Discovery of Small Compounds that Target FOXO Transcription Factors and Modulate their Transcriptional Activity and Physiological Function

Abstract

FOXO transcription factors are critical regulators of cell homeostasis that control a plethora of different target genes and thereby steer cell death, differentiation, longevity and senescence in mammalian cells. The objective of the present study was to identify novel chemical compounds that bind to the DNA-binding domain of FOXO3 and modulate its transcriptional activity. The strategy was to combine pharmacophore-modelling-based in silico compound screening with fluorescence polarization protein-DNA binding assays and cell-based compound validation. By this approach, we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO transcription factors with highest affinity to human FOXO3. These compounds do not act as chemical inhibitors, but modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed by NMR spectroscopy and docking studies. We demonstrate that compound S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, modulate the transcription of distinct gene subsets and interfere with the physiological program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators by directly affecting protein-DNA interaction in normal and malignant cells.