File S1 - Inhibition of Human Dyskerin as a New Approach to Target Ribosome Biogenesis

Supporting Information. Methods S1, Pseudouridylation assay on synthetic rRNA lacking E3/U68 binding sequences. Figure S1, Sequence alignment of human dyskerin and yeast cbf5 primary structures. Figure S2, Backbone RMSD fluctuation of the human dyskerin model presented and used for structure based virtual screening during 10 ns of molecular dynamics simulation. The higher fluctuation between 7–8.5 ns is due to the movement of the loop covering the pseudouridylation site. The protein remains folded for the complete simulation time demonstrating the good quality of the assembled human dyskerin model. Figure S3, Chemical structures of tested compounds for inhibition of pseudouridylation catalytic activity of human dyskerin. Figure S4, Real time RT-PCR assay used in the pseudouridylation assay selectively amplifies full length products (5′ TAA TAC GAC TCT CTA TAG GGC GTC GCT ATG AAC GCT TGG CCG GTT CAG GTT CGG GAT 3′), while predicted truncated products (5′ TAA TAC GAC TCT CTA TAG GGC GTC GCT 3′) originating from pseudouridylated templates are not amplified. Figure S5, Dose-Response curve of compound 1 on MCF7 cells. The experiment was performed treating the cells with DMSO (CTRL) or 5, 10, 50 and 100 µM of compound 1 for up to 96 hours. Alamar Blue assay was performed each 24 hours. Means and SDs from three independent experiments are represented. Figure S6, Effect of compound 1 on telomerase RNA component (TERC) expression. The experiment was performed treating the nuclear lysate with DMSO (CTRL) or 100 µM of compound 1 for 120 minutes in the reaction mixture. Histograms represent means and SDs from three independent experiments. The final results were determined by the 2∧−ΔΔCt method. Differences between the groups are not significant. Figure S7, Effect of treatment with compound 9 on MCF7 endogenous U4393/U4390 rRNA pseudouridylation. Cells were treated with 1 µM, 10 µM and 100 µM of compound 9 or DMSO for 24 hours. The pseudouridylation reaction was carried out at 30°C. Histograms represent means and SEMs from three independent experiments. Differences between the groups are not significant. Figure S8, (A) Contour maps generated with the software SiteMap (http://www.schrodinger.com), thereby presenting hydrophobic (yellow regions), donor (blue regions) and acceptor (red regions) potentials. Contour maps represent the ideal region of the space where a corresponding ligand feature should be located in order to interact optimally with the human dyskerin catalytic site. (B) Molecular surface of the human dyskerin coloured by electrostatic potential. Blue regions represent positively charged residues while red regions negatively charged residues. Figure S9, (A) Comparative analysis of the available structures of related pseudouridine-synthase PUS1 and PUS10. On the left column, compound 1 complexes were minimized into the active site of PUS1 and PUS10 starting from the binding pose obtained by overlaying these structures with the human Dyskerin – compound 1 complex (Yellow lines indicate hydrogen bonds); On the right column, a ligand interaction diagram for each complex is presented (Grey highlight - solvent exposed; blue arrow - side chain hydrogen bond; blue dotted arrow - backbone hydrogen bond; red ball - negative charged residue; blue ball - positive charged residue; green ball - hydrophobic residue; light blue ball - polar residue). (B) Specific inhibitory effect of compound 1 on dyskerin activity. A different synthetic substrate RNA depleted of the sequences for snoRNAs U68 and E3 recognition and for U4390, but conserving U4393 was used in the in vitro assay developed. The experiment was performed treating the nuclear lysate with DMSO (CTRL) or 100 µM of compound 1 for 120 minutes in the reaction mixture. Histograms represent means and SEMs from three independent experiments. Correlations between the groups are not significant. Table S1, List of NCI human tumor cell lines with confirmed bioactivity of compound 1 and their relative expression profiles of human dyskerin classified as overexpressed, underexpressed or not differentially expressed. Data were collected as described in the materials and methods paragraph. Table S2, List of NCI human tumor cell lines with confirmed inactivity of compound 1 and their relative expression profiles of human dyskerin classified as overexpressed, underexpressed or not differentially expressed. Data were collected as described in the materials and methods paragraph. (PDF)