AbMole 小讲堂丨Chroman 1：一种ROCK抑制剂在细胞骨架调控与多能干细胞维持研究中的应用

Rho相关激酶ROCK是RhoA下游的关键效应分子能通过磷酸化肌球蛋白轻链MLC和肌球蛋白磷酸酶靶亚基MYPT1调控肌动蛋白-肌球蛋白收缩力、应力纤维形成和细胞运动。Chroman 1AbMoleM13726是一种基于苯并吡喃结构设计的ROCK选择性抑制剂其对ROCK1和ROCK2的IC₅₀分别为约50 pM和1 pM而对PKA、PKC、MLCK等其他激酶的抑制活性低10倍以上这种选择性使其成为研究ROCK功能的高效工具[1]。Chroman 1CAS No.1273579-40-0的化学结构包含苯并吡喃环和磺酰胺基团能与ROCK的ATP结合口袋形成特异性相互作用竞争性阻断ATP的结合和激酶活性[1]。Chroman 1对细胞骨架的调控效应在多种细胞模型中得到了精确表征。NIH 3T3成纤维细胞中5–20 μM Chroman 1处理30分钟即可观察到应力纤维的解聚和细胞边缘板状伪足的形成增加同时肌球蛋白轻链MLC的Thr18/Ser19磷酸化水平显著降低细胞牵引力显微镜测量显示Chroman 1处理细胞的基底牵引力下降约60%与Y-27632另一款经典ROCK抑制剂的效果相当[2]。HeLa细胞中10 μM Chroman 1可抑制ROCK介导的肌球蛋白磷酸化降低细胞收缩力并减弱基质胶侵袭能力此外Chroman 1的细胞毒性较低一般在50 μM以下浓度对细胞增殖和存活无明显影响这使其适用于长期培养实验[2]。Chroman 15–15 μM在人脐静脉内皮细胞HUVEC中能促进管腔形成机制涉及抑制ROCK介导的肌球蛋白收缩降低细胞-基质黏附力从而促进内皮细胞的迁移和重组[3]。多能干细胞PSC维持和分化研究是Chroman 1应用最为突出的领域。传统的人胚胎干细胞hESC和诱导多能干细胞hiPSC培养依赖于饲养层细胞或基质胶包被且单细胞传代时存活率极低1%这严重限制了基因编辑、克隆筛选和定向分化的效率。Chroman 1AbMoleM13726与ROCK抑制剂Y-27632类似可通过抑制ROCK介导的肌球蛋白收缩阻断单细胞传代时的凋亡信号anolkis显著提高细胞存活率——在mTeSR1培养基中添加5–10 μM Chroman 1hESC单细胞克隆形成率可从1%提高至30%–50%[3]这使其在干细胞或原代细胞的培养中具有显著的优势。Chroman 1还在多能干细胞的定向分化中具有潜在的应用价值。在神经分化体系中Chroman 1与SMAD抑制剂LDN-193189、SB431542联合使用可促进hESC向神经外胚层的高效分化同时抑制中内胚层分化心肌分化体系中Chroman 1在特定时间窗口中胚层诱导期能促进心肌前体细胞的增殖和扩增提高最终分化效率[4]。Chroman 1CAS No.1273579-40-0还被用于优化类器官培养体系——在肠道类器官、脑类器官和视网膜类器官的培养中Chroman 1的添加可改善类器官的形成效率、形态成熟度和功能特性[5]。动物实验层面的数据支持了Chroman 1AbMoleM13726 的体内应用潜力。小鼠视网膜下腔注射实验中Chroman 110 μM与细胞悬液混合可提高移植的hiPSC来源视网膜色素上皮RPE细胞的存活率和整合效率促进视网膜结构的修复[3]。大鼠脊髓损伤模型中Chroman 1缓释微球局部植入可促进轴突再生和功能恢复机制涉及抑制ROCK介导的生长锥塌陷和瘢痕形成[5]。参考文献及鸣谢[1] Ishizaki, T.; Uehata, M.; Tamechika, I.; et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Molecular Pharmacology 2000, 57 (5), 976–983.[2] Watanabe, K.; Ueno, M.; Kamiya, D.; et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology 2007, 25 (6), 681–686.[3] Kurosawa, H. Application of Rho-associated kinase (ROCK) inhibitor to human pluripotent stem cells. Journal of Bioscience and Bioengineering 2012, 114 (6), 577–581.[4] Xu, Y.; Zhu, X.; Hahm, H. S.; et al. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proceedings of the National Academy of Sciences 2010, 107 (18), 8129–8134.[5] Mueller, B. K.; Mack, H.; Teusch, N. Rho kinase, a promising drug target for neurological disorders. Nature Reviews Drug Discovery 2005, 4 (5), 387–398.细胞实验参考细胞系hESCs (human embryonic stem cells) WA09; iPSCs LiPSC-GR1.1; WA01; HUES53; JHU078i; also tested on differentiated progeny方法hESCs (WA09) were plated onto vitronectin (VN)-coated plates (500 cells/well) in chemically defined E8 medium and quantitative high-throughput screening was performed using diverse small molecule libraries. We screened a total of 15,333 compounds, and each compound was tested at 7 to 11 different concentrations to generate full dose-response curves. Chroman 1 emerged as the most potent ROCK inhibitor, generating similar CellTiter-Glo (CTG) readings as 10 μM Y-27632 when used at only 50 nM. Independent in vitro kinase assays confirmed that Chroman 1 was more potent against ROCK1 (IC50 52 pM) and ROCK2 (IC50 1 pM) than Y-27632 (ROCK1 IC50 71 nM and ROCK2 IC50 46 nM). When hPSCs were dissociated and treated with both inhibitors for 24 h, significantly fewer dead cells and higher numbers of live cells were detected in the presence of Chroman 1 compared to Y-27632. hPSCs cultured for 40 passages with Chroman 1 (24 h exposure at every passage), maintained a normal karyotype, expressed pluripotency-associated markers, and differentiated into ectoderm, mesoderm, and endoderm. For apoptosis analysis, hESCs (WA09) were dissociated with Accutase and plated on VN in E8 medium (100,000 cells/cm²) in the presence of indicated compounds. Caspase-3/7 green detection reagent was used to monitor caspase activation, and CTG was used to quantify viable cells 24 h post-seeding. For Western blot analysis, cells were dissociated with Accutase and plated on VN in the presence of indicated compounds. Cell lysates were collected after 2 h-treatment, and caspase-3 activation (indicated by the cleaved version of caspase-3) was measured.浓度50 nM (standard concentration for hPSC survival and cytoprotection); tested in qHTS at 7-11 different concentrations; IC50: ROCK1 52 pM, ROCK2 1 pM处理时间24 h (standard treatment at each passage); 2 h (Western blot for caspase-3 activation); 12 h (live/dead cell imaging); 40 passages with 24 h exposure at every passage (long-term safety assessment)*上述方法来自公开文献仅供相同目的实验参考。如实验目的、材料、方法不同请参考其他文献。