Fig. 1
The compound CBX interacts with the recombinant FOXO3-DBD protein. a Screening of the 1120 substance Prestwick Chemical Library® for compounds that bind to the FOXO3-DBD by FPA. 100 nM recombinant FOXO3-DBD protein, 5 nM fluorescence-labeled IRE oligonucleotide (IRE-FAM), and 1 µl of each compound were incubated for 30 min at room temperature and analyzed in the chameleon plate reader. a, b 10 compounds that potentially bind to the FOXO3-DBD protein were identified (cut-off < 31% mP-value). c Chemical structure of CBX. d Analyses of the dose-dependent interaction of CBX with the FOXO3-DBD protein (20 nM) by FPA using 5 nM IRE-FAM oligonucleotide in combination with 0.5 µM unlabeled IRE oligonucleotide or with indicated concentrations of CBX (µM). Shown are means ± s.e.m. of three independent experiments. Statistical analysis was done using the Student’s unpaired t test; ***P < 0.01 compared with the negative control (CTR). e Analyses of specific binding of CBX to the FOXO3-DBD by an alternative FPA system that measures the binding of an R18 peptide (10 nM) to recombinant 14–3–3 sigma protein (100 nM). In this setting CBX did not quench the mP-value at a concentration of 163 µM, indicating specific binding of CBX to the FOXO3-DBD protein
