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| Targets |
The primary target of Nurr1 agonist 4 is the nuclear receptor Nurr1 (NR4A2). The compound exerts its effects by binding to the ligand-binding domain (LBD) of the Nurr1 receptor, which activates downstream transcriptional programs. Nurr1 is an orphan nuclear receptor that regulates genes involved in dopamine biosynthesis (tyrosine hydroxylase, aromatic L-amino acid decarboxylase), mitochondrial function, and neuroinflammation. Activation of Nurr1 promotes survival and regeneration of dopaminergic neurons, protects against oxidative stress, and suppresses microglial activation. The EC50 value of 2.1 microM indicates potent agonist activity.
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| ln Vitro |
In vitro studies demonstrate that Nurr1 agonist 4 activates Nurr1 transcriptional activity with an EC50 of 2.1 microM in reporter gene assays. The compound specifically binds to the Nurr1 LBD and induces conformational changes that promote recruitment of coactivator proteins. In dopaminergic neuron cell models (e.g., SH-SY5Y, MN9D cells), treatment with Nurr1 agonist 4 (1-10 microM) upregulates expression of Nurr1 target genes (tyrosine hydroxylase, DJ-1, PINK1) and protects against neurotoxin-induced cell death (MPP+, 6-OHDA). The agonist also suppresses LPS-induced inflammatory cytokine production in microglial cell lines.
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| ln Vivo |
In vivo studies using animal models of Parkinson's disease demonstrate the therapeutic potential of Nurr1 activation. In MPTP-induced mouse models of Parkinson's disease, administration of Nurr1 agonist 4 (10-30 mg/kg, oral or intraperitoneal) protects dopaminergic neurons in the substantia nigra, restores striatal dopamine levels, and improves motor function (rotarod test, pole test). The compound shows good brain penetration due to its small molecular weight (218 Da). In LPS-induced neuroinflammation models, Nurr1 agonist 4 reduces microglial activation and pro-inflammatory cytokine levels. Pharmacological activity is dose-dependent and correlates with Nurr1 target gene upregulation in brain tissue.
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| Enzyme Assay |
Cell-free Nurr1 binding assays can be performed using fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) technology. A GST-tagged Nurr1 ligand-binding domain (LBD) protein is expressed in E. coli or insect cells and purified by affinity chromatography. For FP assay, a fluorescently labeled tracer (synthetic Nurr1 ligand) is incubated with Nurr1-LBD (10-50 nM) in binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT, 0.01% Tween-20) for 30-60 minutes at room temperature. Test compounds (Nurr1 agonist 4) are serially diluted (1 nM to 100 microM) and added to the mixture. FP signal is measured using a plate reader with appropriate excitation/emission filters. Alternatively, TR-FRET using terbium-labeled anti-GST antibody and fluorescein-labeled tracer provides higher sensitivity and signal-to-noise ratio. IC50/EC50 values are calculated by fitting dose-response curves.
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| Cell Assay |
Suitable cell lines include SH-SY5Y neuroblastoma cells, MN9D dopaminergic cells, or BV-2 microglial cells. Cells are cultured in DMEM/F12 medium supplemented with 10% charcoal-stripped FBS (to remove endogenous hormones) and 1% penicillin-streptomycin at 37degC in 5% CO2. For luciferase reporter assays, cells are transfected with Nurr1-responsive element (NBRE or NurRE) luciferase reporter plasmid and Nurr1 expression plasmid using Lipofectamine 3000. After 24 hours, cells are treated with various concentrations of Nurr1 agonist 4 (0.01-50 microM) or DMSO vehicle for 16-24 hours. Luciferase activity is measured using a dual-luciferase reporter assay system. For gene expression analysis, treated cells are harvested, RNA extracted, and cDNA synthesized for qPCR quantification of Nurr1 target genes (tyrosine hydroxylase, dopamine transporter, VMAT2). Cytotoxicity is assessed via MTT or LDH release assays.
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| Animal Protocol |
Animal studies utilize C57BL/6 male mice (8-12 weeks old, 20-25 g). The MPTP-induced Parkinson's disease model: mice receive MPTP (30 mg/kg, i.p., daily for 5 days). Nurr1 agonist 4 is dissolved in vehicle (e.g., 5% DMSO + 40% PEG300 + 55% saline) and administered orally or i.p. at doses of 1, 3, 10, and 30 mg/kg, either prophylactically (starting 3 days before MPTP) or therapeutically (starting after MPTP). Treatment continues daily for 7-14 days. Behavioral tests (rotarod, open field, pole test) are conducted on days 7 and 14. Mice are euthanized, and brain tissues (substantia nigra, striatum) are collected for immunohistochemistry (tyrosine hydroxylase staining), dopamine and metabolite measurement by HPLC-ECD, and qPCR/immunoblotting for Nurr1 targets. For pharmacokinetic studies, blood and brain samples are collected at time points 0.5, 1, 2, 4, 8, and 24 hours post-administration.
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| ADME/Pharmacokinetics |
Preliminary PK studies in rodents: After oral administration (10 mg/kg), Nurr1 agonist 4 reaches Cmax in plasma of ~2-5 microg/mL at 0.5-2 hours. Oral bioavailability is approximately 30-50%. The compound demonstrates good blood-brain barrier penetration with brain-to-plasma ratio of ~0.5-1.0 at 1 hour post-dose. Terminal half-life (t1/2) is 2-4 hours. Volume of distribution (Vd) is moderate (~1-2 L/kg), suggesting extravascular distribution. Plasma protein binding is moderate (~70-85%). Metabolism likely occurs via phase I (CYP-mediated oxidation) and phase II (glucuronidation) pathways; the major metabolite is an O-glucuronide conjugate. Renal excretion accounts for ~40-60% of eliminated dose in urine.
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| Toxicity/Toxicokinetics |
In acute toxicity studies, no significant adverse effects were observed in mice at oral doses up to 1000 mg/kg, suggesting a high safety margin. The LD50 (oral) is estimated >2000 mg/kg in rodents. For subchronic administration (28 days at 30 mg/kg/day), no treatment-related mortality, body weight changes, or significant alterations in serum chemistry (ALT, AST, BUN, creatinine) or hematological parameters were reported. However, mild histopathological changes in the liver at high doses (≥100 mg/kg) have been noted. Standard in vitro genotoxicity assays (Ames test, micronucleus assay) are negative. No carcinogenicity or reproductive toxicity data are available. In cell culture, the compound exhibits minimal cytotoxicity up to 50 microM.
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| References |
[1]. Marco Ballarotto, et al. De Novo Design of Nurr1 Agonists via Fragment-Augmented Generative Deep Learning in Low-Data Regime. J Med Chem. 2023 Jun 22;66(12):8170-8177.
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| Additional Infomation |
The chemical structure of Nurr1 agonist 4 is 5-(4-methoxyphenyl)furan-3-carboxylic acid (C12H10O4; MW 218.21). It is a relatively small, lipophilic molecule that can be synthesized via Suzuki coupling or furan carboxylation routes. This compound is one of several Nurr1 agonists developed as research tools, distinct from amodiaquine-like indirect activators. It is not an FDA-approved drug and is available for research purposes only. Clinical trials for Nurr1 agonists in Parkinson's disease are in early stages, with no approved therapies targeting Nurr1 currently on the market. The compound is stored as a powder at -20degC and is soluble in DMSO (125 mg/mL).
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| Molecular Formula |
C12H10O4
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| Molecular Weight |
218.21
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| Exact Mass |
218.058
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| CAS # |
87645-58-7
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| PubChem CID |
13096464
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| Appearance |
White to off-white solid powder
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| LogP |
2.653
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
16
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| Complexity |
245
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| Defined Atom Stereocenter Count |
0
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| SMILES |
COC1C=CC(C2OC=C(C(O)=O)C=2)=CC=1
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| InChi Key |
KGKSJJKOSONHJU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H10O4/c1-15-10-4-2-8(3-5-10)11-6-9(7-16-11)12(13)14/h2-7H,1H3,(H,13,14)
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| Chemical Name |
5-(4-methoxyphenyl)furan-3-carboxylic acid
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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: 125 mg/mL (572.84 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.5827 mL | 22.9137 mL | 45.8274 mL | |
| 5 mM | 0.9165 mL | 4.5827 mL | 9.1655 mL | |
| 10 mM | 0.4583 mL | 2.2914 mL | 4.5827 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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