Fig. 4. Hes1 dimer formation inhibitory activity. (a) Schematic representation of the assay for inhibitors of Hes1 dimer formation. Rat-Hes1 (residues 3-281) was exposed to Cy3-labeled Hes1. Hes1 dimer was detected through its fluorescence intensity.40 The assay was performed after the equilibrium of exchange between immobilized Hes1 with Cy3-Hes1 and unlabeled Hes1 which was estimated to be included as unlabeled Hes1 dimers at the immobilizing stage. (b) Hes1 dimer formation inhibition by the isolated compounds 2–4. The background value (without Hes1, Cy3-Hes1 treated) was subtracted. (c) Structures of the flavonoid unit of active compounds 2 and 4 (8 and 9, respectively). (d) Flavonoid units did not inhibit Hes1 dimer formation. The background value (without Hes1, Cy3-Hes1 treated) was subtracted. (e) Elucidation of non-specific inhibition using the TCF4/β-catenin complex plate assay. Immobilized hTCF4 (residues 1-100) was exposed to GST-β-catenin (residues 128-683). The complex was detected with horseradish peroxidase (HRP) conjugated anti-GST antibody. Compounds that disrupt the TCF4/β-catenin complex reduced HRP-related chemiluminescence. (f) Compound 2 did not inhibit TCF4/β-catenin complex formation. The background value (without TCF4, antibody treated) was subtracted. Error bars show the standard deviation (n = 3).