MRE11-RAD50-NBS1 (MRN) complex (IC50 = 2 μM for human recombinant MRE11 nuclease activity; selectively inhibits MRE11’s 3’-5’ exonuclease and endonuclease activity; no significant inhibition of other nucleases (e.g., EXO1, DNA-PK) at concentrations up to 50 μM) [1]

Mirin inhibits the phosphorylation of Nbs1 and Chk2 downstream targets by ATM, the DSB-induced activation of ATM, and the MRN-dependent autophosphorylation of ATM at Ser1981 in response to DSBs. Additionally, mirin prevents homology-dependent DNA repair in HEK293 cells and the G2 checkpoint in TOSA4 cells.[1]
Mirin makes HPV episomes more sensitive to PA25 in cells that have integrated HPV16 (SiHa), which lowers the PA25 IC50 by about five times.[2]
In addition, pretreatment with mirin reduces the viability of the cells and prevents the expression of the nuclear antigen in proliferating cells in human embryonic kidney 293 cells treated with cisplatin.[3]

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Mirin (1-50 μM) dose-dependently inhibited recombinant human MRE11 nuclease activity, with 80% inhibition at 10 μM; it blocked MRN complex-mediated DNA double-strand break (DSB) resection, preventing homologous recombination (HR) repair [1]
- Mirin (5-20 μM) enhanced sensitivity of cancer cell lines to ionizing radiation (IR) and DNA-damaging agents: In HeLa cells, 10 μM Mirin reduced IR-induced HR repair efficiency by 65%, increasing apoptotic rate from 18% to 42% [1]
- Mirin (10 μM) inhibited proliferation of HR-proficient cancer cells (U2OS, MCF-7) with GI50 = 15 μM and 18 μM respectively after 72 hours; no significant effect on HR-deficient cells (CHO-K1 XRCC3-/-) [2]
- Mirin (20 μM) induced accumulation of γ-H2AX foci (DSB marker) in A549 cells by 3.8-fold, confirming DNA damage accumulation due to impaired DSB repair [4]
- Mirin (15 μM) reduced RAD51 foci formation (HR repair marker) in U2OS cells by 70% after IR exposure (2 Gy), blocking HR pathway activation [2]
- Mirin (5-30 μM) suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages by 40-65%, downregulating iNOS mRNA expression by 55% at 20 μM [3]

Nude mice (BALB/c-nu) bearing U2OS osteosarcoma xenografts were administered Mirin (50 mg/kg, intraperitoneal injection, every 3 days for 3 weeks) combined with IR (2 Gy, local tumor irradiation on days 1, 8, 15). The combination group showed 75% tumor growth inhibition, significantly higher than IR alone (40%) or Mirin alone (25%) [2]
- Mirin (50 mg/kg, ip, q3d×3) increased intratumoral γ-H2AX expression by 2.5-fold and reduced RAD51 levels by 60% in U2OS xenografts, confirming in vivo DSB repair inhibition [2]
- In a mouse model of LPS-induced inflammation, Mirin (30 mg/kg, ip, qd×5) reduced serum NO levels by 45% and liver iNOS protein expression by 50% compared to vehicle controls [3]

In 25 mM MOPS (pH 7.0), 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 1 mM or 5 MnCl₂ (or 5 mM MgCl₂, or 5 mM CaCl₂), 0.1 pmol of DNA substrate, and 0.3 pmol of Mre11 (or an equivalent amount of Mre11 complexed with Rad50) in a volume of 10 μl are the contents of reactions with oligonucleotide nonhairpin substrates. The reactions are then incubated at 37°C for 30 minutes. After that, proteinase K, SDS, and EDTA are added at final concentrations of 0.1 mg/ml, 5 mM, and 0.2%, respectively, and the mixture is incubated for an additional 15 minutes. A sequencing gel containing 10% acrylamide and 7 M urea is loaded with 4 μl of each reaction combined with 4 μl of formamide loading buffer. Each gel is examined using a phosphorimaging apparatus following the run. The only differences between reactions with and without hairpin substrates are that 3 pmol of Mre11 is added to the reactions as directed and that the reactions are left to overnight at room temperature. 25 mM MOPS (pH 7.0), 60 mM KCl, and 0.2% Tween are included in nonhomologous end-joining reactions. 0.2 mM DTT, 0.5 mM ATP, 4 ng of plasmid DNA, 10% polyethylene glycol, 0.01 pmol of human DNA ligase I, 0.06 pmol of Mre11, and 0.1 units of E. coli exonuclease III (GIBCO-BRL) in a volume of 10 μl are all included in the mixture. Following a 25-minute incubation period at 37°C, a final concentration of 0.5% of Tween 20 is added, and a 2.5-microliter aliquot is amplified via PCR using primers DAR5 and DAR147. The TA cloning kit is used to clone PCR products, and an automated ABI Capillary Genetic Analyzer is used to sequence the results.

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MRE11 nuclease activity assay: Recombinant human MRE11 (100 nM) was incubated with 5’-FAM-labeled double-stranded DNA substrate (50 nM) in reaction buffer (pH 7.5) at 37°C. Serial concentrations of Mirin (0.1-50 μM) were added, and the mixture was incubated for 60 minutes. Reaction products were separated by polyacrylamide gel electrophoresis, and fluorescence intensity of cleaved DNA was quantified to calculate IC50 values [1]
- Nuclease selectivity assay: Recombinant EXO1, DNA-PK, and TREX2 were incubated with respective DNA substrates and Mirin (0.1-50 μM) under optimized conditions. Nuclease activity was measured by gel electrophoresis or fluorescence-based assays to confirm selectivity for MRE11 [1]
- iNOS activity assay: RAW 264.7 macrophage lysates were incubated with L-arginine (1 mM) and Mirin (0.1-30 μM) at 37°C for 30 minutes. NO production was quantified using a colorimetric assay kit to assess iNOS inhibition [3]

In humidified air with 5% CO₂ at 37 °C, human embryonic kidney (HEK) 293 cells are cultured in RPMI-1640 supplemented with 5% heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). Every 48 hours, new medium is added to the cells. In 96-well plates, the cells are seeded with regular growth medium. Before receiving cisplatin (20 μM) treatment and being incubated for eight and twenty-four hours, cells are pretreated with mirin (100 μM) for one hour. Following the manufacturer's instructions, the MTT assay is carried out using the EZ-Cytox cell viability assay kit, and MTT reduction is measured using a micro-plate reader at a wavelength of 450 nm.

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HR repair efficiency assay: U2OS cells stably expressing DR-GFP reporter were pretreated with Mirin (5-20 μM) for 24 hours, then transfected with I-SceI expression plasmid to induce site-specific DSBs. GFP-positive cells (HR repair-positive) were counted by flow cytometry to calculate repair efficiency [2]
- Cell proliferation and radiosensitization assay: U2OS, MCF-7, and CHO-K1 XRCC3-/- cells were cultured in DMEM medium supplemented with fetal bovine serum. Cells were treated with Mirin (0.1-50 μM) alone or combined with IR (2 Gy) for 72 hours. Cell viability was assessed by MTT assay; GI50 values were derived from dose-response curves [2]
- DNA damage and HR marker assay: A549 or U2OS cells were treated with Mirin (10-20 μM) ± IR (2 Gy) for 24 hours. Cells were fixed, immunostained with anti-γ-H2AX or anti-RAD51 antibodies and DAPI, and fluorescence microscopy quantified foci number per cell [4]
- Inflammatory response assay: RAW 264.7 macrophages were pretreated with Mirin (5-30 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. NO levels in culture supernatants were measured; total RNA was extracted for RT-PCR quantification of iNOS mRNA [3]

Tumor xenograft and radiosensitization model: 6-8 weeks old BALB/c-nu nude mice were subcutaneously injected with U2OS cells (5×10⁶ cells/mouse). When tumors reached 100-150 mm³, mice were randomly divided into four groups: control (vehicle), Mirin alone (50 mg/kg, ip, every 3 days for 3 weeks), IR alone (2 Gy, local tumor irradiation on days 1, 8, 15), and combination group. Mirin was dissolved in DMSO and diluted with normal saline (final DMSO ≤5%) for intraperitoneal injection. Tumor volume was measured every 3 days; mice were euthanized on day 22, and tumor tissues were collected for immunohistochemical analysis [2]
- LPS-induced inflammation model: C57BL/6 mice were intraperitoneally injected with LPS (10 mg/kg) to induce systemic inflammation. Concurrently, Mirin (30 mg/kg, ip, once daily for 5 days) was administered. Serum and liver tissues were collected at endpoint for NO quantification and iNOS protein detection [3]

Mirin (≤30 μM) showed low cytotoxicity to normal human fibroblasts (CCD-18Co) and mammary epithelial cells (MCF-10A), with cell survival >80% after 72 hours [2]
- Acute toxicity in mice: A single intraperitoneal injection of up to 200 mg/kg of mirin did not cause death or significant weight loss (<5%) [2]
- Subchronic toxicity study in mice (21 days): Mirin (50 mg/kg, intraperitoneal injection, once every 3 days for a total of 3 times) did not cause significant changes in serum ALT, AST or creatinine levels, and no pathological damage was observed in the liver, kidneys, heart or lungs [2]
- Mirin (30 mg/kg/day, intraperitoneal injection for a total of 5 times) did not induce significant inflammatory cell infiltration in the liver or spleen of mice, confirming its low immunotoxicity [3]

[1]. Nat Chem Biol . 2008 Feb;4(2):119-25.

[2]. PLoS One . 2013 Oct 2;8(10):e75406.

[3]. Environ Toxicol Pharmacol . 2015 Jul;40(1):12-7.

[4]. Cell Death Dis . 2018 Aug 30;9(9):895.

Mirin is a selective small-molecule MRN complex inhibitor that specifically targets the nuclease activity of MRE11[1][2]. Its core mechanism includes blocking MRN-mediated DNA double-strand break (DSB) excision, inhibiting homologous recombination (HR) repair, and making cancer cells with HR repair capabilities more sensitive to DNA damaging agents (ionizing radiation, chemotherapy)[1][2]. Mirin also has anti-inflammatory activity and can exert its effects by downregulating the expression of iNOS and the production of NO in LPS-stimulated macrophages[3]. It is widely used as a research tool to study the function of MRN complexes, DNA repair pathways, and the efficacy of DNA damage-enhancing cancer therapies[1][4]. Mirin has minimal off-target effects on other nucleases and normal cells, which supports its potential as a radiosensitizer or chemosensitizer in cancer treatment[2].

C10H8N2O2

220.25

220.03

C, 54.53; H, 3.66; N, 12.72; O, 14.53; S, 14.56

1198097-97-0

1206243

Light yellow to yellow solid powder

1.5±0.1 g/cm3

441.6±55.0 °C at 760 mmHg

298 °C

220.8±31.5 °C

0.0±1.1 mmHg at 25°C

1.718

1.36

2

3

1

15

330

0

S1/C(=N/[H])/N([H])C(C1=C([H])C1C([H])=C([H])C(=C([H])C=1[H])O[H])=O

YBHQCJILTOVLHD-YVMONPNESA-N

InChI=1S/C10H8N2O2S/c11-10-12-9(14)8(15-10)5-6-1-3-7(13)4-2-6/h1-5,13H,(H2,11,12,14)/b8-5-

(5Z)-5-[(4-hydroxyphenyl)methylidene]-2-imino-1,3-thiazolidin-4-one

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: ~44 mg/mL (~199.8 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (4.54 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (4.54 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 10.0 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: ≥ 1 mg/mL (4.54 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 10.0 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.)