Modulating FOXO3 transcriptional activity by small, DBD-binding molecules
- Medical University Innsbruck, Austria
- The Czech Academy of Sciences, Czech Republic
- University of Innsbruck, Austria
- Tyrolean Cancer Research Institute, Austria
- Charles University, Czech Republic
- Paracelsus Medical University Salzburg, Austria
- Biocenter, Medical University Innsbruck, Austria
- Independent Data Lab UG, Germany
Figures
Figure 1 with 2 supplements
Strategy to identify small molecule compounds that interact with FOXO3-DBD.
(a) Overview of the workflow employed to identify FOXO3 inhibitors. (b) Interactions between FOXO3-DBD and the 13 bp DNA strand were represented as pharmacophore features. DNA is shown as sticks and …
Figure 1—figure supplement 1
Overview of combined fluorescence polarization assay (FPA)- and live/dead flow cytometry-based validation of candidate compounds.
FPAs were performed with 125 nM FOXO3-DBD and 25 nM FAM-labeled IRE-oligonucleotide as described in Materials and methods. Compound concentration was 1 μM. mP value of freely rotating IRE-FAM …
Figure 1—figure supplement 2
Effect of S9 on protein-DNA interaction of different FOX family members.
Recombinant FOXO1-DBD (159 - 272) FOXO4-DBD (82-207) and FOXO6-DBD (87 - 200) were prepared as described for FOXO3-DBD in Materials and methods. FPAs to measure binding of FAM-labeled IRE …
Figure 2 with 2 supplements
Compounds S9/S9OX block the DNA binding surface of FOXO3-DBD.
(a) 1D 1H STD-NMR experiments for S9OX compound in the presence of the 15 µM FOXO3-DBD. The reference spectrum of S9OX is shown in black, the corresponding STD-NMR spectrum is shown in red. …
Figure 2—figure supplement 1
S9 binding to FOXO3-DBD.
(a) 1D 1 H STD-NMR experiments for S9 compound in the presence of the 15 µM FOXO3-DBD. The reference spectrum of S9 is shown in black, the corresponding STD-NMR spectrum is shown in red. Chemically …
Figure 2—figure supplement 2
Comparison of S9OX binding to various FOXO proteins.
The comparison of chemical shift perturbation (CSP) obtained for 100 μM FOXO1- DBD (red), FOXO3-DBD (blue) and FOXO4-DBD (yellow) in the presence of 3 mM S9OX. Lines indicate changes greater than …
Figure 3
Compounds S9/S9OX affect induction of FOXO3 target gene mRNAs.
(a) Heatmaps of Affymetrix microarray analyses (U133 plus 2.0 expression profiling chips). Total RNA was prepared of SH-EP/FOXO3 cells treated with 100 nM 4OHT alone or in combination with 50 µM S9 …
Figure 4
Compounds S9 and S9OX inhibit protein expression of FOXO3-regulated proteins and prevent binding of FOXO3 to target promoters.
SH-EP/FOXO3 cells were treated for eight hours (a,b) or 24 hr (c,d) with 100 nM 4OHT alone or in combination with 50 µM S9 or S9OX. Cell lysates were subjected to immunoblot analyses using …
Figure 5
Selectivity assessment of S9 and S9OX for FOXO3.
SH-EP cells expressing a conditionally activated FOXO3(A3)-ERtm-H212R mutant or FOXO(A3)-ERtm were analyzed by immunoblot, treated with 20 nM 4OHT with or without 50 µM S9 or S9OX and subjected to …
Figure 6
Effects of compounds S9/S9OX on FOXO3-induced ROS and sphere growth in 3D cell culture models.
SH-EP/FOXO3 cells were treated either for four hours with 100 nM 4OHT (a) or for two hours with 20 µg/ml etoposide (b). 50 µM S9 or S9OX were pre-incubated for 15 min. ROS accumulation was analyzed …
Additional files
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Supplementary file 1
Synthesis of S9 and S9OX.
- https://cdn.elifesciences.org/articles/48876/elife-48876-supp1-v4.pdf
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Supplementary file 2
Key Resources Table.
- https://cdn.elifesciences.org/articles/48876/elife-48876-supp2-v4.doc
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Transparent reporting form
- https://cdn.elifesciences.org/articles/48876/elife-48876-transrepform-v4.pdf
