Cell lines

A549 cells

Preparation Method

A549 cells were transfected with a firefly luciferase reporter (Fluc) construct driven by a minimal TATA promoter with upstream NRF2-specific antioxidant response element (ARE) enhancer sequence from human NQO1 promoter ARE and clones stably expressing ARE-FLuc7 were screened and validated.

Reaction Conditions

Cells were cultured in the presence of ML385 (5 μM) for 48h and 72 h and measured the changes in the expression levels of NRF2 and its target genes.

Applications

ML385 treatment to A549 cells demonstrated a reduction in glutathione synthesis and recycling enzymes, members of the thioredoxin family, and glucose metabolism-related genes with a time-dependent manner. ML385 treatment significantly attenuated NQO1 enzyme activity and reduced GSH levels along with cellular antioxidant capacity.

Animal models

1-8-week-old C57B/6 male mice

Preparation Method

Mice received a daily intraperitoneal injection of ML385 (30 mg/kg) dissolved in PBS with 5% Dimethyl Sulfoxide (DMSO) for 7 d.

Dosage form

30 mg/kg

Applications

ML385 could inhibit Nrf2 and used to explore the mechanism of Nrf2-NF-κB signaling pathway involved in the inhibition of astrocyte activation. Mice treated with ML385 had a significant decrease in Keap1, HO-1, Nrf2, and p-P65/P-65 (P < 0.05) expression compared to Sham or Vehicle control groups.

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ML385 is a specific nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor. ML385 binds to NRF2 and inhibits its downstream target gene expression as a probe molecule. Specifically, ML385 binds to the Neh1, the Cap ‘N’ Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation leading to gain of NRF2 function.^[1][2]

In vitro study demonstrated that ML385 potentially inhibits NRF2 through direct interaction. The NRF2 signaling was decreased in a time-dependent manner and the maximum decline was at 72 h. A reduction in NRF2 mRNA levels was also observed. In addition, ML385 caused global inhibition of NRF2 signaling in lung cancer cells with KEAP1 mutations, and other target genes. ML385 treatment also significantly attenuated NQO1 enzyme activity and reduced GSH levels along with cellular antioxidant capacity.^[2]

In vivo study of ML385 indicated that it inhibits NRF2 and showed promising anti-tumor activity. ML385 in combination with carboplatin showed a significant reduction in tumor growth compared to vehicle. Although the treatment with a single agent (either ML385 or carboplatin) led to a reduction in tumor growth, the magnitude of these effects was variable between cell lines and did not reach statistical significance. Moreover, tumor samples treated with ML385 showed a significant reduction in NRF2 protein level and its downstream target genes. Besides, the addition of ML385 decreased anisotropy in a dose-dependent manner, with an IC50 of 1.9 μM, suggesting that the NRF2-MAFG protein complex was dissociated from fluorescein-labeled ARE-DNA.^[2]

References:
[1]. Xian P, et al. Mesenchymal stem cell-derived exosomes as a nanotherapeutic agent for amelioration of inflammation-induced astrocyte alterations in mice. Theranostics. 2019 Aug 14;9(20):5956-5975.
[2]. Singh A, et al. Small Molecule Inhibitor of NRF2 Selectively Intervenes Therapeutic Resistance in KEAP1-Deficient NSCLC Tumors. ACS Chem Biol. 2016 Nov 18;11(11):3214-3225.