Rational design and chemical modification of TEAD coactivator peptides to target hippo signaling pathway against gastrointestinal cancers.

Human Hippo signaling pathway has been recognized as a new and promising therapeutic target of gastrointestinal cancers, which is regulated by the intermolecular recognition between the TEA domain (TEAD) transcription factor and its prime coactivators. The coactivator proteins adopt two hotspot sites, namely α-helix and Ω-loop, to interact with TEAD. Here, we demonstrate that both the α-helix and Ω-loop peptides cannot maintain in structured state when splitting from the full-length coactivator proteins; they exhibit a large intrinsic disorder in free state that prevents the coactivator peptide recognition by TEAD. Rational design is used to optimize the interfacial residues of coactivator α-helix peptides, which can effectively improve the favorable direct readout effect upon the peptide binding to TEAD. Chemical modification is employed to constrain the free α-helix peptide into native ordered conformation. The method introduces an all-hydrocarbon bridge across and  + 4 residues to stabilize the helical structure of a free coactivator peptide, which can considerably reduce the unfavorable indirect readout effect upon the peptide binding to TEAD. The all-hydrocarbon bridge is designed to point out of the TEAD-peptide complex interface, which would not disrupt the direct intermolecular interaction between the TEAD and peptide. Therefore, the stapling only improves peptide affinity, but does not alter peptide specificity, to TEAD. Affinity assay confirms that the binding potency of coactivator α-helix peptides is improved substantially by >5-fold upon the rational design and chemical modification. Structural analysis reveals that the optimized/stapled peptides can form diverse nonbonded interactions such as hydrogen bonds and hydrophobic contacts with TEAD, thus conferring stability and specificity to the TEAD-peptide complex systems.