ML385

NRF2 (IC50 = 1.9 μM)

体外活性：ML385 与 NRF2 相互作用并影响 NRF2-MAFG 蛋白复合物的 DNA 结合活性。添加 ML385 会剂量依赖性地降低各向异性，IC50 为 1.9 μM。观察到 NRF2 转录活性呈剂量依赖性降低，ML385 的最大抑制浓度为 5 μM。 ML385 处理导致 KEAP1 突变 H460 细胞中 NRF2 和下游靶基因表达选择性显着降低。 ML385 选择性影响肺癌细胞的集落形成能力或生长，并获得 NRF2 功能激酶测定：ML385 是一种特异性核因子红细胞 2 相关因子 2 (NRF2) 抑制剂，IC50 为 1.9 μM。细胞测定：首先用ML385处理细胞36小时，然后加入等量的CellTiter-Blue试剂，30分钟后测量荧光。弃去 CellTiter-Blue 试剂，将 Caspase-Glo (100 μL) 试剂添加到细胞中，并在 37°C 下再孵育 60-90 分钟。记录产生的发光并将半胱天冬酶活性标准化为细胞数。 MCE 尚未独立证实这些方法的准确性。它们仅供参考。

ML385 与 DNA 烷化剂卡铂联合使用可显着减少肿瘤细胞增殖，Ki-67 阳性细胞减少就证明了这一点。用 ML385 处理的肿瘤样本显示 NRF2 蛋白水平及其下游靶基因显着降低。

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ML385在非小细胞肺癌中显示出抗肿瘤活性，无论是单独使用还是与卡铂联合使用[1]
为了确定ML385是否具有适合体内研究的药代动力学（PK）特征，我们以30mg/kg IP给CD-1小鼠服用。PK曲线显示，ML385在IP注射后具有半衰期（t1/2=2.82小时），支持其在体内疗效研究中的使用（补充图7）。为了确定在细胞培养中观察到的ML385和卡铂的组合是否可以在体内重现，我们使用A549和H460细胞进行了皮下异种移植物实验。小鼠服用ML385、卡铂或ML385与卡铂联合用药3-4周，每两周测量一次肿瘤体积。与载体相比，用ML385联合卡铂治疗的A549和H460肿瘤在两种细胞系中的肿瘤生长均显著减少。尽管使用单一药物（ML385或卡铂）治疗导致肿瘤生长减少，但这些影响的程度在细胞系之间是可变的，没有达到统计学意义（图6a-d，补充图8a-b）。这些结果与NRF2 siRNA18与化疗药物联合使用的先前发现一致。在最后一次用ML385治疗后4-6小时，分析肿瘤样本对ML385的暴露情况。我们在单药和联合治疗队列中检测到肿瘤内浓度约为1μM的ML385。小鼠耐受了联合治疗，血清样本中肝脏和毒性相关标志物的分析显示没有明显的毒性迹象（补充表3）。ML385与卡铂联合使用导致肿瘤细胞增殖显著减少，表现为Ki-67阳性细胞减少（图6e，补充图8c）。肿瘤样本的RT-PCR和免疫印迹分析用于确定抗肿瘤活性是否与ML385对NRF2信号传导的药效学标志物的调节相关。用ML385处理的肿瘤样本显示NRF2蛋白水平及其下游靶基因显著降低（图6f-g）。通过电感耦合等离子体质谱法（ICP-MS）测定单独使用卡铂或ML385与卡铂联合治疗的A549和H460肿瘤中的铂水平，发现联合治疗的肿瘤中铂水平高出约2倍（图6h）。总的来说，这些结果表明，ML385部分通过阻断NRF2依赖的药物解毒途径来增强卡铂的细胞毒性活性，从而导致药物在肿瘤中的滞留增加。ML385与卡铂联合的抗肿瘤活性在独立研究者的实验室中使用H460异种移植物进行了复制。

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ML385和卡铂联合治疗阻断原位人肺肿瘤生长[1]
我们评估了ML385单独和联合卡铂在人类NSCLC原位模型中的治疗效果，该模型密切再现了肺癌进展的临床模式。在我们的模型中，我们试图在左肺或右肺内建立一个单一的肿瘤。动物接受了显微CT成像，具有约3-7mm结节的小鼠被随机分配到治疗组。在A549肺癌原位模型中，与治疗前相比，用载体治疗的小鼠在3周时的肺容量为34%。单药卡铂或ML385治疗组在3周时的肺容量分别为治疗前的42%和57%（图7a-b）。尽管载体和单一药物（卡铂和ML385）治疗组之间的肺体积差异没有达到统计学意义，但载体治疗组的小鼠在3周后立即死亡，而ML385或卡铂治疗组的老鼠存活下来。根据无肿瘤肺容量确定，卡铂和ML385联合治疗的抗肿瘤和抗转移作用明显高于赋形剂或卡铂单一治疗，联合治疗开始后3周肺容量保留率为74%

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H460荷瘤小鼠肺部的显微CT分析显示，接受ML385单一疗法治疗的小鼠在治疗后2周的肺体积为治疗前的64%。与治疗前相比，卡铂治疗产生了50%的肺体积，这与ML385组的肺体积没有显著差异（图7c–d）。同样，当ML385和卡铂联合使用时，抗肿瘤作用比卡铂单一疗法显著增强，在治疗后2周，小鼠的肺容量为治疗前的73%（图7d；补充图10b）。总体而言，这些数据表明，ML385与卡铂联合使用在原位非小细胞肺癌模型中具有显著的体内疗效[1]。

镍下拉链霉抗生物素-HRP测定[1]
将全长NRF2（1-605 AA）、Neh1、NRF2的Cap-n-collar（CNC）bZip结构域（434-561 AA）和ΔNeh1片段克隆到pET14B表达载体中。将过量纯化的组氨酸标记的NRF2蛋白结合到预充电和预平衡的Ni-NTA珠上，并在冰上孵育30分钟。孵育后，用PBS洗涤NRF2结合的NTA树脂（3次）。随后，以10μM的浓度加入生物素标记的ML385或对照化合物。在冰上孵育1小时后，用PBS洗涤含有蛋白质的珠粒（3次）。对于竞争试验，以10μM的浓度加入ML385和化合物3，在冰上孵化，并用PBS洗涤（3倍）。接下来，将5μg辣根过氧化物酶（HRP）偶联的链霉抗生物素蛋白加入试管中，然后在冰上温育30分钟，然后用PBS洗涤8次。最后，用含有10 mM EDTA的PBS洗脱结合的蛋白质-药物复合物，与SuperSignal West PICO溶液1:1混合，在Flexistation-3中使用井扫描模式测量HRP活性。

NQO1酶活性测定[1]
用载体或ML385处理细胞72小时。如前所述测定总蛋白裂解物中的酶活性。

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总抗氧化能力和谷胱甘肽测定[1]
细胞用载体或ML385处理72小时。分别使用抗氧化剂和谷胱甘肽测定试剂盒测量总抗氧化能力和谷胱甘肽水平。

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半胱天冬酶活性测定[1]
按照制造商的说明，使用Caspase-Glo®3/7检测试剂盒测量Caspase活性。CellTiter Blue测定用于量化细胞密度并使胱天蛋白酶活性正常化。简而言之，用ML385处理细胞36小时。将等量的CellTiter Blue试剂加入孔中，30分钟后测量荧光。丢弃CellTiter蓝色试剂，将Caspase-Glo（100μL）试剂加入细胞中，在37°C下再孵育60-90分钟。记录产生的发光，并将胱天蛋白酶活性归一化为细胞数。

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

109Ų

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.89 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.07 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: 6% DMSO+40% PEG 300+5%Tween80+ 49%ddH2O: 1.5mg/ml

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配方 4 中的溶解度: 10 mg/mL (19.55 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: 9.01 mg/mL (17.61 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 6 中的溶解度: 5 mg/mL (9.77 mM) in 15% Solutol HS 15 10% Cremophor EL 35% PEG 400 40% water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。