Integrated Strategic Design and In Situ Click Chemistry Approach to Rapid Discovery of Novel Potent Covalent TEAD Inhibitors.

Transcriptional enhanced associated domain (TEAD) proteins, activated by YAP/TAZ, are oncogenic drivers. While parallel synthesis has advanced lead discovery, most libraries are assembled randomly or in a scaffold-centric manner, which results in low efficiency for inhibitor discovery. In this study, we leveraged a strategy integrating structure-based design with CuAAC-enabled parallel synthesis to build a covalent, hydrophobic-fragment library. Combined with in situ screening, this approach rapidly identified hits at 8.33%, among which LC-TEAD01 emerged as a selective inhibitor showing a 17-fold preference for NF2-deficient NCI-H226 cells. Biochemical and structural studies confirmed covalent engagement of the conserved cysteine and occupancy of the hydrophobic channel, disrupting YAP-TEAD interaction and suppressing TEAD-dependent transcription. In vivo, LC-TEAD01 inhibited tumor growth in NF2-deficient xenografts. Collectively, this work integrates structure-based design with CuAAC-enabled parallel synthesis and in situ screening, enabling rapid discovery of TEAD inhibitors and offering a generalizable route for targets with structurally defined pockets.