The oncoprotein YAP (Yes-associated protein) requires the TEAD family of transcription factors for the up-regulation of genes important for cell proliferation. Disrupting YAP–TEAD interaction is an attractive strategy for cancer therapy. Targeting TEADs using small molecules that either bind to the YAP-binding pocket or the palmitate-binding pocket is proposed to disrupt the YAP–TEAD interaction. There is a need for methodologies to facilitate robust and reliable identification of compounds that occupy either YAP-binding pocket or palmitate-binding pocket. Here, using NMR spectroscopy, we validated compounds that bind to these pockets and also identify the residues in mouse TEAD4 (mTEAD4) that interact with these compounds. Flufenamic acid (FA) was used as a positive control for validation of palmitate-binding pocket-occupying compounds by NMR. Furthermore, we identify a hit from a fragment screen and show that it occupies a site close to YAP-binding pocket on the TEAD surface. Our results also indicate that purified mTEAD4 can catalyze autopalmitoylation. NMR studies on mTEAD4 revealed that exchanges exist in TEAD as NMR signal broadening was observed for residues close to the palmitoylation site. Mutating the palmitoylated cysteine (C360S mutant) abolished palmitoylation, while no significant changes in the NMR spectrum were observed for the mutant which still binds to YAP. We also show that FA inhibits TEAD autopalmitoylation. Our studies highlight the utility of NMR spectroscopy in identifying small molecules that bind to TEAD pockets and reinforce the notion that both palmitate-binding pocket and YAP-binding pocket are targetable.

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