ETC159 (ETC192215)

ETC-159 (ETC-1922159) targets PORCN (Porcupine O-Acyltransferase); [1]

所有 Wnt 活性和分泌均被 ETC-159 抑制。在几种 Wnt 信号传导升高的癌症模型中观察到 ETC-159 的强活性。当 R-spondin 易位存在于分子定义的结直肠癌 (CRC) 中时，ETC-159 非常有效[1]。

---

1. 在STF3A细胞中，ETC-159可抑制Wnt/β-连环蛋白报告基因活性及Wnt3a的分泌；100 nM的ETC-159处理后，免疫印迹检测显示细胞培养上清中Wnt3A蛋白水平显著降低 [1]
2. 在瞬时表达Wnt3A-V5的HeLa细胞中，细胞经炔基棕榈酸（Alk-C16）代谢标记16小时（同时加入100 nM ETC-159），通过生物素-叠氮化物点击反应检测棕榈酰化水平（上样）及Wnt3a-V5免疫印迹（下样），证实100 nM ETC-159可抑制Wnt3A的棕榈酰化 [1]
3. 转染Wnt3a、PORCN表达质粒及Super 8xTOPFLASH报告基因的HT1080细胞经ETC-159处理16小时后，过表达PORCN可逆转ETC-159对β-连环蛋白报告基因活性的抑制作用（P⩽0.01，P⩽0.0001） [1]
4. 瞬时表达Wnt3A-V5的HeLa细胞经100 nM ETC-159过夜处理后，内源性Wntless的免疫沉淀实验证实ETC-159可阻止Wnt与Wntless的相互作用 [1]
5. 稳定表达Wnt3a的小鼠L细胞经100 nM ETC-159处理后，不同时间点收集细胞并进行免疫印迹检测，结果显示ETC-159可促进β-连环蛋白降解 [1]
6. 转染Super 8xTOPFLASH报告基因及不同Wnt表达质粒的HT1080细胞经100 nM ETC-159处理24小时后，多种Wnt诱导的Wnt/β-连环蛋白报告基因活性均被抑制（P⩽0.05，P⩽0.01，P⩽0.001） [1]
7. 转染小鼠（1 ng）或爪蟾porcn（0.75 ng）表达质粒及Super 8xTOPFLASH报告基因的PORCN敲除HT1080细胞中，ETC-159对哺乳动物PORCN（小鼠PORCN）的抑制作用强于爪蟾PORCN，且呈剂量依赖性降低β-连环蛋白报告基因活性（以DMSO处理组为对照计算百分比） [1]
8. 转染Super 8xTOPFLASH报告基因的人畸胎瘤PA-1细胞中，ETC-159可剂量依赖性特异性抑制自分泌Wnt信号通路（结果以相对于DMSO对照组的均值±标准差表示） [1]
9. 100 nM ETC-159处理PA-1畸胎瘤细胞24小时后，免疫印迹分析显示LRP6和Dvl2的下游激活水平降低 [1]
10. 接种于软琼脂中的PA-1细胞经不同浓度ETC-159处理后，2~3周计数克隆形成数（每个数据点为两个孔的平均计数），证实ETC-159可剂量依赖性抑制细胞锚定非依赖性生长 [1]
11. 转染RSPO融合蛋白表达质粒及STF报告基因的HEK293细胞经100 nM ETC-159处理16小时后，RSPO融合蛋白诱导的Wnt信号通路被显著抑制（P⩽0.001，P⩽0.0001） [1]

ETC-159 抑制小鼠 PORCN 的 IC50 为 18.1 nM，而 Xenopus Porcn 的 IC50 大约高四倍 (70 nM)。当用于治疗源自携带 RSPO 易位的结直肠癌 (CRC) 患者的异种移植物时，ETC-159 显示出显着的疗效。 ETC-159 在小鼠中显示出良好的口服药代动力学，使得口服给药能够用于临床前评估。 ETC-159的口服生物利用度为100%，单剂量5 mg/kg后Tmax约为0.5小时，迅速吸收到血液中[1]。

---

1. BALB/c裸鼠第4脂肪垫接种MMTV-Wnt1肿瘤组织片段（原位模型）后，每日口服ETC-159（药效相关具体剂量未详述，但检测了5、30、100 mg/kg剂量下的血浆药物浓度）可有效抑制肿瘤生长；实验终点收集肿瘤组织，免疫组化显示β-连环蛋白在细胞核和细胞质中表达减少，且Wnt/β-连环蛋白靶基因表达下调（末次给药6小时后检测，P⩽0.01，P⩽0.001，P⩽0.0001，n=8/组） [1]
2. BALB/c裸鼠接种PA-1畸胎瘤细胞（平均肿瘤体积~150 mm³）后，每日口服30 mg/kg ETC-159可显著抑制肿瘤生长（n=10个肿瘤/组，数据以均值±标准误表示） [1]
3. BALB/c裸鼠接种NCCIT畸胎瘤细胞（平均肿瘤体积~150 mm³）后，每日口服30 mg/kg ETC-159可抑制肿瘤生长（n=9个肿瘤/组）；末次给药4小时后收集肿瘤组织，qRT-PCR检测显示Axin2 mRNA水平显著降低（P⩽0.01，P⩽0.0001，n=8个肿瘤/组） [1]
4. 携带结直肠癌（CRC）患者来源异种移植物（CR-1、CR-2，均含PTPRK-RSPO融合基因）的雌性BALB/c裸鼠，每日口服75 mg/kg ETC-159可有效抑制肿瘤生长（n=12个肿瘤/组，数据以均值±标准误表示）；肿瘤组织经苏木精-伊红（HE）和阿尔辛蓝染色显示分化程度增加 [1]
5. BALB/c裸鼠接种HPAF-II胰腺肿瘤细胞（平均肿瘤体积~150 mm³）后，每日口服ETC-159（药效相关具体剂量未详述）可抑制肿瘤生长（n=9/组，数据以均值±标准误表示）；末次给药6小时后收集肿瘤组织，AXIN2 mRNA水平显著降低（P⩽0.0001，n=6/组） [1]
6. BALB/c裸鼠接种AsPC-1胰腺肿瘤细胞后，每日两次口服15 mg/kg剂量的ETC-159可抑制肿瘤生长（n=16/组，数据以均值±标准差表示）；肿瘤组织中AXIN2 mRNA水平降低（P⩽0.0001，n=16/组），黏蛋白基因表达升高（P⩽0.001，P⩽0.0001） [1]
7. 携带AsPC-1异种移植物的BALB/c裸鼠经21天每日两次15 mg/kg ETC-159处理后停药，随访6周显示肿瘤未出现复发生长（n=20/组，数据以均值±标准差表示） [1]

1. Wnt3A棕榈酰化检测实验：将HeLa细胞瞬时转染以表达Wnt3A-V5，随后在100 nM ETC-159存在下，用炔基棕榈酸（Alk-C16）对细胞进行16小时代谢标记；标记完成后，检测生物素-叠氮化物点击反应后的棕榈酸水平（上样），并通过免疫印迹检测Wnt3a-V5（下样），以此评估ETC-159对棕榈酰化的抑制作用 [1]
2. β-连环蛋白报告基因活性挽救实验：向HT1080细胞中转染Wnt3a表达质粒、PORCN表达质粒及Super 8xTOPFLASH报告基因质粒；转染后的细胞经ETC-159（该挽救实验中未明确具体浓度）处理16小时，随后收集细胞并检测荧光素酶活性，以此评估PORCN过表达是否可逆转ETC-159对β-连环蛋白活性的抑制作用 [1]
3. 不同Wnt诱导的报告基因活性抑制实验：向HT1080细胞中转染Super 8xTOPFLASH报告基因质粒及多种Wnt表达质粒；细胞经100 nM ETC-159处理24小时后，检测荧光素酶活性，量化ETC-159对Wnt诱导的报告基因活性的抑制效果 [1]
4. PORCN物种特异性抑制实验：向PORCN敲除的HT1080细胞中转染小鼠（1 ng）或爪蟾porcn（0.75 ng）表达质粒及Super 8xTOPFLASH报告基因质粒；细胞经不同浓度ETC-159处理后，以DMSO处理组为对照，检测β-连环蛋白报告基因活性（百分比），对比ETC-159对小鼠和爪蟾PORCN的抑制效力 [1]

1. Wnt/β-连环蛋白报告基因活性及Wnt3a分泌实验：将STF3A细胞经不同浓度ETC-159处理24小时，检测荧光素酶活性以评估Wnt/β-连环蛋白报告基因活性（数据以均值±标准差表示）；另取STF3A细胞经100 nM ETC-159处理后，通过免疫印迹检测细胞培养上清中Wnt3A蛋白水平，评估Wnt3a分泌情况 [1]
2. Wnt3A棕榈酰化实验：将HeLa细胞瞬时转染以表达Wnt3A-V5，随后在100 nM ETC-159存在下，用炔基棕榈酸（Alk-C16）孵育细胞16小时；通过免疫印迹检测生物素-叠氮化物点击反应后的棕榈酸及Wnt3a-V5水平，评估棕榈酰化程度 [1]
3. β-连环蛋白降解实验：将稳定表达Wnt3a的小鼠L细胞胰酶消化后，经DMSO或100 nM ETC-159处理再接种；在指定时间点收集细胞，通过免疫印迹检测总β-连环蛋白水平，评估其降解情况 [1]
4. 畸胎瘤细胞自分泌Wnt信号抑制实验：将PA-1细胞转染Super 8xTOPFLASH报告基因质粒，随后经不同浓度ETC-159处理24小时；检测荧光素酶活性并以相对于DMSO对照组的均值±标准差表示，评估自分泌Wnt信号的抑制效果 [1]
5. LRP6和Dvl2激活水平检测实验：将PA-1畸胎瘤细胞经100 nM ETC-159处理24小时，制备细胞裂解液并进行免疫印迹分析，检测LRP6和Dvl2的激活水平 [1]
6. 锚定非依赖性生长实验：将PA-1细胞接种于软琼脂中，并经不同浓度ETC-159处理；2~3周后计数克隆总数（每个数据点为两个孔的平均计数），评估ETC-159对锚定非依赖性生长的抑制作用 [1]
7. RSPO融合蛋白诱导的Wnt信号抑制实验：将HEK293细胞转染指定的RSPO表达质粒及STF报告基因质粒，随后经DMSO或100 nM ETC-159处理16小时；检测荧光素酶活性，评估ETC-159对RSPO融合蛋白诱导的Wnt信号的抑制作用 [1]

Formulated in 50% PEG400 (vol/vol) in water
Mice bearing colorectal cancer (CRC) patient-derived xenografts

---

1. MMTV-Wnt1 orthotopic tumor model: BALB/c nude mice were implanted with a fragment of MMTV-Wnt1 tumor from a transgenic mouse into the 4th fat-pad; after palpable tumors developed, mice were randomized into four groups (matched for tumor size) and treated daily (unblinded) with vehicle or ETC-159 at indicated doses (oral administration); tumor growth was monitored, and at study end, tumors were harvested for β-catenin staining and Wnt target gene expression analysis [1]
2. PA-1 teratocarcinoma xenograft model: BALB/c nude mice were inoculated with PA-1 cells; 3 weeks post-inoculation (average tumor volume ~150 mm³), mice were divided into two groups (matched for tumor volume) and treated daily (unblinded) with vehicle or 30 mg/kg ETC-159 (oral administration); tumor volume was measured, and data were presented as mean±s.e.m. (n=10 tumors/group) [1]
3. NCCIT teratocarcinoma xenograft model: BALB/c nude mice were inoculated with NCCIT cells; 3 weeks post-inoculation (average tumor volume ~150 mm³), mice were divided into treatment groups (matched for tumor volume) and treated daily (unblinded) with vehicle or 30 mg/kg ETC-159 (oral administration); tumor volume was measured (n=9 tumors/group), and tumors were harvested 4 h after last dose for Axin2 mRNA analysis [1]
4. CRC patient-derived xenograft (PDX) model (CR-1 and CR-2): Female BALB/c nude mice with established subcutaneous CR-1/CR-2 tumors (PTPRK-RSPO fusions) were randomized into groups (matched for tumor size) and treated daily (unblinded) with vehicle or 75 mg/kg ETC-159 (oral administration); tumor growth was monitored (n=12 tumors/group, data as mean±s.e.m.), and tumors were harvested for hematoxylin/eosin and Alcian blue staining [1]
5. HPAF-II pancreatic tumor xenograft model: BALB/c nude mice were inoculated with HPAF-II cells; 3 weeks post-inoculation (average tumor volume ~150 mm³), mice were divided into four groups (matched for tumor volume) and treated daily (unblinded) with vehicle or ETC-159 at indicated doses (oral administration); tumor volume was measured (n=9/group, data as mean±s.e.m.), and tumors were harvested 6 h after last dose for AXIN2 mRNA analysis [1]
6. AsPC-1 pancreatic tumor xenograft model: BALB/c nude mice were inoculated with AsPC-1 cells; 3 weeks post-inoculation, mice were randomized into two groups (matched for tumor size) and treated twice daily (unblinded) with 15 mg/kg/dose ETC-159 or vehicle (oral administration); tumor volume was measured (n=16/group, data as mean±s.d.), and tumors were harvested for AXIN2 and mucin gene expression analysis [1]
7. AsPC-1 xenograft post-treatment follow-up: BALB/c nude mice with matched AsPC-1 tumors were treated twice daily with 15 mg/kg/dose ETC-159 or vehicle for 21 days (oral administration), then treatment was stopped; tumor volumes were measured for up to 6 weeks post-treatment (n=20/group, data as mean±s.d.) [1]
8. ETC-159 oral bioavailability assay: Mice were given a single oral dose of ETC-159 at 5, 30, or 100 mg/kg; plasma levels of ETC-159 were measured, and data were presented as mean±s.d. [1]

ETC-159 is orally bioavailable in mice; a single oral dose of 5, 30, or 100 mg/kg results in a dose-proportional increase in plasma levels of the drug [1]

[1]. Wnt addiction of genetically defined cancers reversed by PORCN inhibition. Oncogene. 2015 Aug 10. doi: 10.1038/onc.2015.280.

Porcupine Inhibitor ETC-1922159 is an orally bioavailable inhibitor of the membrane-bound O-acyltransferase (MBOAT) porcupine (PORCN), with potential antineoplastic activity. Upon oral administration, ETC-1922159 binds to and inhibits PORCN in the endoplasmic reticulum (ER), which blocks post-translational palmitoylation of Wnt ligands and inhibits their secretion. This prevents the activation of Wnt ligands, interferes with Wnt-mediated signaling, and inhibits cell growth in Wnt-driven tumors. Porcupine catalyzes the palmitoylation of Wnt ligands, and plays a key role in Wnt ligand secretion. Wnt signaling is dysregulated in a variety of cancers.

---

1. ETC-159 (also named ETC-1922159) is a novel, potent, orally available PORCN inhibitor developed to block the secretion and activity of all Wnts by inhibiting Wnt palmitoleation (an essential post-translational modification for Wnt secretion) [1]
2. In RSPO3-translocated cancers, inhibition of PORCN by ETC-159 causes marked remodeling of the transcriptome, with downregulation of cell cycle, stem cell, and proliferation-related genes, and upregulation of differentiation markers [1]
3. ETC-159 is the first example of an effective targeted therapy for CRC with RSPO translocations [1]
4. In RNF43 mutant pancreatic tumors (HPAF-II, AsPC-1), ETC-159 treatment induces differentiation (increased mucin expression, Alcian blue staining) [1]

C19H17N7O3

391.38

391.139

1638250-96-0

86280523

White to off-white solid powder

1.5±0.1 g/cm3

1.732

1.2

113

1

6

4

29

654

0

QTRXIFVSTWXRJJ-UHFFFAOYSA-N

InChI=1S/C19H17N7O3/c1-24-17-16(18(28)25(2)19(24)29)26(11-20-17)10-15(27)21-14-9-8-13(22-23-14)12-6-4-3-5-7-12/h3-9,11H,10H2,1-2H3,(H,21,23,27)

2-(1,3-dimethyl-2,6-dioxopurin-7-yl)-N-(6-phenylpyridazin-3-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 (6.39 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。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (6.39 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。

---

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。