NRF2 (IC50 = 1.9 μM)

ML385 exhibits anti-tumor activity in NSCLC (subcutaneous and orthotopic NSCLC models) both as a single agent and in combination with carboplatin. ML385 has a half-life (t1/2 = 2.82 h) after IP injection (30 mg/kg), according to the PK profile examined in CD-1 mice[1]. The pancreatic injury might be reduced by ML385[2].

ML385 in combination with DNA alkylating agent carboplatin leads to a significant reduction in tumor cell proliferation, as demonstrated by fewer Ki-67 positive cells. NRF2 protein levels and its downstream target genes are significantly decreased in tumor samples treated with ML385.

Nickel pull-down streptavidin-HRP assay[1]
Full-length NRF2 (1-605 AA), Neh1, the Cap-n-collar (CNC) bZip domain of NRF2 (434–561 AA) and ΔNeh1 fragments were cloned in a pET14B expression vector. The excess amount of purified histidine-tagged NRF2 proteins was bound to the pre-charged and pre-equilibrated Ni-NTA beads and was incubated for 30 min on ice. After incubation, the NRF2-bound NTA-resin was washed (3×) with PBS. Subsequently, biotin-labeled ML385 or control compounds were added at a concentration of 10 μM. After 1 h incubation on ice, beads with protein were washed (3×) with PBS. For the competition assay, ML385 and compound 3 were added at a concentration of 10 μM, incubated on ice, and washed (3×) with PBS. Next, 5 μg of horseradish peroxidase (HRP)-conjugated streptavidin was added to the tube, followed by a 30-min incubation on ice, followed by an 8× wash with PBS. Lastly, bound protein-drug complex was eluted with PBS containing 10 mM EDTA, mixed 1:1 with SuperSignal West PICO solution, and the HRP activity was measured using well-scan mode in a Flexistation-3.

NQO1 enzyme activity measurement[1]
Cells were treated with vehicle or ML385 for 72 h. The enzyme activity in the total protein lysate was determined as described previously.

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Total antioxidant capacity and GSH measurement[1]
Cells were treated with vehicle or ML385 for 72 h. The total antioxidant capacity and glutathione levels were measured using antioxidant and glutathione assay kits, respectively.

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Caspase activity assay[1]
Caspase activity was measured using the Caspase-Glo® 3/7 Assay kit as per the manufacturer’s instructions. The CellTiter-Blue assay was utilized to quantify cell density and to normalize caspase activity. Briefly, cells were treated with ML385 for 36 h. An equal amount of CellTiter-Blue reagent was added to the wells and the fluorescence was measured after 30 min. The CellTiter-Blue reagent was discarded and the Caspase-Glo (100 μL) reagent was added to the cells and incubated at 37°C for an additional 60–90 min. The resulting luminescence was recorded and the caspase activity was normalized to cell number.

8-week-old C57B/6 male mice
30 mg/kg; 7 days
Intraperitoneal injection

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Pharmacokinetic analysis of ML385 in CD-1 mice[1]
For pharmacokinetic analysis, male CD-1 mice (n=3/time point) were administered a 30 mg/kg intraperitoneal (IP) dose of (vehicle: Solutol/Cremophor EL/polyethylene glycol 400/water [15/10/35/40,v/v/v/v]) of ML385. Blood samples were collected at pre-treatment, 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h and plasma samples were harvested. Plasma concentration of ML385 was determined using a qualified LC-MS/MS. A simulation was conducted to predict the in vivo exposure after a multiple-dose treatment based on the single-dose study results.

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Determination of ML385 concentration in tumor samples[1]
An UPLC-MS/MS method was developed to determine the concentration of ML385 in tumor samples. The details are included in the supplementary methods section.

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Establishment of tumor xenografts and treatment[1]
Tumor xenografts were established as described previously18. A549 cells (5.0×106) and H460 cells (1.0×106) were injected subcutaneously into the flank of athymic nude mice and the tumor dimensions were measured by caliper at an interval of 3–5 days18. The tumor volumes were calculated using the following formula: [length (mm) × width (mm) × width (mm) × 0.5]. Once the tumor volumes were approximately 50–100 mm3, mice were randomly allocated into 4 groups: vehicle, ML385, carboplatin, and ML385 in combination with carboplatin. Vehicle, carboplatin (5 mg/kg daily Monday to Friday)18, ML385 (30 mg/kg daily Monday to Friday), or ML385 in combination with carboplatin were administered intraperitoneally for 3 weeks. At the end of treatment period, mice were sacrificed and the tumor, blood, lung, and liver samples were collected.[1]

For the orthotopic lung tumor model, A549 (1.0×106) and H460 cells (1.0×106) were diluted 1:1 in matrigel (30 μL) and were injected directly into the lungs. Ten days post-cell implantation, mice were imaged. Mice with visible localized lung tumor were randomly divided into 4 groups: vehicle, ML385, carboplatin, and ML385+carboplatin. Vehicle, carboplatin, ML385, or ML385 in combination with carboplatin were administered intraperitoneally for 2 weeks using the same regimen as described above. High-resolution lung micro-computed tomography (CT) images were acquired in 512 projections (270 μA, 75 kVp), and the data were reconstructed using the ordered subsets-expectation maximization algorithm. Volume-rendered whole lung images were generated using Amira 5.3.0 software. For each mouse, pretreatment available lung volume was defined as 100% compared to post-treatment lung volumes.

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Treatment with a Nrf2/HO-1 Pathway Inhibitor[2]
A Nrf2 inhibitor (ML385) or a HO-1 inhibitor (ZnPP) was used to inhibit the Nrf2/HO-1 antioxidant pathway in vivo. ML385 was dissolved in 100% DMSO to prepare a stock solution and then diluted it into 5% DMSO solution with PBS before being used. ZnPP was dissolved as follows: 2.5 mg ZnPP was dissolved in 0.33 ml NaOH (0.2 M) in a dark room, and 0.2 M HCl was added to adjust the pH to 7.0. Finally, saline was added to 5 ml (0.5 mg/ml).[2]

ML385 (30 mg/kg) or ZnPP (5 mg/kg) pretreatment was administered intraperitoneally 1 h before administration of caerulein, and the mice in the control group were treated with vehicle. In the MAP model, high-dose ISL (200 mg/kg) was administered after the first caerulein injection immediately to identify the underlying molecular mechanisms of ISL on AP.[2]

[1]. Small molecule inhibitor of NRF2 selectively intervenes therapeutic resistance in KEAP1-deficient NSCLC tumors. ACS Chem Biol. 2016 Nov 18;11(11):3214-3225.

[2]. Isoliquiritigenin Ameliorates Acute Pancreatitis in Mice via Inhibition of Oxidative Stress and Modulation of the Nrf2/HO-1 Pathway. Oxid Med Cell Longev. 2018 Apr 26:2018:7161592.

[3]. Nrf2 protects against seawater drowning-induced acute lung injury via inhibiting ferroptosis. Respir Res . 2020 Sep 9;21(1):232.

Loss of function mutations in Kelch Like ECH Associated Protein 1 (KEAP1) or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) are common in non-small cell lung cancer (NSCLC) and is associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ~400,000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. 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. In clonogenic assays, when used in combination with platinum-based drugs such as doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells compared to single agents alone. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant anti-tumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.[1]

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Oxidative stress plays a crucial role in the pathogenesis of acute pancreatitis (AP). Isoliquiritigenin (ISL) is a flavonoid monomer with confirmed antioxidant activity. However, the specific effects of ISL on AP have not been determined. In this study, we aimed to investigate the protective effect of ISL on AP using two mouse models. In the caerulein-induced mild acute pancreatitis (MAP) model, dynamic changes in oxidative stress injury of the pancreatic tissue were observed after AP onset. We found that ISL administration reduced serum amylase and lipase levels and alleviated the histopathological manifestations of pancreatic tissue in a dose-dependent manner. Meanwhile, ISL decreased the oxidative stress injury and increased the protein expression of the Nrf2/HO-1 pathway. In addition, after administering a Nrf2 inhibitor (ML385) or HO-1 inhibitor (zinc protoporphyrin) to block the Nrf2/HO-1 pathway, we failed to observe the protective effects of ISL on AP in mice. Furthermore, we found that ISL mitigated the severity of pancreatic tissue injury and pancreatitis-associated lung injury in a severe acute pancreatitis model induced by L-arginine. Taken together, our data for the first time confirmed the protective effects of ISL on AP in mice via inhibition of oxidative stress and modulation of the Nrf2/HO-1 pathway.[2]

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Background: Ferroptosis is a new type of nonapoptotic cell death model that was closely related to reactive oxygen species (ROS) accumulation. Seawater drowning-induced acute lung injury (ALI) which is caused by severe oxidative stress injury, has been a major cause of accidental death worldwide. The latest evidences indicate nuclear factor (erythroid-derived 2)-like 2 (Nrf2) suppress ferroptosis and maintain cellular redox balance. Here, we test the hypothesis that activation of Nrf2 pathway attenuates seawater drowning-induced ALI via inhibiting ferroptosis. Methods: we performed studies using Nrf2-specific agonist (dimethyl fumarate), Nrf2 inhibitor (ML385), Nrf2-knockout mice and ferroptosis inhibitor (Ferrostatin-1) to investigate the potential roles of Nrf2 on seawater drowning-induced ALI and the underlying mechanisms. Results: Our data shows that Nrf2 activator dimethyl fumarate could increase cell viability, reduced the levels of intracellular ROS and lipid ROS, prevented glutathione depletion and lipid peroxide accumulation, increased FTH1 and GPX4 mRNA expression, and maintained mitochondrial membrane potential in MLE-12 cells. However, ML385 promoted cell death and lipid ROS production in MLE-12 cells. Furthermore, the lung injury became more aggravated in the Nrf2-knockout mice than that in WT mice after seawater drowning. Conclusions: These results suggested that Nrf2 can inhibit ferroptosis and therefore alleviate ALI induced by seawater drowning. The effectiveness of ferroptosis inhibition by Nrf2 provides a novel therapeutic target for seawater drowning-induced ALI.[3]

C29H25N3O4S

511.60

511.156

C, 68.08; H, 4.93; N, 8.21; O, 12.51; S, 6.27

846557-71-9

1383822

White to yellow solid powder

1.4±0.1 g/cm3

1.693

5.47

1

6

5

37

844

0

O=C(N1CCC2C1=CC=C(C1=C(C)SC(NC(CC3C=C4C(OCO4)=CC=3)=O)=N1)C=2)C1C(C)=CC=CC=1

LINHYWKZVCNAMQ-UHFFFAOYSA-N

InChI=1S/C29H25N3O4S/c1-17-5-3-4-6-22(17)28(34)32-12-11-20-15-21(8-9-23(20)32)27-18(2)37-29(31-27)30-26(33)14-19-7-10-24-25(13-19)36-16-35-24/h3-10,13,15H,11-12,14,16H2,1-2H3,(H,30,31,33)

2-(1,3-benzodioxol-5-yl)-N-[5-methyl-4-[1-(2-methylbenzoyl)-2,3-dihydroindol-5-yl]-1,3-thiazol-2-yl]acetamide

ML385; ML 385; ML385; 846557-71-9; ML-385; 2-(benzo[d][1,3]dioxol-5-yl)-N-(5-methyl-4-(1-(2-methylbenzoyl)indolin-5-yl)thiazol-2-yl)acetamide; 2-Benzo[1,3]dioxol-5-yl-N-{5-methyl-4-[1-(2-methyl-benzoyl)-2,3-dihydro-1H-indol-5-yl]-thiazol-2-yl}-acetamide; SMR000173724; 2-(1,3-benzodioxol-5-yl)-N-[5-methyl-4-[1-(2-methylbenzoyl)-2,3-dihydroindol-5-yl]-1,3-thiazol-2-yl]acetamide; N-[4-[2,3-Dihydro-1-(2-methylbenzoyl)-1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide; ML-385

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.89 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.07 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 20.8 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.

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Solubility in Formulation 3: 6% DMSO+40% PEG 300+5%Tween80+ 49%ddH2O: 1.5mg/ml

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Solubility in Formulation 4: 10 mg/mL (19.55 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 9.01 mg/mL (17.61 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 6: 5 mg/mL (9.77 mM) in 15% Solutol HS 15 10% Cremophor EL 35% PEG 400 40% water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)