Drug discovery of pyrazolo-piperidinone inhibitors targeting YAP:TEAD interaction and development of a fluorescence anisotropy assay for high-throughput screening

Drug discovery of pyrazolo-piperidinone inhibitors targeting YAP:TEAD interaction and development of a fluorescence anisotropy assay for high-throughput screening Giulia Malpezzi1,2, Laura Scalvini 3, Lorenzo Tagliazucchi 1, Annalisa Chiaravalle 3, Daniele Aiello 1, Giulia Saporito1, Davide Illuminati 4, Salvatore Pacifico 4, Remo Guerrini 4, Cecilia Pozzi 5, Alberto Venturelli 1, Glauco Ponterini 1, Marco Mor 3, Sheraz Gul6, Maria Paola Costi 1 1 Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy; 2 Clinical and Experimental Medicine (CEM) PhD Program, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy; 3 Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Parco Area delle Scienze 27/A, I, 43124 Parma, Italy; 4 Dept. of Medical Sciences and Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, 44121 Ferrara, Italy; 5 Department of Biotechnology, Chemistry and Pharmacy – Department of Excellence 2018-2020, University of Siena, via Aldo Moro 2, 53100 Siena, Italy; 6 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) The Hippo signaling pathway (HP) is a critical regulator of cell proliferation, apoptosis, and tissue homeostasis. Central to its oncogenic deregulation is the interaction between Yes-associated protein (YAP) and TEAD transcription factors (TEAD1–4), which drives the expression of genes involved in tumor progression. Disrupting the YAP:TEAD (Y:T) complex has therefore emerged as a valuable strategy in anticancer drug discovery, though few small-molecule inhibitors with validated activity exist. In this work, we describe the discovery and optimization of a novel class of pyrazolo-piperidinone derivatives designed to interfere with the Y:T complex. Structure-based virtual screening targeting Interface 3 of the TEAD surface yielded a focused set of candidate molecules, followed by synthesis and biological evaluation of over twenty derivatives. Several compounds exhibited micromolar IC₅₀ values in colorectal and ovarian cancer cell lines, including drug-resistant models, and modulated the expression of key Y:T downstream targets such as CTGF and CYR61 as shown by RT-PCR and mass spectrometry-based proteomics. A key component of this study was the design and validation of a fluorescence anisotropy (FA) displacement assay to quantitatively assess compound binding at the YAP-binding domain (YBD) of TEAD4. The assay uses a tetramethylrhodamine-labeled peptide (P6-TMR) that mimics the YAP Ω-loop, whose anisotropy increases upon binding to TEAD4 and decreases upon displacement by candidate inhibitors. This approach enabled accurate determination of binding affinities (Kd values) and offered a reliable readout of target engagement, with strong correlation to in vitro antiproliferative activity. Given its robustness, sensitivity, and quantitative nature, the assay has been selected for high-throughput screening (HTS) optimization at the Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP) in Hamburg. The objective is to adapt it to screen large chemical libraries and accelerate the identification of next-generation Y:T complex inhibitors. Overall, this work introduces a promising new chemotype, validates a specialized assay for YAP:TEAD disruption, and lays the foundation for its application in large-scale screening campaigns to expand the repertoire of PPI-targeting oncologic drugs. References [1] J. C. Hau et al., “The TEAD4-YAP/TAZ Protein-Protein Interaction: Expected Similarities and Unexpected Differences,” ChemBioChem, vol. 14, no. 10, pp. 1218–1225, 2013, doi: 10.1002/cbic.201300163. [2] W. Zhou, Y. Li, J. Song, and C. Li, “Fluorescence polarization assay for the identification and evaluation of inhibitors at YAP–TEAD protein–protein interface 3,” Anal. Biochem., vol. 586, pp. 1–24, 2019, doi: 10.1016/j.ab.2019.113413. [3] P. Furet et al., “Structure-based design of potent linear peptide inhibitors of the YAP-TEAD protein-protein interaction derived from the YAP omega-loop sequence,” Bioorganic Med. Chem. Lett., vol. 29, no. 16, pp. 2316–2319, 2019, doi: 10.1016/j.bmcl.2019.06.022. [4] Z. X. Wang, “An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule,” FEBS Lett., vol. 360, no. 2, pp. 111–114, 1995, doi: 10.1016/0014-5793(95)00062-E. [5] Chapeau, E. et al, (2024). Direct and selective pharmacological disruption of the YAP–TEAD interface by IAG933 inhibits Hippo-dependent and RAS–MAPK- altered cancers. Nature Cancer, 5(7), 1102–1120. https://doi.org/10.1038/s43018-024-00754-9