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| Targets |
(R,S,R)-ML334 itself is the inactive isomer and does not bind to Keap1 or activate NRF2. The active isomer, ML334 (LH601A), targets the Kelch domain of Keap1 (Kelch-like ECH-associated protein 1). Keap1 is a substrate adaptor protein for the CUL3-based E3 ubiquitin ligase that targets NRF2 for ubiquitination and proteasomal degradation. ML334 is a potent, cell-permeable inhibitor of the Keap1-NRF2 protein-protein interaction, binding to Keap1 with a Kd of 1 microM and stimulating NRF2 expression and nuclear translocation. (R,S,R)-ML334 is the isomer of ML334 and can be used as an experimental control.
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| ln Vitro |
In vitro, (R,S,R)-ML334 is the inactive isomer and has no significant activity in NRF2 activation assays. ML334 (active isomer) binds to the Keap1 Kelch domain with a Kd of 1 microM, stimulates NRF2 expression and nuclear translocation, and induces antioxidant response elements (ARE) activity. (R,S,R)-ML334 serves as an essential negative control in cellular assays to confirm that the observed NRF2 activation and downstream effects are due to specific Keap1 binding and not to non-specific or off-target effects. (R,S,R)-ML334 is the inactive isomer of ML334.
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| ln Vivo |
As the inactive isomer, (R,S,R)-ML334 has no relevant in vivo activity. The active S-enantiomer, ML334 (LH601A), has been studied in animal models of oxidative stress and inflammation. By activating NRF2, ML334 induces the expression of antioxidant enzymes such as NQO1, HO-1, and GCL, providing protection against oxidative damage. (R,S,R)-ML334 is used as an inactive control in such studies to validate the specific mechanism of action of the active compound. The compound is the isomer of ML334 and can be used as an experimental control.
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| Enzyme Assay |
An in vitro biochemical assay for Keap1-NRF2 interaction is a fluorescence polarization (FP) competition binding assay. A FITC-labeled NRF2 peptide (containing the ETGE motif) and recombinant Keap1 Kelch domain protein are mixed. The test compound is serially diluted and added to the mixture. The degree of polarization is measured. The half-maximal inhibitory concentration (IC50) is calculated. For (R,S,R)-ML334, the IC50 is expected to be high (weak binding), while the active ML334 binds with high affinity (Kd = 1 microM). The compound can be used as a control.
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| Cell Assay |
A cellular assay for NRF2 activation is a reporter gene assay. HEK293 cells are co-transfected with an antioxidant response element (ARE)-luciferase reporter plasmid and a control Renilla plasmid. After 24 hours, cells are treated with varying concentrations of ML334 (active) or (R,S,R)-ML334 (inactive control) for 16-24 hours. Luciferase activity is measured. For active ML334, an increase in ARE-luciferase activity is expected, while (R,S,R)-ML334 should show minimal activity, confirming the stereospecificity of Keap1 binding. (R,S,R)-ML334 can be used as an experimental control.
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| Animal Protocol |
(R,S,R)-ML334 is not intended for in vivo use as a therapeutic agent. The active ML334 (LH601A) can be studied in mouse models of oxidative stress, such as a model of acute kidney injury (AKI) induced by cisplatin or ischemia-reperfusion (I/R). Mice are treated with ML334 (5-20 mg/kg, i.p. or oral) before or after the injury. (R,S,R)-ML334 is used as an inactive control. Endpoints include serum creatinine and BUN levels (for kidney function), renal histology (H&E, PAS), markers of oxidative stress (malondialdehyde, glutathione levels), and expression of NRF2 target genes (NQO1, HO-1, GCLC) in kidney tissue by qPCR and Western blot. The active compound protects against injury, while (R,S,R)-ML334 should have no effect.
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| ADME/Pharmacokinetics |
(R,S,R)-ML334 has a molecular weight of 446.50 and a molecular formula of C26H26N2O5. It is a solid powder with a purity of ≥98.5%. It is soluble in DMSO (e.g., 10 mM stock). For in vivo studies, the active ML334 can be formulated in vehicles such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, or 0.5% CMC-Na. The pharmacokinetic properties (half-life, bioavailability) of the active ML334 have been reported; it has good oral bioavailability and a moderate half-life in rodents. (R,S,R)-ML334 is expected to have similar PK properties but no efficacy.
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| Toxicity/Toxicokinetics |
As the inactive isomer of an NRF2 activator, (R,S,R)-ML334 is expected to have lower toxicity than the active compound. The active NRF2 activator, ML334, is generally well-tolerated in animal studies at therapeutic doses. Since (R,S,R)-ML334 does not activate NRF2, it is unlikely to produce the on-target effects of the active compound, which include NRF2 activation and upregulation of antioxidant enzymes. Standard safety precautions for handling small molecules should be followed, including the use of personal protective equipment (gloves, lab coat, safety goggles).
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| References |
[1]. Wen X, et al. Activation of NRF2 Signaling in HEK293 Cells by a First-in-Class Direct KEAP1-NRF2 Inhibitor. J Biochem Mol Toxicol. 2015 Jun;29(6):261-6.
[2]. Hu L, et al. Discovery of a small-molecule inhibitor and cellular probe of Keap1-Nrf2 protein-protein interaction. Bioorg Med Chem Lett. 2013 May 15;23(10):3039-43. |
| Additional Infomation |
(R,S,R)-ML334 (CAS 1432065-33-2) is the inactive stereoisomer of ML334 (LH601A), a potent, cell-permeable activator of the transcription factor NRF2. NRF2 is a master regulator of the cellular antioxidant response, and its activation is a therapeutic strategy for diseases involving oxidative stress, such as chronic kidney disease, chronic obstructive pulmonary disease, and neurodegenerative disorders. ML334 acts as a direct inhibitor of the Keap1-NRF2 protein-protein interaction, binding to the Kelch domain of Keap1 with a Kd of 1 microM. This technology (direct PPI inhibitors) represents a new class of NRF2 activators, distinct from electrophilic activators like sulforaphane or bardoxolone methyl. (R,S,R)-ML334 is used as a control compound in research to demonstrate stereoselectivity and confirm on-target mechanisms. It is strictly a research chemical and is not approved for any therapeutic use.
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| Molecular Formula |
C26H26N2O5
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| Molecular Weight |
446.50
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| CAS # |
1432065-33-2
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| Related CAS # |
ML334;1432500-66-7
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| Appearance |
White to off-white solid powder
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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: 100 mg/mL (223.96 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (11.20 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 50.0 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. Solubility in Formulation 2: ≥ 5 mg/mL (11.20 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (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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