Wnt/β-catenin (IC50 = 129.8 μM, NCI-H295 cell)

PNU-74654 has a 450 nM KD for binding β-catenin. A distinct hotspot is present on the Tcf3/Tcf4 binding surface of β-catenin, specifically at residues K435 and R469. Two little pockets on either side of this hotspot are part of the binding design of PNU-74654 [2]. PNU-74654 dramatically inhibited cell proliferation, enhanced early and late cell counts, decreased nuclear β-catenin accumulation, hampered CTNNB1/β-catenin expression 96 hours post-treatment, and enhanced early and late cell counts 48 hours post-treatment in NCI-H295 cells. Hours rise in target genes of β-catenin. On HeLa cells, no impacts were seen. PNU-74654 lowers amygdalone and soy alcohol in NCI-H295 cells 24 and 48 hours after treatment. STAR, aldosterone synthase protein, and SF1 and CYP21A2 mRNA expression in NCI-H295 cells following a 48-hour treatment period. level falls. PNU-74654 decreases corticosterone in Y1 cells 24 hours post-treatment, but does not lower cell survival [1].

The CRC in vivo models were created through injection of CT-26 cells into the flank of the mouse, and then these models were treated with PNU-74654 /5-FU/ admixture of PNU-74654 − 5-FU (Fig5A). For further study, the histological staining of tumor samples exhibited vascular disturbance as well as substantial RBC extravasation (Fig5B, a), cell necrosis (Fig5B,b), and tumor stroma (Fig5B, c) besides inflammation area (Fig5B, d). We observed that the tumor growth was significantly inhibited in the mice receiving PNU-74654 plus 5-FU, compared to the control group as well as in the groups treated with either PNU-74654or 5-FU (Fig. 5C). Also, the histological staining of tumor tissues illustrated that the combination induced significantly higher tissue necrosis.[3]

NMR Screening[2]
WaterLOGSY is a method for the identification of compounds that interact with macromolecules. In this experiment, the large bulk water magnetization is partially transferred via the protein-ligand complex to the free ligand in a selective manner. Binding compounds (e.g. PNU-74654) are easily identified in the spectra by looking at the sign of their resonances. Compounds that do not bind show negative or no peaks at all, whereas binders show positive peaks. The compounds were screened in mixtures consisting of four or five molecules. Mixtures that gave a positive signal were deconvoluted to identify individual binders. The β-catenin armadillo repeat concentration used in the NMR experiments was 2 μM in 5 mM Tris, 10 mM NaCl pH 7.3. The Tcf4 concentration was 25 μM. Compounds were dissolved in deuterated dimethyl sulfoxide (DMSO) at a concentration of 40 mM. They were first screened at 20°C in mixtures at 50 μM concentration. The samples for the competition experiments were buffered in PBS. NMR experiments were performed with a Varian Inova 600 MHz spectrometer equipped with a 5-mm triple-resonance probe and a Sample Management System (SMS) autosampler. For each sample a reference spectrum and a WaterLOGSY spectrum was recorded.

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ITC Measurements[2]
ITC measurements were carried out at 20°C by using a VP-ITC titration calorimeter (MicroCal). Samples were extensively dialyzed against PBS, 1 mM DTT. Each titration experiment consisted of an initial 2-μL injection, followed by 20 injections of 5 μL. Heats of dilution were measured in blank titrations by injecting Tcf4 (1-53 W-His6) into the buffer used for each particular experiment. These were subtracted from the binding heats. Direct binding experiments were carried out at 3 μM of β-catenin and 60 μM for the Compounds (e.g. PNU-74654). Concentrations for competition assays were 100 μM for Tcf4 (1-53 W-His6) and 5 μM for β-catenin. Each titration experiment was performed on mixtures of four compounds, where each compound was present in a concentration of 100 μM. Binding heats were determined by a nonlinear least-squares fit of the experimental measurements using the Origin software package (MicroCal) and assuming a single binding site model. Compound mixtures that showed at least a threefold reduction in Tcf4 binding affinity were selected for further characterization.

Treatment of cell lines with PNU-74654[1]
NCI-H295 cells were plated at 200,000 cells per well in 24-well plates for gene expression, protein analysis and adrenal steroid measurements. After 48 h, cells were treated with vehicle (0.1%-0.4% DMSO) or 10, 50, 100 and 200 μM PNU-74654. After 24 and 48 h, medium supernatants were collected for adrenal steroid measurements. Adherent cells were fixed for immunofluorescence or harvested for RNA and protein isolation. At least three independent experiments were performed.
Y1 cells were plated at 200,000 cells per well in 24-well plates for gene expression and corticosterone measurement. After 24 h, cells were treated with vehicle or 10, 50, 100 and 200 μM PNU-74654. After 24 and 48 h, medium supernatants were collected for corticosterone measurements and adherent cells were harvested for RNA isolation. At least two independent experiments were performed.
HeLa cells were plated at 50,000 cells per well in 24-well plates for gene expression and protein analysis. After 24 h, cells were treated with vehicle or 50, 100 and 200 PNU-74654. After 24 and 48 h, adherent cells were harvested for RNA and protein isolation. At least two independent experiments were performed.

Female inbred BALB/c mice were purchased from Pasteur Institute, and divided into 4 groups of 3 mice as follows: administrated with PNU-74654, 5-FU, and 5-FU/ PNU-74654 admixture, and untreated (i.e, control group). The mice were housed under standard laboratory conditions. CT-26 cells were injected subcutaneously in the flanks of the mice. The treatments were administrated when the tumor grew up to a volume of 100mm3. Every 2 days, the size of the tumors was measured. [3]

[1]. Inhibition of the Tcf/beta-catenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis. Oncotarget. 2015 Dec 15;6(40):43016-32.

[2]. Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening. Proteins. 2006 Jul 1;64(1):60-7.

[3]. Inhibition of the Wnt/b-catenin pathway using PNU-74654 reduces tumor growth in in vitro and in vivo models of colorectal cancer. Tissue Cell . 2022 Aug:77:101853.

PNU-74654 is beta-catenin - TCF interaction inhibitor
Background: To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target. Aim: To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells. Methods: Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5-200 μM) for 24-96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot). Results: In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability. Conclusions: Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC.[1]

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The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.[2]

C19H16N2O3

320.348

320.116

C, 71.24; H, 5.03; N, 8.74; O, 14.98

113906-27-7

113906-27-7;

9836739

White to off-white solid powder

1.2±0.1 g/cm3

1.593

3.9

1

4

5

24

434

0

CC1=CC=C(O1)/C=N/NC(=O)C2=CC=CC=C2OC3=CC=CC=C3

JJEDWBQZCRESJL-DEDYPNTBSA-N

InChI=1S/C19H16N2O3/c1-14-11-12-16(23-14)13-20-21-19(22)17-9-5-6-10-18(17)24-15-7-3-2-4-8-15/h2-13H,1H3,(H,21,22)/b20-13+

N-[(E)-(5-methylfuran-2-yl)methylideneamino]-2-phenoxybenzamide

PNU-74654; 113906-27-7; Benzoic acid, 2-phenoxy-, 2-[(5-methyl-2-furanyl)methylene]hydrazide; PNU 74654; CHEMBL254381; N-[(E)-(5-Methylfuran-2-yl)methylideneamino]-2-phenoxybenzamide; PNU74654; (E)-N'-((5-methylfuran-2-yl)methylene)-2-phenoxybenzohydrazide;

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)

DMSO : ≥ 30 mg/mL (~93.65 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.80 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 (7.80 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.)