PORCN (IC50 = 74 pM)[1]

As determined by blocking Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of β-catenin reporter activity, Wnt-C59 (C59) reduces PORCN function in vitro at nanomolar concentrations. With an IC50 of 74 pM, Wnt-C59 blocks WNT3A-mediated activation of a multimerized TCF-binding site that drives luciferase[1].

In mice, wnt-C59 exhibits good bioavailability. Wnt-C59 downregulates Wnt/β-catenin target genes while preventing the growth of breast cancers in MMTV-WNT1 transgenic mice[1]. In human cancers, Wnt-C59 has the ability to eliminate cancer stem cells. Wnt-C59 stops the creation of spheres in both HNE1 and SUNE1 cells, which suppresses the stemness characteristics of NPC cells in a dosage-dependent manner[2].

Wnt Secretion, TOPFlash Assays, Dvl2 mobility shift, and PORCN Rescue[1]
For determination of C59 IC50, STF3a cells were treated with compounds diluted 1/1000 into culture media, and luciferase activity measured 48 hrs later. To monitor Wnt secretion into culture media, STF3a cells were treated with C59 diluted in fresh 1% FBS containing media added at half volume. Lysates were collected in 4% SDS 24 hours later. For SuperTOPflash assays in HT1080 cells, experiments were transfected in 24-well plates with 50 ng Wnt, 100 ng mCherry, and 650 ng SuperTOPflash. For PORCN rescue experiments, 100 ng 3xHA-mPORCN-D was added. For measurement of Dvl2 mobility shift, transfection was with 200 ng of each Wnt plasmid and 600 ng mCherry. Lysates were prepared in 100 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1% IGEPAL-CA630, complete protease inhibitor cocktail and Western blotting was performed to detect Dvl2.

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Wnt-WLS interaction [1]
HeLa cells in 6 well dishes were transfected with C-terminal V5-tagged WNT3A or WNT8A. Six hours after transfection, the media was replaced with addition of either 0.1% DMSO as a control or C59 at a final concentration of 10 nM. Following overnight incubation, the cells were lysed with the buffer: 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40 and complete protease inhibitors). A total of 500 μg lysate was immunoprecipitated with the rabbit polyclonal antibody against the C-terminus of WLS. Western blotting was performed for V5 and WLS (using mouse antibody to WLS).

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Metabolic Labeling with Alk-C16 and Click Chemistry[1]
HT1080 cells plated at a density of 3 x 106 cells in 10 cm plates were transfected with C-terminally V5-tagged WNT3A. After six hours of transfection the media was replaced with DMEM containing 5% fatty acid free BSA (Sigma) with or without 100 μM ω-alkynyl palmitic acid (Alk-C16), synthesized as previously reported (3,4). Cells were simultaneously treated with 0.1% DMSO as a control or 100 nM C59. Following overnight incubation, cells were washed twice with cold PBS and cells’ lysates were prepared in the buffer: 50mM HEPES, 150nM NaCl, 1mM EDTA, 1% NP-40 and protease inhibitors (Roche). The protein lysates were incubated overnight with anti-V5 antibody followed by addition of 30 μL of Protein A/G plus agarose (50% slurry) for 2 hours. Pellets were washed four times with lysis buffer and resuspended in sodium phosphate buffer (100 mM sodium phosphate, pH 7.5, 1% Nonidet P-40, 150mM NaCl). The immunoprecipitated proteins were then subjected to the click labeling reaction in a 50 μl volume for 1 hr RT at final concentrations of the following reagents added serially: 0.1 mM biotin-azide (Life Technologies), 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 0.2 mM Tris[(1-benzyl- 1H-1,2,3-triazol-4- yl)methyl]amine (TBTA) dissolved in DMSO/tert-butanol (20%/80%), and 1 mM CuSO4 (freshly prepared in water). Labeled immunoprecipitated proteins were resolved by SDS–PAGE, transferred to PVDF membrane, blotted with anti-V5 primary antibody followed by Dylight 680 conjugated anti-mouse antibody and Dylight 800 conjugated streptavidin, then visualized in respective channels

In vitro C59 Toxicity Studies[1]
HCT116, K562, HL60, A549, U-2-OS, U266, HCC1937, HCC38, MCF7, BT-20, MB157, MDA-MB-435S, MDA-MB-231, MDA-MB-468, DLD1, SW480, T98G, A172, U-87MG, KATO III, SNU1, SNU5, SNU16, SK-MEL-5, SK-MEL-28, HS294T, A375, A2058, MEWO, FU97, IM95, IM95M, NUGC-3, NUGC-4, OCUM-1, SCH, MKN1, MKN7, MKN45, MKN74, AZ521, JHH2, SNU216, YCC-3 (Korean Cell Line Bank, Korea), EOL-1 were grown in the culture conditions as specified by suppliers. For the assay, 5000 cells in 75 μl culture medium were seeded in each well of black 96 well plates and incubated overnight at 37°C. C59 was serially diluted in medium as 4x stock and then 25 μl of each dilution was added to the cells to give the final concentration of 50 μM to 1.5 nM. After treatment for 2 days, 100 μl of CellTiter-Glo® Luminescent Cell Viability Assay reagent was added to each well and incubated for 10 minutes at room temperature. The luminescence was measured using Tecan Safire instrument.

Pharmacokinetic Analysis[1]
C57/BL6 mice were used. C59 was dissolved in 30% propylene glycol for intravenous tail-vein administration or a mixture of 0.5% methylcellulose and 0.1% tween-80 for oral administration. After single doses (volume 5 ml/kg IV, 10 ml/kg po), mice were sacrificed at indicated times. Blood samples were collected tubes. Plasma was obtained and analyzed using validated bioanalytical LC/MS/MS method. To determine the concentration of C59 in samples, the standard curve of C59 was established in the linear-quadratic manner (r2 = 0.99) over the range of 1 – 1000 ng/ml in mouse plasma. The accuracy and precision of the triplicate quality control samples were within 15% of deviation and 25% of relative standard deviation, respectively. The lower limit of quantification of C59 in mouse plasma was 1 ng/ml. Pharmacokinetic parameters were calculated by the non-compartmental method (Gibaldi and Peter, 1982) using Phoenix WinNonlin 6.0 software.

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Animal studies [1]
MMTV-WNT1 mice were obtained from Jackson Laboratories and backcrossed at least six generations to C57/Bl6 mice. Female virgin mice were placed into control or treatment groups at random as tumors appeared. C59 treatment or vehicle administration was daily by oral gavage, with alternate day caliper-based tumor measurement. The study was conducted for 21 days or till the tumors reached preset size limits. At sacrifice, tumors were resected and weighed, then separated for RNA isolation and histology.

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Dissolved in 0.5% methylcellulose and 0.1% Tween-80; 10 mg/kg; Oral gavage

Female nude mice orthotopically transplanted with independent MMTV-WNT1 tumors

[1]. Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer. Cancer Res. 2013 Jan 15;73(2):502-7.

[2]. Wnt-C59 arrests stemness and suppresses growth of nasopharyngeal carcinoma in mice by inhibiting the Wnt pathway in the tumor microenvironment. Oncotarget. 2015 Jun 10;6(16):14428-39.

C25H21N3O

379.45

379.168

C, 79.13; H, 5.58; N, 11.07; O, 4.22

1243243-89-1

Wnt-C59;1243243-89-1

57519544

White to yellow solid

1.2±0.1 g/cm3

628.3±55.0 °C at 760 mmHg

333.8±31.5 °C

0.0±1.8 mmHg at 25°C

1.648

4.19

1

3

5

29

508

0

O=C(NC1=CC=C(C2=CC=CN=C2)C=C1)CC3=CC=C(C4=CC(C)=NC=C4)C=C3

KHZOJCQBHJUJFY-UHFFFAOYSA-N

InChI=1S/C25H21N3O/c1-18-15-22(12-14-27-18)20-6-4-19(5-7-20)16-25(29)28-24-10-8-21(9-11-24)23-3-2-13-26-17-23/h2-15,17H,16H2,1H3,(H,28,29)

2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)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 (6.59 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 (6.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.59 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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Solubility in Formulation 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5 mg/mL

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