Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.
Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.
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DOI：
10.1016/j.chembiol.2005.01.015
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发表时间：
2005-03
影响因子：
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通讯作者：
J. Yarrow;G. Totsukawa;G. Charras;T. Mitchison
中科院分区：
文献类型：
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作者：
J. Yarrow;G. Totsukawa;G. Charras;T. Mitchison
小分子激酶抑制剂主要在纯蛋白质测定中发现。我们通过基于图像的细胞单层伤口愈合高通量筛选发现了Rho激酶（ROCK）的抑制剂。使用自动显微镜，我们筛选了一个16,000种化合物的库，发现许多影响细胞迁移或细胞形态的化合物以及阻断有丝分裂进程的化合物。我们在一系列子测定中测试了10200种化合物，并选择了一种3-（4-吡啶基）吲哚（Rockout）进行更详细的表征。Rockout抑制起泡并导致肌动蛋白应力纤维溶解，表型模仿Rho激酶抑制剂。在体外测试Rho激酶活性时，Rockout抑制的IC 50为25 μM（比Y-27632的效力低105倍），但具有相似的特异性特征。我们还用具有已知生物活性的化合物库描述了伤口愈合测定，揭示了生物学中涉及的多种途径。
Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of ∼16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested ∼200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50of 25 μM (∼5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.