Identification of pyrazolo-piperidinone derivatives targeting YAP-TEAD interface 3 as anticancer agents through...

The transcriptional activity of TEAD4 (transcriptional enhancer associated domain proteins), one of the final effectors of the Hippo pathway, can be dysregulated or mutated in cancer. Consequently, targeting the interaction between TEAD and its co-activator YAP (Yes Associated Protein) to disrupt the YAP:TEAD (Y:T) heterodimer has emerged as a promising anti-cancer strategy. Therefore, in this study, we aimed to identify novel scaffolds targeting the TEAD Interface 3 surface as effective anticancer agents against colorectal and ovarian cancer. Employing virtual screening, molecular dynamics, computational ADME-T prediction, and synthetic chemistry, we initially identified novel pyrazolo-piperidinone compounds exhibiting binding affinities (K_d) between 0.6 and 10 μM towards TEAD4 Interface 3 in a fluorescence anisotropy assay, with 6a as the initial lead for a library of 20 compounds. A few of them demonstrated effective and well-characterized cellular antiproliferative activity against HCT116 and A2780 cancer cells. They demonstrated efficacy against colorectal cancer cells resistant to the KRAS-dependent clinical candidate IAG933 and confirmed the ability to inhibit TEAD4 target genes expression. Mass spectrometry-based proteomics of HCT116 cells treated with verteporfin and 6a analogs revealed a proteome modulation consistent with an anti-TEAD mechanism of action, revealing the downregulation of CTGF and TGF-β1. Bioinformatic metabolic pathway analysis further differentiated the mechanism of action of the 6a analogs from verteporfin, a known indirect modulator of the Y:T complex. These findings establish the pyrazolo-piperidinone class as novel Y:T disruptors and provide insights into their metabolic impact on downstream effectors, offering a valuable framework for future hit-to-lead optimization and mechanism of action studies.