ML-162

GPX4/glutathione peroxidase 4

ML162（化合物 1a）对表达 HRASG12V 的细胞系和野生型 BJ 母细胞成纤维细胞的 IC50 值分别为 25 nM 和 578 nM，对两种表达 HRASG12V 的细胞系表现出摩尔效应 [1]。用 ML162（8 μM；24 小时）处理可使 Keap1 失活，增加磷酸化 PERK-ATF4-SESN2 色素，并提高 p62 和 Nrf2 以化学方式补充细胞中的 HN3R 和 HN3-rslR 表达 [2]。 ML162 在不同程度上导致头颈癌 (HNC) 细胞死亡。除了顺铂试剂 (HN3R) 和获得性 RSL3 试剂 (HN3-rslR) 细胞比例较低之外，亲代 HN3 细胞更容易受到影响 [2]。

合成致死筛选是一种化学生物学方法，用于识别选择性杀死癌基因表达细胞系的小分子，目的是识别提供针对癌症细胞的特异性靶点的途径。我们对来自美国国立卫生研究院分子库小分子库（NIH-MLSMR）的303282种化合物进行了高通量筛选，以对抗表达HRAS（G12V）的永生化BJ成纤维细胞，然后在一系列缺乏HRAS（G10V）癌基因的同源细胞中对致命化合物进行了反筛选。这项工作导致了两种新型分子探针（PubChem CID 3689413，ML162和CID 49766530，ML210）的鉴定，它们具有纳摩尔电位和4-23倍的选择性，可以潜在地用于鉴定癌症细胞中的致癌特异性途径和靶点[1]。

蛋白质印迹分析 [2]
细胞类型： HN3R 细胞
测试浓度： 8 μM
孵育时间： 24 hrs（小时）
实验结果：化疗耐药的HN3R和HN3-rslR细胞中p62和Nrf2的表达增加。

[1]. Development of small-molecule probes that selectively kill cells induced to express mutant RAS. Bioorg Med Chem Lett. 2012 Feb 15;22(4):1822-6.

[2]. Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer. Free Radic Biol Med. 2018 Dec;129:454-462.

Glutathione peroxidase 4 (GPX4) is a regulator of ferroptosis (iron-dependent, non-apoptotic cell death); its inhibition can render therapy-resistant cancer cells susceptible to ferroptosis. However, some cancer cells develop mechanisms protective against ferroptosis; understanding these mechanisms could help overcome chemoresistance. In this study, we investigated the molecular mechanisms underlying resistance to ferroptosis induced by GPX4 inhibition in head and neck cancer (HNC). The effects of two GPX4 inhibitors, (1S, 3R)-RSL3 and ML-162, and of trigonelline were tested in HNC cell lines, including cisplatin-resistant (HN3R) and acquired RSL3-resistant (HN3-rslR) cells. The effects of the inhibitors and trigonelline, as well as of inhibition of the p62, Keap1, or Nrf2 genes, were assessed by cell viability, cell death, lipid ROS production, and protein expression, and in mouse tumor xenograft models. Treatment with RSL3 or ML-162 induced the ferroptosis of HNC cells to varying degrees. RSL3 or ML-162 treatment increased the expression of p62 and Nrf2 in chemoresistant HN3R and HN3-rslR cells, inactivated Keap1, and increased expression of the phospho-PERK-ATF4-SESN2 pathway. Transcriptional activation of Nrf2 was associated with resistance to ferroptosis. Overexpression of Nrf2 by inhibiting Keap1 or Nrf2 gene transfection rendered chemosensitive HN3 cells resistant to RSL3. However, Nrf2 inhibition or p62 silencing sensitized HN3R cells to RSL3. Trigonelline sensitized chemoresistant HNC cells to RSL3 treatment in a mouse model transplanted with HN3R. Thus, activation of the Nrf2-ARE pathway contributed to the resistance of HNC cells to GPX4 inhibition, and inhibition of this pathway reversed the resistance to ferroptosis in HNC.[2]

C23H22CL2N2O3S

477.4034

476.072

C, 57.87; H, 4.65; Cl, 14.85; N, 5.87; O, 10.05; S, 6.72

1035072-16-2

3689413

Off-white to light yellow solid powder

1.3±0.1 g/cm3

699.3±55.0 °C at 760 mmHg

376.7±31.5 °C

0.0±2.2 mmHg at 25°C

1.630

4

86.9

1

4

9

31

591

0

UNVKYJSNMVDZJE-UHFFFAOYSA-N

InChI=1S/C23H22Cl2N2O3S/c1-30-19-10-9-17(14-18(19)25)27(21(28)15-24)22(20-8-5-13-31-20)23(29)26-12-11-16-6-3-2-4-7-16/h2-10,13-14,22H,11-12,15H2,1H3,(H,26,29)

2-(3-chloro-N-(2-chloroacetyl)-4-methoxyanilino)-N-(2-phenylethyl)-2-thiophen-2-ylacetamide

ML162; 1035072-16-2; ML-162; 2-[(chloroacetyl)(3-chloro-4-methoxyphenyl)amino]-N-(2-phenylethyl)-2-thien-2-ylacetamide; SMR000206941; 2-(3-chloro-N-(2-chloroacetyl)-4-methoxyanilino)-N-(2-phenylethyl)-2-thiophen-2-ylacetamide; MLS000583955; alpha-[(2-chloroacetyl)(3-chloro-4-methoxyphenyl)amino]-N-(2-phenylethyl)-2-thiopheneacetamide;

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)

DMSO : ~100 mg/mL (~209.47 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.24 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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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。