1. Small-Molecule Inhibitors of the SOX18 Transcription Factor
Frank Fontaine, Jeroen Overman, Mehdi Moustaqil, Sreeman Mamidyala, Angela Salim, Kamesh Narasimhan, Nina Prokoph, Avril A.B. Robertson, Linda Lua, Kirill Alexandrov, Peter Koopman, Robert J. Capon, Emma Sierecki, Yann Gambin, Ralf Jauch, Matthew A. Cooper, Johannes Zuegg, Mathias Francois 
Cell Chemical Biology 
Volume 24, Issue 3, Pages (March 2017)
DOI: /j.chembiol
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2. Figure 1 Natural Products and Inhibitors of SOX18-DNA Binding
(A) Representative dataset from high-throughput FP screen of 2,688 marine extracts at 0.25 mg/mL. The screen was run on full-length mouse SOX18 with FAM-labeled SOX-responsive element (mouse Prox1 promoter). Competitive binding of a ligand to SOX18 reduces the FP index (arrow points to the active extract [red dot]).
(B) Chemical structure of Sm1 and Sm2.
(C) FP concentration-response curve of Sm1 and Sm2 (full-length mouse SOX18, mean ± SD; n = 3).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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3. Figure 2
Focused Library of Structural Analogs and Counter-Screen with In Silico Aggregation Predictor and Critical Micelle Concentration Assay
(A) The first group is based on the ortho-hydroxybenzoic (salicylic acid) motif apparent in compounds Sm1 and Sm2. The second group is based on a similar resorcinol scaffold. The third group consists of approved NSAIDs that contain a similar salicylic acid or anthranilic acid scaffold.
(B) Typical CMC data for neutral detergent Triton X-100 control and two compounds Sm4 and Sm10.
(C) SOX18-DNA-binding inhibition by Sm4 and meclofenamic, niflumic, and flufenamic acids.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 3 Compounds Interact with SOX Protein but Not DNA
(A and B) Biotinylated double-strand DNA probes, approximately 40 bp long, with a SOX18 consensus element (A) or a scrambled sequence (B), and flanked with genomic DNA, were used to test small-molecule DNA binding. Probes were immobilized on an SPR streptavidin chip. Positive controls DAPI, ethidium bromide, and actinomycin D bind to DNA in a manner consistent with the literature results. Small-molecule inhibitors (Sm4, 5, and 14) do not bind to consensus or scrambled DNA.
(C) Thermostability of SOX18[109] HMG fragment in the presence of either Prox1-DNA, Sm4, Sm5, or Sm14, as measured by differential static light scattering of protein complex heated from 25°C to 80°C. The binding of small molecules promotes protein stability (ΔTagg > 3°C is considered a significant stabilization). Boltzmann curve fits of normalized light scattering triplicate data (goodness of fit R2 > 0.97).
(D) Sm4 inhibits DNA binding of SOX2-, 6-, 9-, 11-, 15-, and 18-HMG fragments as measured with FP-based DNA-binding competition assay.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 4
Effect of Sm4 and Niflumic, Flufenamic, and Meclofenamic Acids on SOX18 Protein-Protein Interactions
(A) Left: heatmap of SOX18 pairwise protein-protein interactions as tested by ALPHAScreen on XRCC6 (negative control) and two proteins known to interact with SOX18, RBPJ, and MEF2C. Right: Co-immunoprecipitation of protein complex. SOX18-mCherry-cMyc was co-expressed with either GFP-RBPJ, GFP-MEF2C, or GFP only (negative control) under cell-free conditions and immunoprecipitated with GFP Nanotrap beads. Bands: 1, RBPJ-GFP; 2, MEF2C-GFP; 3, SOX18-mCherry; 4, GFP.
(B) Effect of Sm4 and niflumic, flufenamic, and meclofenamic acids on SOX18 interaction with MEF2C and RBPJ.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 5
Contribution of Sm4 Chemical Motives to SOX18 DNA-Binding Inhibition, SOX18-RBPJ-Binding Inhibition, Cytotoxicity, and Aggregation
The table (top) depicts compounds Sm14–Sm44 and summarizes in a color-coded manner results obtained for four activity markers, namely, protein-DNA and protein-protein binding inhibition, cytotoxicity, and aggregation risk. The bar graph (bottom) details SOX18-RBPJ protein-protein binding inhibition results at 50 μM and 5 μM where available (n = 4, mean ± SD). DNA-binding inhibition, cytotoxicity, and cLogP raw data are summarized in Table S2.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 6
In Silico Modeling of Sm4 at the Interface between SOX18 HMG and RBPJ; Inhibition of SOX18-Dependent Transactivation In Vitro
(A and B) Stable binding pose for Sm4 in the SOX18/Prox1 DNA X-ray crystal structure, putting the inhibitor in an “open” pocket between protein and DNA.
(C) Docking of the SOX18/DNA structure into the structure of the Notch transcription complex.
(D) Luciferase reporter assay in COS7 cells transiently transfected with Sox18 and a vector containing Vcam1 promoter merged to firefly luciferase gene (Hosking et al., 2004). Cells were treated with small molecules at concentrations below CC10 (10% cytotoxicity) for 24 hr in culture medium containing a maximum of 1% DMSO (v/v). Results are depicted for Sm4 and niflumic acid. Meclofenamic and flufenamic acids were inactive at concentrations below CC10.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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