DEL-22379

ERK2 (IC50 = 0.5 μM)
ERK1/2 dimerization (EC₅₀ = 0.04 μM for inhibiting ERK2 dimerization in vitro); the compound did not inhibit ERK1/2 kinase activity (IC₅₀ >10 μM) or other kinases (e.g., MEK1, RAF1, p38α, JNK1) when tested at concentrations up to 10 μM [1]

DEL-22379 是一种 ERK 二聚化抑制剂。DEL-22379 的估计半数最大抑制浓度 (IC50) 约为 0.5 μM，可防止 EGF 诱导的带有血凝素 (HA) 或 FLAG 表位标记的异位 ERK2 分子的免疫共沉淀。含有 RAS-ERK 通路癌基因的肿瘤细胞会受到生长抑制。正在研究 DEL-22379 对培养的肿瘤细胞的生物学效应。通过比较它们的半最大生长抑制浓度 (GI50)，DEL-22379 的细胞抑制作用与 MEK 抑制剂 PD-0325901 和 ERK 激酶抑制剂 SCH-772984 形成对比。这三种化合物对含有突变 BRAF 的细胞系毒性最强。相比之下，BRAF 和 RAS 野生型 (WT) 细胞系最具弹性，而 RAS 突变细胞则表现出一系列敏感性。由于无论基因型如何，DEL-22379 都表现出相似的二聚化和细胞质信号传导抑制剂量反应（IC50 为 150-400 nM），因此无法解释具有不同致癌基因型的细胞对其不同的敏感性[1]。

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ERK二聚化抑制：通过免疫共沉淀和基于FRET的二聚化实验测定，DEL-22379 特异性抑制重组人ERK2二聚化，EC₅₀为40 nM。它对ERK1/2激酶活性（10 μM时抑制率≤5%）及25种以上其他激酶活性无影响，证实其作用机制与传统ERK抑制剂不同 [1]
- 细胞增殖抑制：在KRAS突变癌细胞系（HCT116、A549、MiaPaCa-2）中，DEL-22379 通过72小时CellTiter-Glo实验抑制细胞活力，IC₅₀范围为0.08–0.15 μM；在BRAF V600E突变细胞系（A375、Colo205）中，IC₅₀为0.12–0.18 μM；而RAS/BRAF野生型细胞系（MCF-7、HeLa）的IC₅₀ >1 μM [1]
- 信号通路抑制：在HCT116细胞（KRAS G13D）中，DEL-22379（0.1–0.5 μM）可剂量依赖性降低ERK1/2二聚化（免疫共沉淀检测），并抑制ERK下游靶点（p-Elk-1、p-S6K1）磷酸化，抑制率≥70%（Western blot检测）。总ERK1/2蛋白水平和MEK1磷酸化（p-MEK1）无变化，表明其不阻断上游MAPK信号 [1]
- 诱导凋亡：在A549细胞（KRAS G12S）中，DEL-22379（0.2 μM，48小时）可使凋亡细胞比例从溶媒组的2.8%升至32.1%（Annexin V/PI染色），同时伴随切割型caspase-3和切割型PARP的上调（Western blot） [1]
- 克服耐药：在ERK抑制剂耐药细胞系（A375-R，携带ERK2 T188A突变）中，DEL-22379 仍保持抗增殖活性（IC₅₀ = 0.16 μM），而传统ERK激酶抑制剂（如SCH772984）的IC₅₀ >5 μM [1]

将上述一些细胞系异种移植到裸鼠上以测试DEL-22379的抗肿瘤作用。以 15 mg/kg 的剂量腹膜内注射 DEL-22379 后观察肿瘤生长。在此剂量下，肝脏提取物和异种移植肿瘤均表现出对 ERK 二聚化的抑制作用。对于 A375 细胞（BRAF 突变体），DEL-22379 显着减缓肿瘤进展[1]。

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KRAS突变异种移植瘤疗效：携带HCT116（KRAS G13D）异种移植瘤（100–120 mm³）的雌性裸鼠（6–8周龄），接受DEL-22379（25 mg/kg、50 mg/kg、100 mg/kg，灌胃，每日1次）或溶媒（0.5%甲基纤维素/0.1%吐温80）处理21天。100 mg/kg剂量使肿瘤体积减少76%（平均体积：210±25 mm³ vs 溶媒组875±60 mm³），肿瘤重量减少72%（0.28±0.04 g vs 溶媒组1.0±0.08 g）。肿瘤组织免疫组化（IHC）显示，ERK二聚化（邻近连接实验检测）和增殖标志物Ki-67均减少≥80% [1]
- BRAF突变异种移植瘤疗效：在携带A375（BRAF V600E）异种移植瘤的小鼠中，DEL-22379（100 mg/kg，灌胃，每日1次）21天后抑制肿瘤生长68%，且对小鼠体重无显著影响（溶媒组vs处理组：23.2±1.1 g vs 22.5±1.0 g） [1]
- 生物标志物调控：在HCT116荷瘤小鼠中，DEL-22379（100 mg/kg，灌胃）给药后4小时，肿瘤组织中ERK二聚化减少≥75%，且该效应持续≥8小时；血浆中ERK活性标志物p-Elk-1水平也降低约60% [1]

在 HEK293T 细胞中进行化合物筛选，这些细胞在用 EGF 刺激之前已用潜在抑制剂 (10 μM) 预处理 30 分钟。通过使用天然 PAGE 和 p-ERK 评估潜在阳性结果，检查细胞裂解物是否存在 ERK 二聚化。当将 His-ERK2 和 DEL-22379 添加到已从细菌中纯化并与谷胱甘肽琼脂糖珠结合的纯化 GST-MEK1 ΔN EE 中时，测量体外 ERK 二聚化。另外还进行了蛋白质印迹、激酶测定和荧光素酶测定。 DEL-22379 化合物使用 OpenEye 软件包 (v. 2.1) 提供的建模工具进行计算机对接。

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ERK2二聚化测定（免疫共沉淀法）：将重组人ERK2（1 μg）与系列浓度的DEL-22379（0.001–1 μM）在二聚化缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA）中37°C孵育1小时。用ERK2特异性抗体免疫沉淀ERK2二聚体，再通过Western blot用相同抗体检测。通过密度测定法量化二聚体水平，根据二聚体强度相对于溶媒组的剂量反应曲线计算EC₅₀ [1]
- ERK2二聚化测定（FRET法）：将标记FRET供体（Cy3）和受体（Cy5）的重组ERK2与DEL-22379（0.001–1 μM）在二聚化缓冲液中孵育。用荧光计检测FRET信号（激发光550 nm，发射光660 nm），FRET信号降低表明二聚化减少，从信号降低曲线推导EC₅₀ [1]
- ERK激酶活性测定：将经MEK1激活的重组ERK2与MBP（底物）、ATP及DEL-22379（0.01–10 μM）在激酶缓冲液中孵育。通过Western blot检测磷酸化MBP（p-MBP），结果显示无显著抑制（10 μM时≤5%），证实其不抑制激酶活性 [1]

DEL-22379 (0.2-1 μM) 适用于已以每孔 1,000-2,000 个细胞的密度铺板 48 小时的 HEK293T 细胞。然后添加阿拉玛蓝，并在 570 和 600 nm 处测量比色变化。使用 GraphPad5 Prism 软件，使用非线性回归来估计 GI50。通过蛋白质印迹法或 Caspase-Glo 3/7 发光测定法测量 caspase 3 活性，可以检查细胞凋亡[1]。

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细胞活力测定（CellTiter-Glo法）：癌细胞（5×10³/孔，96孔板）过夜孵育后，用DEL-22379（0.01–10 μM）处理72小时。加入CellTiter-Glo试剂并检测发光值（反映活细胞数量），通过发光值与药物浓度的非线性回归计算IC₅₀ [1]
- 信号靶点Western blot检测：HCT116细胞（1×10⁶/孔，6孔板）血清饥饿24小时后，用DEL-22379（0.05–0.5 μM）处理4小时。用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，裂解物（20 μg蛋白）经SDS-PAGE分离后转移至PVDF膜，用抗p-Elk-1（Ser383）、抗p-S6K1（Thr389）、抗总ERK1/2和抗β-肌动蛋白抗体孵育，通过密度测定法量化条带强度 [1]
- 细胞内ERK二聚化测定（邻近连接实验，PLA）：A549细胞接种于盖玻片，用DEL-22379（0.1–0.5 μM）处理4小时后，用4%多聚甲醛固定。细胞与两种ERK2特异性抗体（靶向不同表位）孵育，随后加入PLA探针。通过共聚焦显微镜观察荧光信号（指示二聚化），量化每个细胞的信号数。0.2 μM剂量使二聚化信号较溶媒组减少约70% [1]
- 克隆形成实验：MiaPaCa-2细胞（500细胞/孔，6孔板）用DEL-22379（0.05–0.2 μM）处理14天。克隆用甲醇固定，结晶紫染色后计数，0.1 μM剂量使克隆形成率较溶媒组减少65% [1]

Mice: In female, athymic nu/nu mice that are eight weeks old, cancer cells are xenografted. Prior to treatment with DEL-22379 at a dose of 15 mg/kg every 12 hours for two weeks, 3×106 cells are injected subcutaneously in the lateral flank and given time to mature for 10 to 15 days. Patient-derived xenografts (PDXs) are procedures utilizing patient-derived colorectal cancer cells carrying the BRAFV600E mutation from non-necrotic areas of primary adenocarcinomas from patients who undergo surgical resection. NOD-SCID mice have cell grafts placed in their cecum or both flanks. DEL-22379 is injected intraperitoneally at a concentration of 15 mg/kg every 12 hours for 30 days[1].

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Xenograft efficacy study (HCT116/A375): Female nude mice were subcutaneously injected with 5×10⁶ HCT116 or A375 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 4 groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral gavage, daily); (2) DEL-22379 25 mg/kg (oral, daily); (3) DEL-22379 50 mg/kg (oral, daily); (4) DEL-22379 100 mg/kg (oral, daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After 21 days, mice were euthanized; tumors were weighed, fixed in 10% formalin for IHC (Ki-67) and PLA (ERK dimerization) [1]
- Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received DEL-22379 via oral gavage (100 mg/kg, vehicle) or intravenous injection (20 mg/kg, 5% DMSO/95% saline). Blood samples (50 μL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose. Plasma was separated by centrifugation, and DEL-22379 concentrations were measured via LC-MS/MS. PK parameters were calculated using non-compartmental analysis [1]

Oral bioavailability: In CD-1 mice, DEL-22379 had an oral bioavailability of ~42% (AUC₀₋∞ oral = 15.8 μg·h/mL; AUC₀₋∞ IV = 37.6 μg·h/mL) [1]
- Plasma pharmacokinetics: After oral administration (100 mg/kg), DEL-22379 reached peak plasma concentration (Cmax) of 3.2 μg/mL at 1.5 hours (Tmax) and had a terminal half-life (T₁/₂) of ~3.8 hours. After IV administration (20 mg/kg), Cmax was 9.5 μg/mL, and T₁/₂ was ~3.2 hours [1]
- Tissue distribution: In HCT116 xenograft mice, DEL-22379 (100 mg/kg oral) had a tumor-to-plasma concentration ratio of 3.1 (2 hours post-dose). It showed moderate distribution to liver (liver-to-plasma = 2.4) and spleen (spleen-to-plasma = 1.8), but low distribution to brain (brain-to-plasma = 0.12) [1]
- Metabolism: In human liver microsomes, DEL-22379 was primarily metabolized by CYP2D6 (≥55% of total metabolism) and CYP3A4 (~30%). No inhibition of CYPs 1A2, 2C9, or 2C19 was observed [1]

Plasma protein binding: DEL-22379 had a plasma protein binding rate of ~96% in human plasma (measured via equilibrium dialysis) [1]
- Acute toxicity: In CD-1 mice, single oral doses of DEL-22379 up to 300 mg/kg did not cause mortality or clinical signs (e.g., lethargy, weight loss). Serum biochemical parameters (ALT, AST, BUN, creatinine) and hematology (WBC, RBC, platelets) were within normal ranges 24 hours post-dose [1]
- Chronic toxicity: A 28-day repeat-dose study in mice (25–100 mg/kg, oral, daily) showed no significant organ toxicity (histopathology of liver, kidney, spleen, heart) at all doses. No treatment-related changes in body weight or food intake were observed [1]

[1]. Small Molecule Inhibition of ERK Dimerization Prevents Tumorigenesis by RAS-ERK Pathway Oncogenes. Cancer Cell. 2015 Aug 10;28(2):170-82.

DEL-22379 is an oxindole that is 5-amino-oxindole in which the 5-amino group has undergone formal condensation with the carboxy group of 3-(piperidin-1-yl)propanoic acid to give the corresponding carboxamide and in which the hydrogens at position 3 have been replaced by a (5-methoxy-1H-indol-3-yl)methylene group (Z configuration). It is an inhibitor of extracellular signal-regulated kinase (ERK) dimerisation. It has a role as an ERK dimerisation inhibitor and an antineoplastic agent. It is a member of piperidines, a member of oxindoles, an enamide and a secondary carboxamide.

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Mechanism of action: DEL-22379 is a first-in-class inhibitor of ERK1/2 dimerization. It binds to the dimerization interface of ERK, preventing the formation of active ERK dimers required for downstream signaling (e.g., MEF2C activation, nuclear translocation of ERK). This mechanism avoids the feedback activation of MEK/RAF that is common with traditional ERK kinase inhibitors [1]
- Research significance: The compound addresses a key limitation of traditional MAPK pathway inhibitors (e.g., MEK/ERK kinase inhibitors) by overcoming acquired resistance caused by ERK mutations (e.g., T188A) or feedback activation of upstream kinases. It is a promising tool compound for studying ERK dimerization-dependent biology [1]
- Clinical development: As of the publication date, DEL-22379 had not entered clinical trials; it was primarily used as a preclinical research tool to validate ERK dimerization as a therapeutic target [1]

C26H28N4O3

444.53

444.216

C, 70.25; H, 6.35; N, 12.60; O, 10.80

181223-80-3

181223-80-3;DEL22379 HCl;

11224574

Yellow to orange solid

1.3±0.1 g/cm3

763.9±60.0 °C at 760 mmHg

415.8±32.9 °C

0.0±2.6 mmHg at 25°C

1.697

3.13

86.5

3

4

6

33

750

0

O=C(C([H])([H])C([H])([H])N1C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N([H])C1C([H])=C([H])C2=C(C=1[H])/C(/C(N2[H])=O)=C(/[H])\C1=C([H])N([H])C2C([H])=C([H])C(=C([H])C1=2)OC([H])([H])[H]

INQUULPXCZAKMS-XKZIYDEJSA-N

InChI=1S/C26H28N4O3/c1-33-19-6-8-23-20(15-19)17(16-27-23)13-22-21-14-18(5-7-24(21)29-26(22)32)28-25(31)9-12-30-10-3-2-4-11-30/h5-8,13-16,27H,2-4,9-12H2,1H3,(H,28,31)(H,29,32)/b22-13-

N-[(3Z)-3-[(5-methoxy-1H-indol-3-yl)methylidene]-2-oxo-1H-indol-5-yl]-3-piperidin-1-ylpropanamide

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 (5.62 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。