Identification of Novel Small Molecule Inhibitors of YAP-TEAD Binding within the Hippo Signaling Pathway

Abstract

The Hippo signaling pathway controls organ size by regulating cell proliferation, apoptosis, and cell differentiation. The Hippo pathway ultimately regulates the concentration of the coactivator yes-associated protein 1 (YAP1) in the nucleus, which binds the transcription factor TEA domains (TEAD), activating genes related to cell proliferation. In several cancers, increased YAP1 activity is linked to increased cellular proliferation, de-differentiation and survival to drive tumor progression and spreading. The development of inhibitors against YAP-TEAD binding are consequently a research topic of much interest. While several inhibitors of the YAP-TEAD interaction are reported, none possess both high specificity and anti-tumor activity. The work described here is based upon a collaboration with AtomWise, Inc to find a best in class competitive inhibitor of YAP-TEAD binding. AtomWise completed a computational screen of ten million compounds for potential binding to a TEAD pocket that is essential for interaction with YAP1. This master’s thesis encompasses both intracellular and biochemical validation of these compounds. Initially, a luciferase YAP/TEAD dependent reporter assay was used to identify compounds with potential intracellular activity. This data was compared with the action of these compounds on the metabolic activity (MTT assay) across three cancer cell lines. Three sulfonamide-based compounds, 4, 22, and 59 were identified as top 10 YAP/TEAD inhibitors. Six additional compounds were synthesized that combined specific moieties in these three compounds. Dose response curves for inhibition of TEAD reporter activity showed that compounds 22 and 59 exhibited both high inhibition and low IC50 values relative to the derivatives. Further, compound 22 also significantly reduced the levels of CTGF transcript (measured by RT-qPCR), a surrogate measure for endogenous YAP1: TEAD activity. Preliminary fluorescent polarization data also suggests that compound 22 inhibits the binding of a 5(6)-FAM labeled YAP1 peptide (residues 58-74) to a purified TEAD fragment. In this work, compounds 22 and 59 are found to inhibit TEAD dependent transcription and the growth of multiple cancer cell lines. In addition, promising preliminary biochemical data indicates that compound 22 may inhibit the interaction of YAP1 with TEAD.