Compound 30, identified through iterative medicinal chemistry optimization, represents a potent and selective small-molecule inhibitor of the β-catenin/BCL9 protein-protein interaction (PPI). This study presents a comprehensive biological evaluation to characterize its cellular activity, target specificity, and on-target mechanism in multiple cancer models.
In MTS viability assays, compound 30 exhibited dose-dependent growth inhibition across Wnt-hyperactive cancer cell lines, including colorectal HCT116 and SW480 cells, and triple-negative breast cancer MDA-MB-468 and MDA-MB-231 cells. IC50 values ranged from 14 to 30 μM, indicating strong antiproliferative effects. Notably, normal mammary epithelial MCF10A cells showed significantly less sensitivity, with IC50 values exceeding 100 μM, demonstrating a >5-fold selectivity for cancer cells with dysregulated Wnt signaling.
Functional assessment using the TOPFlash/FOPFlash luciferase reporter system confirmed that compound 30 specifically inhibits Wnt/β-catenin transcriptional activity. It suppressed TOPFlash signal with an IC50 of 6.6 μM, while showing no significant effect on FOPFlash, confirming pathway specificity. Quantitative real-time PCR analysis revealed robust downregulation of key oncogenic Wnt target genes—Axin2, cyclin D1, and LEF1—in both Wnt-stimulated MDA-MB-231 and SW480 cells, consistent with inhibition of downstream transcriptional output.
To validate target engagement, a biotin-streptavidin pull-down assay was performed using N-terminally biotinylated BCL9 HD2 peptide (residues 350–375) and whole-cell lysates from SW480 cells. Treatment with compound 30 resulted in a concentration-dependent reduction of endogenous β-catenin pulled down by the BCL9 peptide, confirming direct disruption of the β-catenin/BCL9 complex in a physiological context.
Co-immunoprecipitation experiments further demonstrated that compound 30 selectively disrupts the full-length β-catenin/BCL9 interaction in living cells without affecting the β-catenin/E-cadherin complex, which shares overlapping binding surfaces.347841-88-7 InChIKey This selectivity is critical for minimizing off-target effects on adherens junction integrity.58-05-9 Molecular Weight
The most compelling evidence for on-target activity came from a rescue experiment.PMID:29762996 Transfection of constitutively active β-catenin into SW480 cells significantly attenuated the growth-inhibitory effect of compound 30. The IC50 value increased fourfold compared to control cells, directly linking the compound’s cytotoxicity to β-catenin function.
These results collectively demonstrate that compound 30 is a potent, selective, and mechanism-based inhibitor of the β-catenin/BCL9 PPI. Its ability to suppress Wnt signaling, downregulate oncogenic targets, and selectively inhibit cancer cell proliferation—without disrupting essential cell adhesion functions—establishes it as a promising lead compound for the development of targeted therapies against Wnt-driven cancers.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
