The Hippo pathway controls cell-cell contact, cell proliferation as well as organ size by integrating changes in the cellular microenvironment. In recent years, the pivotal role of the Hippo signaling in cancer development has been recognized and brought into focus. Inhibition of the pathway promotes the translocation of the major Hippo pathway effectors, the yes-associated protein (YAP) and its paralog TAZ, to the nucleus, where they interact with transcription factor family transcriptional enhancer associate domain (TEADs) thus coactivate the expression of its downstream genes, leading to cell transformation, tissue overgrowth and tumor development. Therefore, the interruption of the YAP-TEAD transcriptional complex represents a novel opportunity for the treatment of cancer. Here, we established a fluorescence polarization (FP)-based assay for the identification and evaluation of YAP-TEAD PPI inhibitors at the Ω-loop region, which is also called interface 3 at the YAP-TEAD binding interface. The assay was optimized from the aspects of the probe sequences, fluorophore labeling, concentration of each components, buffer effect, temperature effect, incubation time and DMSO tolerance. The application of this FP competitive assay was validated by YAP mutant peptides. Furthermore, a patented small molecule (<b>Patent-22</b>) was evaluated in the FP assay and confirmed that it was a YAP-TEAD PPI inhibitor at interface 3. With great application value, this FP method is reliable, robust and economical for inhibitor assessment and drug discovery.

Development of a fluorescence polarization assay for the identification and evaluation of inhibitors at the Ω-loop region of the YAP-TEAD protein-protein interface (PPI)

2019 Fall National Meeting

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