| Size | Price | Stock | Qty |
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| 5mg |
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| 100mg |
| Targets |
ML334 targets the Keap1-NRF2 protein-protein interaction, specifically binding to the Kelch domain of Keap1 with a Kd of 1 μM. By inhibiting this interaction, ML334 prevents the ubiquitination and proteasomal degradation of NRF2, allowing NRF2 to accumulate and translocate to the nucleus. In the nucleus, NRF2 activates the antioxidant response element (ARE), driving the expression of cytoprotective genes including NQO1, HO-1, and TRX1. This makes ML334 a valuable probe for redox biology and oxidative stress research.
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| ln Vitro |
ML334 (LH601A; 50-100 μM; 6-16 hours; HEK293 cells) treatment elevated NQO1 and TRX1 mRNA by 2- to 3-fold at 6 and 16 hours. It also increased HO-1 mRNA expression four to sevenfold at six hours, with a lesser fold change at sixteen hours [1]. ML334 (LH601A; 50-100 μM; 16 hours) produces HO-1 and TRX1 proteins in HEK293 cells [1]. ML334 (LH601A) activates NRF2 expression and nuclear translocation in HEK293 cells [1].
In vitro, ML334 stimulates NRF2 expression and nuclear translocation and induces antioxidant response element (ARE) activity. In HEK293 cells, treatment with ML334 (50-100 μM) increases NQO1 and TRX1 mRNA 2-3 fold and HO-1 mRNA up to 7 fold, while also inducing HO-1 and TRX1 proteins. The compound binds to the Keap1 Kelch domain with an IC50 of 1 μM and activates ARE in a cell-based assay with an EC50 of 12 μM. ML334 inhibits cardiac fibroblast activation and proliferation, improving myocardial infarction-induced cardiac fibrosis. |
| ln Vivo |
In vivo, ML334 has been investigated for its ability to activate the NRF2 pathway and induce cytoprotective gene expression. By inhibiting the Keap1-NRF2 interaction, ML334 promotes NRF2-mediated antioxidant responses. The compound has been studied for its potential therapeutic effects in conditions associated with oxidative stress, including cardiac fibrosis. However, specific in vivo efficacy data, including dosing regimens and animal models, are not extensively detailed in the available literature. Further studies are needed to fully characterize its in vivo pharmacological profile.
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| Enzyme Assay |
The cell-free assay for ML334 involves evaluating its binding affinity to the Keap1 Kelch domain. Binding assays such as surface plasmon resonance (SPR) or fluorescence polarization are used to determine the Kd value (1 μM). The compound's ability to disrupt the Keap1-NRF2 interaction can be assessed using AlphaScreen or FRET-based assays. IC50 values are determined from dose-response curves by incubating Keap1 with varying concentrations of ML334 and measuring the displacement of labeled NRF2 peptides or the inhibition of complex formation.
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| Cell Assay |
RT-PCR[1]
Cell Types: HEK293 cells Tested Concentrations: 50 μM or 100 μM Incubation Duration: 6 hrs (hours), 16 hrs (hours) Experimental Results: Increased NQO1 and TRX1 mRNAs between 2- and 3-fold at both 6 and 16 h. Enhanced HO- 1 mRNA expression between 4- and 7-fold at 6 h. Western Blot Analysis[1] Cell Types: HEK293 cells Tested Concentrations: 50 μM or 100 μM Incubation Duration: 16 hrs (hours) Experimental Results: Enhanced HO-1 and TRX1 protein expression at 16 h . For in vitro cellular assays, ML334 is typically dissolved in DMSO and diluted in cell culture medium. HEK293 cells or other cell lines are treated with ML334 at concentrations ranging from 50-100 μM. NRF2 nuclear translocation is assessed by immunofluorescence or subcellular fractionation followed by western blotting. Antioxidant response element (ARE) activity is measured using luciferase reporter assays. The expression of NRF2 target genes (NQO1, HO-1, TRX1) is quantified by qPCR and western blotting. |
| Animal Protocol |
In vivo animal studies for ML334 are typically conducted in rodent models of oxidative stress-related diseases. The compound is administered via various routes including intraperitoneal or oral administration. Tissue samples are collected for analysis of NRF2 target gene expression and antioxidant enzyme activities. Disease progression is monitored using appropriate endpoints. However, specific dosing regimens and experimental protocols are not extensively documented in the available literature. Standard protocols for evaluating NRF2 activators would typically be employed.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of ML334 include a molecular weight of 446.50 g/mol and molecular formula C26H26N2O5. The compound has a purity of ≥95%. As a small molecule, it is cell-permeable and is expected to have moderate oral bioavailability. The compound is typically formulated for in vivo administration using appropriate vehicles such as DMSO, PEG300, and saline. Detailed ADME parameters such as half-life, Cmax, and AUC are not extensively reported in the available literature.
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| Toxicity/Toxicokinetics |
The toxicity profile of ML334 has not been extensively characterized in published literature. As an NRF2 activator that upregulates cytoprotective genes, the compound is expected to have a favorable safety profile. Standard preclinical safety studies would include acute and sub-chronic toxicity assessments in rodent models. The compound is intended for research use only and not for therapeutic applications in humans. Standard safety precautions should be followed when handling this compound.
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| References | |
| Additional Infomation |
(1S,2R)-2-[[(1S)-1-[(1,3-dioxo-2-isoindolyl)methyl]-3,4-dihydro-1H-isoquinoline-2-yl]-oxomethyl]-1-cyclohexanecarboxylic acid is a member of the phthalimide class of compounds.
ML334 (LH601A) is a potent, cell-permeable activator of NRF2 that inhibits the Keap1-NRF2 protein-protein interaction by binding to the Keap1 Kelch domain with a Kd of 1 μM. It promotes NRF2 nuclear translocation and induces ARE activity. In HEK293 cells, ML334 (50-100 μM) increases NQO1 and TRX1 mRNA 2-3 fold and HO-1 mRNA up to 7 fold. ML334 is a valuable research probe for redox biology and oxidative stress research. It has not entered clinical trials and is strictly for research purposes. |
| Molecular Formula |
C₂₆H₂₆N₂O₅
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| Molecular Weight |
446.50
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| Exact Mass |
446.184
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| CAS # |
1432500-66-7
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| PubChem CID |
56840728
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| Appearance |
White to off-white solid powder
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| Density |
1.354±0.06 g/cm3(Predicted)
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| Boiling Point |
693.1±55.0 °C(Predicted)
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| LogP |
3
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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 |
4
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| Heavy Atom Count |
33
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| Complexity |
789
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| Defined Atom Stereocenter Count |
3
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| SMILES |
C1CC[C@@H]([C@@H](C1)C(=O)N2CCC3=CC=CC=C3[C@H]2CN4C(=O)C5=CC=CC=C5C4=O)C(=O)O
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| InChi Key |
LNENLABLFGGAFF-BHIFYINESA-N
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| InChi Code |
InChI=1S/C26H26N2O5/c29-23(20-11-5-6-12-21(20)26(32)33)27-14-13-16-7-1-2-8-17(16)22(27)15-28-24(30)18-9-3-4-10-19(18)25(28)31/h1-4,7-10,20-22H,5-6,11-15H2,(H,32,33)/t20-,21+,22-/m1/s1
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| Chemical Name |
(1S,2R)-2-[(1S)-1-[(1,3-dioxoisoindol-2-yl)methyl]-3,4-dihydro-1H-isoquinoline-2-carbonyl]cyclohexane-1-carboxylic acid
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| Synonyms |
ML334; ML-334
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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 : ~44.65 mg/mL (~100.00 mM)
Ethanol : ~44.65 mg/mL (~100.00 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 | 2.2396 mL | 11.1982 mL | 22.3964 mL | |
| 5 mM | 0.4479 mL | 2.2396 mL | 4.4793 mL | |
| 10 mM | 0.2240 mL | 1.1198 mL | 2.2396 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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