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

All Wnt activity and secretion are inhibited by ETC-159. Strong activity of ETC-159 is seen in several cancer models with elevated Wnt signaling. When R-spondin translocations are present in colorectal cancers (CRCs), which are defined molecularly, ETC-159 is very effective[1].

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1. In STF3A cells, ETC-159 inhibits Wnt/β-catenin reporter activity and secretion of Wnt3a; treatment with 100 nM ETC-159 significantly reduces Wnt3A protein levels in culture supernatants as assessed by immunoblot [1]
2. In HeLa cells transiently expressing Wnt3A-V5, 100 nM ETC-159 inhibits Wnt3A palmitoleation when cells are metabolically labeled with alkyne-palmitate (Alk-C16) for 16 h, as shown by biotin-azide-clicked palmitate detection (upper panel) and Wnt3a-V5 immunoblot (lower panel) [1]
3. In HT1080 cells transfected with Wnt3a, PORCN expression plasmids, and Super 8xTOPFLASH reporter, PORCN overexpression rescues the inhibition of β-catenin reporter activity induced by ETC-159 after 16 h of treatment (P⩽0.01, P⩽0.0001) [1]
4. In HeLa cells transiently expressing Wnt3A-V5, overnight treatment with 100 nM ETC-159 prevents the interaction of Wnt with endogenous Wntless, confirmed by immunoprecipitation of Wntless [1]
5. In mouse L cells stably expressing Wnt3a, treatment with 100 nM ETC-159 promotes β-catenin degradation, as shown by immunoblot analysis of total β-catenin levels at different time points after plating [1]
6. In HT1080 cells transfected with Super 8xTOPFLASH and various Wnt expression plasmids, 100 nM ETC-159 inhibits Wnt/β-catenin reporter activity induced by diverse Wnts after 24 h of treatment (P⩽0.05, P⩽0.01, P⩽0.001) [1]
7. In porcupine null HT1080 cells transfected with murine (1 ng) or Xenopus porcn (0.75 ng) expression plasmids and Super 8xTOPFLASH reporter, ETC-159 is a more potent inhibitor of mammalian PORCN (murine PORCN) than Xenopus porcn, with dose-dependent reduction of β-catenin reporter activity (expressed as percentage of DMSO control) [1]
8. In PA-1 human teratocarcinoma cells transfected with Super 8xTOPFLASH reporter, ETC-159 specifically inhibits autocrine Wnt signaling in a dose-dependent manner (results shown as mean±s.d. relative to DMSO control) [1]
9. In PA-1 teratocarcinoma cells treated with 100 nM ETC-159 for 24 h, western blot analysis shows decreased downstream activation of LRP6 and Dvl2 [1]
10. In PA-1 cells plated in soft agar, ETC-159 inhibits anchorage-independent growth in a dose-dependent manner; total colony number was scored after 2–3 weeks (each data point represents average count of two wells) [1]
11. In HEK293 cells transfected with RSPO fusion expression plasmids and STF reporter, 100 nM ETC-159 inhibits Wnt signaling induced by RSPO fusion proteins after 16 h of treatment (P⩽0.001, P⩽0.0001) [1]

With an IC50 of 18.1 nM, ETC-159 inhibits mouse PORCN, while Xenopus Porcn has an IC50 that is roughly four times higher (70 nM). When used to treat xenografts derived from patients with colorectal cancer (CRC) that bear RSPO-translocation, ETC-159 shows remarkable efficacy. ETC-159 shows favorable oral pharmacokinetics in mice, enabling oral administration for preclinical assessment. ETC-159 has an oral bioavailability of 100% and a Tmax of approximately 0.5 hours after a single dose of 5 mg/kg, which is rapidly absorbed into the blood [1].

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1. In BALB/c nude mice implanted with MMTV-Wnt1 tumor fragments in the 4th fat-pad (orthotopic mouse model), daily oral treatment with ETC-159 (doses not specified in detail for efficacy, but plasma levels were measured for 5, 30, 100 mg/kg doses) effectively inhibits tumor growth; tumors harvested at study end show nuclear and cytoplasmic exclusion of β-catenin (immunohistochemistry), and downregulation of Wnt/β-catenin target gene expression (assessed 6 h after last dose, P⩽0.01, P⩽0.001, P⩽0.0001, n=8/group) [1]
2. In BALB/c nude mice inoculated with PA-1 teratocarcinoma cells (average tumor volume ~150 mm³), daily oral treatment with 30 mg/kg ETC-159 prevents tumor growth (n=10 tumors/group, data as mean±s.e.m.) [1]
3. In BALB/c nude mice inoculated with NCCIT teratocarcinoma cells (average tumor volume ~150 mm³), daily oral treatment with 30 mg/kg ETC-159 inhibits tumor growth (n=9 tumors/group); Axin2 mRNA levels in tumors (harvested 4 h after last dose) are significantly decreased (P⩽0.01, P⩽0.0001, n=8 tumors/group) [1]
4. In female BALB/c nude mice with established subcutaneous colorectal cancer (CRC) patient-derived xenografts (CR-1 and CR-2, with PTPRK-RSPO fusions), daily oral treatment with 75 mg/kg ETC-159 effectively inhibits tumor growth (n=12 tumors/group, data as mean±s.e.m.); tumors show increased differentiation (hematoxylin and eosilcian blue staining) [1]
5. In BALB/c nude mice inoculated with HPAF-II pancreatic tumor cells (average tumor volume ~150 mm³), daily oral treatment with ETC-159 (doses not specified in detail for efficacy) inhibits tumor growth (n=9/group, data as mean±s.e.m.); AXIN2 mRNA levels in tumors (harvested 6 h after last dose) are significantly decreased (P⩽0.0001, n=6/group) [1]
6. In BALB/c nude mice inoculated with AsPC-1 pancreatic tumor cells, twice-daily oral treatment with 15 mg/kg/dose ETC-159 inhibits tumor growth (n=16/group, data as mean±s.d.); AXIN2 mRNA levels are decreased (P⩽0.0001, n=16/group), and mucin gene expression is increased (P⩽0.001, P⩽0.0001) [1]
7. In BALB/c nude mice with AsPC-1 xenografts, 21 days of twice-daily 15 mg/kg/dose ETC-159 treatment (followed by treatment cessation) prevents tumor regrowth for up to 6 weeks post-treatment (n=20/group, data as mean±s.d.) [1]

1. For assessment of Wnt3A palmitoleation: HeLa cells were transiently transfected to express Wnt3A-V5, then metabolically labeled with alkyne-palmitate (Alk-C16) for 16 h in the presence of 100 nM ETC-159; after labeling, biotin-azide-clicked palmitate was detected (upper panel) and Wnt3a-V5 was assessed via immunoblot (lower panel) to determine the level of palmitoleation inhibition [1]
2. For β-catenin reporter activity rescue assay: HT1080 cells were transfected with Wnt3a expression plasmid, PORCN expression plasmid, and Super 8xTOPFLASH reporter plasmid; the transfected cells were treated with ETC-159 (concentration not specified for this rescue assay) for 16 h, then harvested to measure luciferase activity (used to evaluate if PORCN overexpression reverses ETC-159-mediated inhibition of β-catenin activity) [1]
3. For Wnt/β-catenin reporter activity inhibition by diverse Wnts: HT1080 cells were transfected with Super 8xTOPFLASH reporter plasmid and various Wnt expression plasmids; the cells were treated with 100 nM ETC-159 for 24 h, then luciferase activity was measured to quantify the inhibition of Wnt-induced reporter activity [1]
4. For PORCN species-specific inhibition assay: Porcupine null HT1080 cells were transfected with murine (1 ng) or Xenopus porcn (0.75 ng) expression plasmid and Super 8xTOPFLASH reporter plasmid; the cells were treated with different concentrations of ETC-159, and β-catenin reporter activity was measured as a percentage of DMSO-treated control to compare inhibitory potency against murine vs Xenopus PORCN [1]

1. Wnt/β-catenin reporter activity and Wnt3a secretion assay: STF3A cells were treated with ETC-159 at indicated concentrations for 24 h, then luciferase activity was measured to assess Wnt/β-catenin reporter activity (data as mean±s.d.); separate STF3A cell cultures were treated with 100 nM ETC-159, and Wnt3a secretion was evaluated by immunoblotting Wnt3A protein in culture supernatants [1]
2. Wnt3A palmitoleation assay: HeLa cells were transiently transfected to express Wnt3A-V5, then incubated with alkyne-palmitate (Alk-C16) for 16 h in the presence of 100 nM ETC-159; biotin-azide-clicked palmitate and Wnt3a-V5 were detected via immunoblot to assess palmitoleation [1]
3. β-catenin degradation assay: Mouse L cells stably expressing Wnt3a were trypsinized and treated with DMSO or 100 nM ETC-159 before plating; cells were harvested at specified time points, and total β-catenin levels were measured by immunoblot to evaluate degradation [1]
4. Autocrine Wnt signaling inhibition assay in teratocarcinoma cells: PA-1 cells were transfected with Super 8xTOPFLASH reporter plasmid, then treated with ETC-159 at indicated concentrations for 24 h; luciferase activity was measured and presented as mean±s.d. relative to DMSO control to assess autocrine Wnt signaling inhibition [1]
5. LRP6 and Dvl2 activation assay: PA-1 teratocarcinoma cells were treated with 100 nM ETC-159 for 24 h, then cell lysates were prepared and western blot analysis was performed to detect the activation levels of LRP6 and Dvl2 [1]
6. Anchorage-independent growth assay: PA-1 cells were plated in soft agar and treated with ETC-159 at indicated concentrations; after 2–3 weeks, the total number of colonies was counted (each data point represents the average count of two wells) to evaluate the inhibition of anchorage-independent growth [1]
7. RSPO fusion-induced Wnt signaling inhibition assay: HEK293 cells were transfected with indicated RSPO expression plasmids and STF reporter plasmid, then treated with DMSO or 100 nM ETC-159 for 16 h; luciferase activity was measured to assess inhibition of RSPO fusion-induced Wnt signaling [1]

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

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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 has oral bioavailability in mice; a single oral dose of 5, 30, or 100 mg/kg resulted in a dose-proportional increase in plasma concentrations[1]

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

ETC-1922159, a hedgehog protein inhibitor, is an orally bioavailable membrane-bound O-acyltransferase (MBOAT) hedgehog protein (PORCN) inhibitor with potential antitumor activity. After oral administration, ETC-1922159 binds to PORCN in the endoplasmic reticulum (ER) and inhibits its activity, thereby blocking the post-translational palmitoylation modification of Wnt ligands and inhibiting their secretion. This prevents Wnt ligand activation, interferes with Wnt-mediated signaling pathways, and inhibits Wnt-driven tumor cell growth. Hedgehog protein catalyzes the palmitoylation modification of Wnt ligands and plays a crucial role in Wnt ligand secretion. The Wnt signaling pathway is dysregulated in various cancers.

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1. ETC-159 (also known as ETC-1922159) is a novel, potent, orally administered PORCN inhibitor that blocks all Wnt secretion and activity by inhibiting Wnt palmitization (a post-translational modification essential for Wnt secretion)[1]
2. In RSPO3 translocation cancers, ETC-159 inhibition of PORCN leads to significant transcriptome remodeling, downregulation of cell cycle, stem cell and proliferation-related genes, and upregulation of differentiation markers[1]
3. ETC-159 is the first effective targeted therapy for RSPO translocation CRC[1]
4. In RNF43 mutant pancreatic tumors (HPAF-II, AsPC-1), ETC-159 treatment induces differentiation (increased mucin expression, alicin 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)