<scp>TAZ</scp>‐<scp>hTrap</scp>: A Rationally Designed, Disulfide‐Stapled Tead Helical Hairpin Trap to Selectively Capture Hippo Signaling Taz With Potent Antigynecological Tumor Activity

Transcriptional enhanced associate domain (Tead)-mediated Hippo signaling pathway regulates diverse physiological processes; its dysfunction has been implicated in an increasing number of human gynecological cancers. The transcriptional coactivator with PDZ-binding motif (Taz) binds to and then activates Tead through forming a three-helix bundle (THB) at their complex interface. The THB is defined by a double-helical hairpin from Tead and a single α-helix from Taz, serving as the key interaction hotspot between Tead and Taz. In the present study, the helical hairpin was derived from Tead protein to generate a hairpin segment, which is a 25-mer polypeptide consisting of a longer helical arm-1 and a shorter helical arm-2 as well as a flexible loop linker between them. Dynamics simulation and energetics characterization revealed that the hairpin peptide is intrinsically disordered when splitting from its protein context, thus incurring a large entropy penalty upon binding to Taz α-helix. A disulfide bridge was introduced across the two helical arms of hairpin peptide to obtain a strong binder termed TAZ-hTrap, which can maintain in a considerably structured, native-like conformation in unbound state, and the entropy penalty was minimized by disulfide stapling to effectively improve its affinity toward the α-helix. These computational findings can be further substantiated by circular dichroism and fluorescence polarization at molecular level, and viability assay also observed a potent cytotoxic effect on diverse human gynecological tumors at cellular level. In addition, we further demonstrated that the TAZ-hTrap has a good selectivity for its cognate Taz over other noncognate proteins that share a high conservation with the Taz α-helix.