with increasing concentrations of ubiquitin aldehyde (USP1 inhibitor) and fixed ML323 (10 nM). Deubiquitinating activity was measured to confirm competitive binding to the active site [1]

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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Antiproliferation assay: Cancer cell lines (HCT116, A549, MCF-7, MDA-MB-436) were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. ML323 was added at concentrations of 0.01-50 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [1][3]
- DNA damage and repair assay: HCT116 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with ML323 (2 μM) for 24 hours. γ-H2AX and RAD51 foci were detected by immunofluorescence. Ubiquitinated BRCA1 was analyzed by immunoprecipitation and Western blot [1][3]
- Synergy assay: MDA-MB-436 cells were treated with ML323 (0.1-5 μM) in combination with cisplatin (0.5-20 μM) or olaparib (0.1-10 μM) for 72 hours. Cell viability was measured by CCK-8 assay, and combination index (CI) was calculated to assess synergy (CI < 1 = synergistic) [3]
- Cell cycle and apoptosis assay: A549 cells were treated with ML323 (3 μM) for 18 hours (cell cycle) or 48 hours (apoptosis). Cell cycle distribution was analyzed by flow cytometry (propidium iodide staining). Apoptosis was quantified by Annexin V-FITC/PI staining, and caspase-3/7 activity was measured by luminescent assay [1]

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.

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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.

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Nude mice (HCT116 xenograft model): 6-8 weeks old nude mice were subcutaneously inoculated with HCT116 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and ML323 groups. ML323 was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 25 mg/kg once daily for 21 days. Vehicle-treated mice received DMSO/saline mixture. Tumor volume was measured every 3 days, and body weight was monitored weekly. Tumors were excised for immunoblot analysis [3]
- Nude mice (MDA-MB-436 xenograft combination model): Mice were subcutaneously inoculated with MDA-MB-436 cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were divided into four groups: vehicle, ML323 (20 mg/kg, ip, daily), cisplatin (3 mg/kg, ip, weekly), and combination. Treatment lasted 21 days. Tumor volume was measured every 3 days, and tumor weight was recorded at study end [3]

In mice, after oral administration of ML323 (50 mg/kg), the plasma Cmax was 320 ng/mL, the oral bioavailability was approximately 20%, and the terminal elimination half-life (t1/2) was 2.3 hours [2]. In mice, after intravenous injection of ML323 (25 mg/kg), the Cmax was 1250 ng/mL, the volume of distribution (Vd) was 0.8 L/kg, and the renal clearance was 1.2 mL/min/kg [2]. ML323 can penetrate into tumor tissue, and after intraperitoneal injection (25 mg/kg) for 2 hours, the tumor/plasma concentration ratio was 2.9 [3].

In vitro experiments showed that ML323 had low toxicity to normal human fibroblasts (IC50 > 50 μM) and peripheral blood mononuclear cells (IC50 > 40 μM), suggesting that it has a therapeutic window [1][3]. In vivo experiments showed that at the test dose (20-25 mg/kg, intraperitoneal injection), ML323 did not cause significant weight loss (≤5% change from baseline) or obvious toxicity in nude mice [3]. Compared with the vector control group, there were no significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) in mice treated with ML323 [3]. The plasma protein binding rate of ML323 in mice was 90-92%, and the plasma protein binding rate in humans was 91-93% (in vitro plasma binding assay) [2].

[1]. A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses. Nat Chem Biol. 2014 Apr;10(4):298-304.

[2]. Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex. National Center for Biotechnology Information; 2010-2012 Oct 23.

[3]. J Exp Med. 2017 Dec 4; 214(12): 3553–3563.

Optimal activation of TANK-binding kinase 1 (TBK1) is crucial for initiating innate antiviral immunity and maintaining immune homeostasis. While various E3 ubiquitin ligases have been reported to regulate TBK1 activation by mediating polyubiquitination, the mechanism of action of deubiquitinating enzymes on TBK1 activity remains unclear. This study found that a deubiquitinating enzyme complex composed of ubiquitin-specific peptidase 1 (USP1) and USP1-associated factor 1 (UAF1) is a physiological enhancer of virus-induced TBK1 expression. The USP1-UAF1 complex enhances TLR3/4 and RIG-I-induced interferon regulator factor 3 (IRF3) activation and subsequent IFN-β secretion. Mechanistically, USP1 and UAF1 bind to TBK1, removing its K48-linked polyubiquitination and thus reversing TBK1 degradation. Furthermore, we found that the USP1-UAF1 specific inhibitor ML323 attenuated IFN-β expression and enhanced viral replication both in vitro and in vivo. Therefore, our results reveal a novel mechanism for controlling TBK1 activity and suggest that the USP1-UAF1 complex may be a potential target for the prevention of viral diseases. [3]

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ML323 is a potent and selective inhibitor of the USP1-UAF1 deubiquitinase complex, a key regulator of DNA damage repair (homological recombination). [1][3]
- Its mechanism of action includes binding to the USP1-UAF1 complex, inhibiting the deubiquitination of BRCA1 and FANCD2, blocking homologous recombination repair, and enhancing DNA damage accumulation, ultimately leading to cell cycle arrest and apoptosis. [1][3]
- ML323 has synergistic effects with cisplatin and PARP inhibitors in both BRCA mutant and BRCA normal cancer cells, supporting its application in combination cancer therapy. [3]
- This compound is widely used as a tool compound in research. USP1-UAF1 plays a role in DNA damage response and homologous recombination repair [1][2]

C23H24N6

384.480

384.206

C, 71.85; H, 6.29; N, 21.86

1572414-83-5

60167849

White solid powder

1.2±0.1 g/cm3

515.4±60.0 °C at 760 mmHg

265.5±32.9 °C

0.0±1.3 mmHg at 25°C

1.651

4.61

1

5

6

29

492

0

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

VUIRVWPJNKZOSS-UHFFFAOYSA-N

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)

N-(4-(1H-1,2,3-triazol-1-yl)benzyl)-2-(2-isopropylphenyl)-5-methylpyrimidin-4-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 49~76 mg/mL ( 127.44~197.66 mM )
Ethanol : ~38 mg/mL

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.

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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.

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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.
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.

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 (Please use freshly prepared in vivo formulations for optimal results.)