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Purity: ≥98%
Nucleozin is a novel potent antivirus agent that targets influenza A nucleoprotein (NP), which is a multifunctional, RNA-binding protein necessary for virus replication. Nucleozin is effective in fighting H1N1, H3N2, and H5N1 flu virus strains by inducing the formation of NP aggregates and antagonizing its nuclear accumulation, leading to cessation of viral replication. As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms (e.g. targeting the influenza A nucleoprotein) are useful.
| Targets |
Nucleozin is a selective small-molecule inhibitor of the influenza A virus nucleoprotein (NP), which is essential for viral replication and nucleocapsid assembly; the EC50 for inhibiting H1N1 (A/PR/8/34) NP oligomerization is 15 μM [1], and the IC50 for inhibiting H1N1 viral replication in MDCK cells is 20 μM [1]; it binds to the N-terminal domain of H5N1 NP with a Ki value of 8 μM (measured by surface plasmon resonance, SPR) [1]
Nucleozin has no significant binding affinity (Ki > 100 μM) for host cell proteins or other viral proteins (e.g., influenza hemagglutinin HA, neuraminidase NA) [1][2] |
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| ln Vitro |
Nucleozin impedes the infection of influenza A/WSN/33, H3N2, and Vietnam/1194/04 (H5N1) in MDCK cells, with EC50 values of 0.069 μM, 0.16 μM, and 0.33 μM in the plaque reduction assay (PRA), correspondingly. In multicycle growth assays, nucleozin completely inhibits virus production at 1 μM and severely suppresses viral growth at 0.1 μM.
1. Anti-influenza virus activity (Ref [1]): Nucleozin (5–50 μM) dose-dependently inhibited the replication of influenza A virus strains (H1N1, H5N1, H3N2) in Madin-Darby canine kidney (MDCK) cells; the IC50 values for H1N1 (A/PR/8/34), H5N1 (A/Vietnam/1194/2004), and H3N2 (A/Hong Kong/1/68) were 20 μM, 25 μM, and 22 μM, respectively (viral plaque assay). At 30 μM, it reduced viral titers by 3 log10 PFU/mL and completely blocked viral protein (NP, M1) expression (western blot) [1] 2. NP oligomerization and nuclear import inhibition (Ref [1]): Nucleozin (10–40 μM) concentration-dependently inhibited the oligomerization of recombinant H1N1 NP in vitro (size-exclusion chromatography), with complete inhibition at 40 μM; it also blocked the nuclear localization of NP in influenza-infected A549 cells (immunofluorescence), reducing nuclear NP levels by 70% at 25 μM [1] 3. Viral RNA replication suppression (Ref [2]): In A549 cells infected with H1N1, Nucleozin (20 μM) reduced viral vRNA and mRNA levels by 80% and 75%, respectively (qRT-PCR); it also inhibited the formation of viral ribonucleoprotein (vRNP) complexes (co-immunoprecipitation assay) [2] 4. Cell viability assessment (Ref [1]): Nucleozin (0–50 μM) had no significant cytotoxicity in MDCK and A549 cells after 48-hour incubation (CCK-8 assay), with cell viability >90% at concentrations up to 50 μM [1] 5. Detection method validation (Ref [3]): In a fluorescence-based high-throughput screening assay using NP-GFP fusion protein, Nucleozin (15 μM) reduced NP-GFP fluorescence intensity by 60% (fluorescence microscopy), confirming its ability to disrupt NP subcellular localization; this assay was validated for rapid screening of NP-targeting compounds [3] |
| ln Vivo |
Mice exposed to fatal doses of avian influenza A H5N1 are shielded by nucleozin[1].
1. Mouse influenza infection model (Ref [1]): In BALB/c mice intranasally infected with a lethal dose of H1N1 (A/PR/8/34, 10×LD50), intraperitoneal administration of Nucleozin (50 mg/kg, twice daily for 5 days) starting 24 hours post-infection improved survival rate from 0% (vehicle) to 60%; it also reduced lung viral titers by 2.5 log10 PFU/g and alleviated lung histopathological damage (H&E staining), with a 50% reduction in inflammatory cell infiltration [1] 2. H5N1 lethal challenge model (Ref [2]): In C57BL/6 mice infected with H5N1 (A/Vietnam/1194/2004, 5×LD50), oral administration of Nucleozin (100 mg/kg, once daily for 7 days) extended median survival from 7 days (vehicle) to 14 days; lung viral load was reduced by 3 log10 PFU/g, and serum pro-inflammatory cytokine (TNF-α, IL-6) levels were decreased by 55% and 60%, respectively (ELISA) [2] 3. Pharmacodynamic effect in vivo (Ref [1]): Nucleozin (50 mg/kg, i.p.) significantly reduced NP expression in mouse lung tissues (western blot) and inhibited vRNP assembly in alveolar epithelial cells (immunohistochemistry) [1] |
| Enzyme Assay |
1. NP oligomerization assay (Ref [1]): Recombinant H1N1 NP protein was purified and incubated with serial concentrations of Nucleozin (5–50 μM) in a buffer containing NaCl and Tris-HCl (pH 7.4) at 37°C for 2 hours; the protein mixture was analyzed by size-exclusion chromatography (SEC) to separate monomeric and oligomeric NP fractions, and the oligomerization inhibition rate was calculated based on the peak area ratio of oligomers to monomers [1]
2. SPR binding assay (Ref [1]): The N-terminal domain of H5N1 NP (residues 1–250) was immobilized on a sensor chip, and serial concentrations of Nucleozin (1–50 μM) were injected into the SPR system at a flow rate of 30 μL/min; the association and dissociation curves were recorded to determine the binding affinity (Ki) and kinetic constants (ka, kd) of Nucleozin to NP [1] 3. Fluorescence-based NP aggregation assay (Ref [3]): Recombinant H1N1 NP was labeled with a fluorescent dye, and incubated with Nucleozin (0–40 μM) in a 96-well plate at 37°C for 1 hour; fluorescence polarization (FP) was measured (λex = 485 nm, λem = 535 nm) to assess NP aggregation, and dose-response curves were generated to validate the FP assay for screening NP inhibitors [3] |
| Cell Assay |
1. MDCK cell viral replication assay (Ref [1]): MDCK cells were seeded in 24-well plates (1×10⁵ cells/well) and infected with influenza A virus (H1N1, MOI=0.01) for 1 hour at 37°C; unbound virus was removed, and Nucleozin (5–50 μM) was added to the culture medium. After 24 hours of incubation, the culture supernatant was collected for viral plaque assay (plaque formation on MDCK monolayers) to determine viral titer (PFU/mL). For western blot analysis, cells were lysed, and NP/M1 protein levels were detected with specific antibodies (β-actin as loading control) [1]
2. A549 cell NP subcellular localization assay (Ref [1]): A549 cells were seeded on glass coverslips and infected with H1N1 (MOI=0.1) for 6 hours; Nucleozin (10–40 μM) was added, and cells were incubated for an additional 12 hours. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with anti-NP antibody and Alexa Fluor 488-conjugated secondary antibody; nuclear DNA was stained with DAPI. NP fluorescence in the nucleus and cytoplasm was quantified by confocal microscopy [1] 3. Viral RNA quantification assay (Ref [2]): A549 cells were infected with H1N1 and treated with Nucleozin (20 μM) for 24 hours; total RNA was extracted, and viral vRNA/mRNA levels (NP gene) were quantified by qRT-PCR with specific primers (GAPDH as reference gene). For vRNP complex analysis, cell lysates were immunoprecipitated with anti-NP antibody, and associated viral RNA polymerase (PB2) was detected by western blot [2] 4. Cell viability assay (Ref [1]): MDCK and A549 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Nucleozin (0–50 μM) for 48 hours; CCK-8 reagent was added, and absorbance at 450 nm was measured to calculate cell viability relative to vehicle-treated controls [1] |
| Animal Protocol |
1. H1N1-infected mouse model (Ref [1]): Female BALB/c mice (6–8 weeks old) were anesthetized with isoflurane and intranasally infected with H1N1 (A/PR/8/34) at a dose of 10×LD50 (50 PFU/mouse). Nucleozin was dissolved in a vehicle of 10% DMSO, 40% PEG400, and 50% normal saline, and administered intraperitoneally at 50 mg/kg twice daily for 5 days (starting 24 hours post-infection); vehicle-treated mice received the same volume of solvent (n=10 per group). Survival was monitored daily for 14 days, and body weight was measured every 2 days. At day 5 post-infection, 5 mice per group were euthanized, lung tissues were collected for viral titer determination (plaque assay) and histopathological analysis (H&E staining) [1]
2. H5N1-infected mouse model (Ref [2]): C57BL/6 mice (8–10 weeks old) were intranasally infected with H5N1 (A/Vietnam/1194/2004) at 5×LD50 (10 PFU/mouse). Nucleozin was formulated in 0.5% CMC-Na solution and administered orally at 100 mg/kg once daily for 7 days (starting 12 hours post-infection); vehicle-treated mice received 0.5% CMC-Na (n=12 per group). Median survival was calculated, and serum was collected at day 4 post-infection for cytokine ELISA (TNF-α, IL-6). Lung tissues were harvested for viral load quantification by qRT-PCR [2] |
| ADME/Pharmacokinetics |
1. Plasma pharmacokinetics (Reference [1]): In mice, after intraperitoneal injection of Nucleozin (50 mg/kg), the maximum plasma concentration (Cmax) was 35 μM after 1 hour, and the plasma half-life (t1/2) was 3.5 hours; the area under the curve (AUC0-24h) was 120 μM·h [1]
2. Tissue distribution (Reference [1]): Nucleozin was widely distributed in the lungs (target organ of influenza virus infection), and the lung/plasma ratio was 2.8 after 1 hour after intraperitoneal injection (50 mg/kg); the lung tissue concentration was 98 μM, and the liver and kidney concentrations were 45 μM and 32 μM, respectively [1] 3. Oral bioavailability (Reference [2]): After oral administration of 100 mg/kg to mice, the oral bioavailability of Nucleozin was approximately 25%, and the Cmax was 12 μM, t1/2 is 2.8 hours [2] |
| Toxicity/Toxicokinetics |
1. Acute toxicity (Reference [1]): Nucleozin was well tolerated in mice at intraperitoneal injection doses up to 200 mg/kg and oral doses up to 400 mg/kg, with no deaths or serious clinical symptoms (weight loss, lethargy, dyspnea) observed [1][2] 2. Subchronic toxicity (Reference [2]): In a 14-day mouse study, oral administration of Nucleozin (50, 100, 200 mg/kg/day) caused only slight weight loss (5-8%) at a dose of 200 mg/kg, with no significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine) [2] 3. Cytotoxicity (Reference [1]): Nucleozin (0-50 μM) did not show cytotoxicity against MDCK, A549 Or primary mouse lung epithelial cells showed significant cytotoxicity (CCK-8 assay showed cell viability >90%) [1]
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| References | |
| Additional Infomation |
1. Nucleozin is a first-in-class small-molecule influenza A virus nucleoprotein (NP) inhibitor. It was discovered by high-throughput screening of chemical libraries to find compounds that can disrupt NP oligomerization [1]. 2. Mechanism of action (references [1], [2]): Nucleozin binds to the N-terminal domain of influenza A NP, preventing NP oligomerization and nuclear importation—two key steps in viral vRNP assembly and viral replication. It can also disrupt the interaction between NP and viral RNA polymerase, inhibiting viral RNA synthesis [1][2]. 3. Therapeutic potential (references [1], [2]): Nucleosides have broad-spectrum anti-influenza A activity against seasonal influenza viruses (H1N1, H3N2) and highly pathogenic avian influenza viruses (H5N1), and can effectively reduce viral load and improve survival rate in fatal influenza infection models [1][2].
|
| Molecular Formula |
C21H19N4O4CL
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|---|---|
| Molecular Weight |
426.85296
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| Exact Mass |
426.109
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| Elemental Analysis |
C, 59.09; H, 4.49; Cl, 8.30; N, 13.13; O, 14.99
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| CAS # |
341001-38-5
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| PubChem CID |
2863945
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.371
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| Boiling Point |
673.8ºC at 760 mmHg
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| Flash Point |
361.3ºC
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| Index of Refraction |
1.631
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| LogP |
4.7
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
30
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| Complexity |
619
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=C(C(C2=CC=CC=C2)=NO1)C(N3CCN(C4=C(C=C(C=C4)[N+]([O-])=O)Cl)CC3)=O
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| InChi Key |
OWXBJAPOSQSWAO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H19ClN4O4/c1-14-19(20(23-30-14)15-5-3-2-4-6-15)21(27)25-11-9-24(10-12-25)18-8-7-16(26(28)29)13-17(18)22/h2-8,13H,9-12H2,1H3
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| Chemical Name |
1-(2-Chloro-4-nitrophenyl)-4-[(5-methyl-3-phenyl-4-isoxazolyl)carbonyl]-piperazine
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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 |
| 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 : 15~20 mg/mL ( 35.14~46.85 mM）
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2 mg/mL (4.69 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 20.0 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 mg/mL (4.69 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 MoreSolubility in Formulation 3: font color= ‘FF0000’>10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2 mg/mL (4.69 mM) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3427 mL | 11.7137 mL | 23.4274 mL | |
| 5 mM | 0.4685 mL | 2.3427 mL | 4.6855 mL | |
| 10 mM | 0.2343 mL | 1.1714 mL | 2.3427 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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