Cell experiment:

The PNU-74654 compound is dissolved in DMSO at stock concentrations of 31.2 mM. For working solutions, PNU-74654 is diluted 100X in growth medium with no serum deprivation. NCI-H295 cells are plated at 200,000 cells per well in 24-well plates for gene expression, protein analysis and adrenal steroid measurements. After 48 h, cells are treated with vehicle (0.1%-0.4% DMSO) or 10, 50, 100 and 200 μM PNU-74654. After 24 and 48 h, medium supernatants are collected for adrenal steroid measurements[1].

PNU-74654 is an inhibitor of Wnt/β-catenin pathway with an IC50 of 129.8 μM in NCI-H295 cell.

PNU-74654 binds to β-catenin with a KD of 450 nM. The Tcf3/Tcf4-binding surface on β-catenin contains a well-defined hot spot around residues K435 and R469. The binding mode of PNU-74654 involves the two narrow pockets on either side of this hot spot[2]. In NCI-H295 cells,PNU-74654 significantly decreases cell proliferation 96 h after treatment, increases early and late apoptosis, decreases nuclear beta-catenin accumulation, impairs CTNNB1/beta-catenin expression and increases beta-catenin target genes 48 h after treatment. No effects are observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreases cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. The SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase are decreased in NCI-H295 cells after 48 h PNU-74654 treatment. In Y1 cells, PNU-74654 impairs corticosterone secretion 24 h after treatment but does not decrease cell viability[1].

References:
[1]. Leal LF, et al. 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]. Trosset JY, et al. 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.