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 1（AbMole，M13726）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 1（CAS 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 1（5–15 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 1（AbMole，M13726）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 1，hESC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 1（CAS 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 1（AbMole，M13726） 的体内应用潜力。小鼠视网膜下腔注射实验中，Chroman 1（10 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。

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