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)辅助信息。方法S1：缺失E3/U68结合序列的合成rRNA假尿苷酸化测定（pseudouridylation assay）。图S1：人类dyskerin与酵母cbf5一级结构的序列比对。图S2：本研究用于基于结构虚拟筛选的人类dyskerin模型，在10 ns分子动力学模拟过程中的主链均方根偏差（RMSD）波动情况。7~8.5 ns时段的波动幅度较高，源于覆盖假尿苷酸化位点的环区运动；整个模拟周期内蛋白始终保持折叠状态，证明组装得到的人类dyskerin模型具备良好质量。图S3：用于测试人类dyskerin假尿苷酸化催化活性抑制效果的受试化合物的化学结构。图S4：本假尿苷酸化测定中采用的实时RT-PCR测定法可选择性扩增全长产物（5′ TAA TAC GAC TCT CTA TAG GGC GTC GCT ATG AAC GCT TGG CCG GTT CAG GTT CGG GAT 3′），而源自假尿苷酸化模板的预测截短产物（5′ TAA TAC GAC TCT CTA TAG GGC GTC GCT 3′）则无法被扩增。图S5：化合物1对MCF7细胞的剂量反应曲线。实验设置二甲基亚砜（DMSO，对照组）与5、10、50、100 µM的化合物1处理组，处理时长最长达96小时，每24小时开展一次Alamar Blue测定。结果展示3次独立实验的均值与标准差（SD）。图S6：化合物1对端粒酶RNA组分（TERC）表达的影响。实验中将细胞核裂解液与二甲基亚砜（对照组）或100 µM的化合物1在反应体系中共孵育120分钟，直方图展示3次独立实验的均值与标准差，最终结果采用2^−ΔΔCt法计算得出，各组间差异无统计学意义。图S7：化合物9处理对MCF7细胞内源性U4393/U4390 rRNA假尿苷酸化的影响。细胞分别用1 µM、10 µM、100 µM的化合物9或二甲基亚砜处理24小时，假尿苷酸化反应于30℃下进行，直方图展示3次独立实验的均值与标准误（SEM），各组间差异无统计学意义。图S8：(A) 采用SiteMap软件（http://www.schrodinger.com）生成的等高线图，展示了疏水势（黄色区域）、供体势（蓝色区域）与受体势（红色区域）。该等高线图标注了配体特征基团与人类dyskerin催化位点实现最优相互作用所需占据的理想空间区域。(B) 基于静电势着色的人类dyskerin分子表面，蓝色区域代表带正电荷的残基，红色区域代表带负电荷的残基。图S9：(A) 相关假尿苷合酶PUS1与PUS10现有结构的对比分析。左栏：将化合物1与PUS1、PUS10活性位点的结合姿势，通过与人类dyskerin-化合物1复合物（黄线表示氢键）叠加后得到，随后进行最小化优化；右栏：展示各复合物的配体相互作用图（灰色高亮：溶剂暴露区域；蓝色箭头：侧链氢键；蓝色虚线箭头：主链氢键；红色球：带负电荷残基；蓝色球：带正电荷残基；绿色球：疏水残基；浅蓝色球：极性残基）。(B) 化合物1对dyskerin活性的特异性抑制效果。本体外实验采用了一段缺失小核仁RNA（snoRNAs）U68和E3识别序列、以及U4390序列但保留U4393的合成底物RNA。实验中将细胞核裂解液与二甲基亚砜（对照组）或100 µM的化合物1在反应体系中共孵育120分钟，直方图展示3次独立实验的均值与标准误，各组间相关性无统计学意义。表S1：确认对化合物1具有生物活性的NCI人类肿瘤细胞系列表，及其人类dyskerin的相对表达谱（分为过表达、低表达或无差异表达）。数据收集方法见材料与方法部分。表S2：确认对化合物1无生物活性的NCI人类肿瘤细胞系列表，及其人类dyskerin的相对表达谱（分为过表达、低表达或无差异表达）。数据收集方法见材料与方法部分。(PDF)