EAI045

EGFR (IC50 = 1.9 μM); EGFRL858R (IC50 = 0.019 μM); EGFRT790M (IC50 = 0.19 μM); EGFRL858R/T790M (IC50 = 0.002 μM)

EAI045（而非 HaCaT 细胞）有效抑制 H1975 细胞中 EGFR Y1173 磷酸化 (EC50=2 nM)。在 1 mM ATP 下，EAI045 是 L858R/T790M 突变体的选择性抑制剂，其选择性是野生型 EGFR 的 1000 倍。与一组 250 种蛋白激酶相比，EAI045 表现出显着的选择性；在 1 μM 浓度下，没有其他激酶被抑制超过 20%[1]。 EAI045 中的 L858R/T790M 突变具有高效力和选择性。 EAI045 可降低 L858R/T790M 突变型 NSCLC 细胞系 H1975 细胞中的 EGFR 自磷酸化，但并不能完全消除它。 EAI045 在表达 L858R/T790M EGFR 突变的稳定转染的 NIH-3T3 细胞中表现出相同的活性。 EAI045 在携带 L858R 突变的 H3255 细胞中具有中等活性。 EAI045 在 HaCaT 细胞（一种具有野生型 EGFR 的角质形成细胞系）中不表现出任何抑制 EGFR 磷酸化的作用。它验证了 EAI045 对 EGFR 突变体的特异性[2]。

用 EAI045 和西妥昔单抗联合治疗的 L858R/T790M 突变小鼠在 L858R/T790M 突变驱动的肺癌基因工程小鼠模型中显示出显着的肿瘤消退。在单独使用 EAI045 治疗的小鼠中，没有明显的反应。携带 L858R/T790M/C797S 肿瘤异种移植物的小鼠和经过这些突变改造的 Ba/F3 细胞表现出相同的效果。这些测试明确证明 EAI045 可以克服获得性 T790M 和 C797S 突变抗性[2]。

磷酸化EGFR（Y1173）靶向调节试验[1]
在室温下用10ng/mL EGF刺激HaCaT细胞5分钟。构成性激活的EGFR突变细胞系（H1975和H3255）不受EGF刺激。使用Bio-Tek ELx 405 SelectTM洗板机将培养基减少到20μL。用20μL含蛋白酶和磷酸酶抑制剂的2X裂解缓冲液（2%Triton X-100、40 mM Tris、pH 7.5、2 mM EDTA、2 mM EGTA、300 mM NaCl、2X完全鸡尾酒抑制剂、2X磷酸酶抑制剂鸡尾酒组II和组III）裂解细胞。将盘子摇晃20分钟。将每个孔中25μL的等分试样转移到制备的ELISA板上进行分析。[1]
为了研究EGF预处理对EAI045靶向调节的影响，收获H1975细胞并将其接种在0.5%FBS/RPMI Pen/Strep中。第二天，用含或不含10ngEGF/mL的0.5%FBS/RPMI培养基预处理细胞5分钟。加入化合物并如上所述进行测定。该实验进行了两次，每次实验中都有重复的样本。

H1975、H3255和HaCaT增殖试验[1]
将H1975、H3255和HaCaT细胞系以每孔500个细胞的速度在10%FBS RPMI P/S培养基中镀在白色384孔板中。使用Pin Tool，将50nL的连续稀释化合物转移到细胞中。3天后，根据制造商的说明，用CellTiter Glo测量细胞存活率。将发光读数归一化为0.1%DMSO处理的细胞和空孔。通过非线性回归曲线拟合对数据进行分析，并报告EC50值。

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Ba/F3细胞增殖模型[1]
先前已经描述了EGFR突变体L858R、L858R/T790M、DelE746_A750/T790M、L858R-T790M/C797S和Del/T790M/C797S-Ba/F3细胞15。根据制造商的说明，使用快速更换定点突变试剂盒通过定点突变引入EGFR I941R突变。所有构建体均通过DNA测序得到证实。使用BD Creator™系统将构建体穿梭到逆转录病毒载体JP1540中。用逆转录病毒感染Ba/F3细胞并按照之前描述的标准方案30。通过在嘌呤霉素（2μg/ml）中选择获得稳定的克隆。[1]
通过MTS测定评估生长和生长抑制，并根据先前建立的方法15进行。将不同EGFR基因型的Ba/F3细胞暴露于治疗72小时，每次实验使用的细胞数量根据经验确定，并且已经预先确定。所有实验点都设置在六个井中，所有实验至少重复三次。使用Windows版GraphPad Prism 5.0以图形方式显示数据（GraphPad软件；www.GraphPad.com）。使用具有S形剂量反应的非线性回归模型拟合曲线。

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NIH-3T3细胞研究[1]
如前所述，按照标准方案，用表达EGFR突变体的逆转录病毒构建体感染NIH-3T3细胞15,19。通过在嘌呤霉素（2μg/ml）中选择获得稳定的克隆。

The EAI045 compound was dissolved in 10% NMP (10% 1-methyl-2-pyrrolidinone: 90% PEG-300), and was dosed at 60 mg/kg daily by oral gavage. Cetuximab was administrated at 1 mg/mouse every other day by intraperitoneal injection. The TL, TD and TLCS mice were monitored by MRI to quantify lung tumor burden before being assigned to various study treatment cohorts, which were non-blinded and not formally randomized. All treated mice had an equal initial tumor burden. MRI evaluation was repeated every 2 weeks during treatment. The animals were imaged with a rapid acquisition with relaxation enhancement sequence (TR = 2000 ms, TE effect = 25 ms) in the coronal and axial planes with a 1-mm slice thickness gating with respiratory rates. The detailed procedure for MRI scanning has been previously described27. The tumor burden volumes were quantified using 3-dimensional Slicer software. Source data for tumor volume measurements are provided in Supplementary Figure 2.[1]

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Formulated in 10% NMP (10% 1-methyl-2-pyrrolidinone:90% PEG-300); 60 mg/kg; Oral gavage

EGFR(TL) (bearing L858R/T790M point mutations) and EGFR(TD) (bearing exon19del/T790M point mutations) mice

[1]. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors. Nature. 2016 Jun 2;534(7605):129-32.

[2]. EAI045: The fourth-generation EGFR inhibitor overcoming T790M and C797S resistance. Cancer Lett. 2017 Jan 28;385:51-54.

The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.[1]

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The third-generation tyrosine kinase inhibitors (TKI), AZD9291 (osimertinib) and CO-1686 (rociletinib) of epidermal growth factor receptor (EGFR) are highly active against T790M positive non-small cell lung cancer (NSCLC). However, resistance develops rapidly. EGFR C797S mutation was reported to be a leading mechanism of resistance to the third-generation inhibitors. The C797S mutation appears to be an ideal target for overcoming the acquired resistance to the third-generation inhibitors. This review summarizes the latest development on the discovery of a fourth-generation EGFR TKI, EAI045.3.[2]

C19H14FN3O3S

383.40

383.074

C, 59.52; H, 3.68; F, 4.96; N, 10.96; O, 12.52; S, 8.36

1942114-09-1

1942114-09-1;

121231412

White to khaki solid powder

1.5±0.1 g/cm3

1.729

2.47

111

2

6

4

27

580

0

0

YTUFHOKUFOQRDF-UHFFFAOYSA-N

InChI=1S/C19H14FN3O3S/c20-12-5-6-15(24)14(9-12)16(17(25)22-19-21-7-8-27-19)23-10-11-3-1-2-4-13(11)18(23)26/h1-9,16,24H,10H2,(H,21,22,25)

2-(5-fluoro-2-hydroxyphenyl)-2-(3-oxo-1H-isoindol-2-yl)-N-(1,3-thiazol-2-yl)acetamide

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 (6.52 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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 2 中的溶解度: 2.5 mg/mL (6.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in 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。
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 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.52 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 储备液加入到 900 μL 玉米油中并混合均匀。

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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；
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