Tanaka Masahiro, Bateman Raynard, Rauh Daniel, Vaisberg Eugeni, Ramachandani Shyam, Zhang Chao, Hansen Kirk C, Burlingame Alma L, Trautman Jay K, Shokat Kevan M, Adams Cynthia L
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, USA.
PLoS Biol. 2005 May;3(5):e128. doi: 10.1371/journal.pbio.0030128. Epub 2005 Apr 5.
We have implemented an unbiased cell morphology-based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase-inhibitor scaffolds. Cellular phenotypes induced by each compound were quantified by multivariate statistical analysis of the morphology, staining intensity, and spatial attributes of the cellular nuclei, microtubules, and Golgi compartments. Principal component analysis was used to identify inhibitors of cellular components not targeted by known protein kinase inhibitors. Here we focus on a hydroxyl-substituted analog (hydroxy-PP) of the known Src-family kinase inhibitor PP2 because it induced cell-specific morphological features distinct from all known kinase inhibitors in the collection. We used affinity purification to identify a target of hydroxy-PP, carbonyl reductase 1 (CBR1), a short-chain dehydrogenase-reductase. We solved the X-ray crystal structure of the CBR1/hydroxy-PP complex to 1.24 A resolution. Structure-based design of more potent and selective CBR1 inhibitors provided probes for analyzing the biological function of CBR1 in A549 cells. These studies revealed a previously unknown function for CBR1 in serum-withdrawal-induced apoptosis. Further studies indicate CBR1 inhibitors may enhance the effectiveness of anticancer anthracyclines. Morphology-based screening of diverse cancer cell types has provided a method for discovering potent new small-molecule probes for cell biological studies and anticancer drug candidates.
我们利用Cytometrix（TM）自动成像与分析系统，开展了一项基于细胞形态学的无偏筛选，以鉴定细胞过程的小分子调节剂。这种检测形式通过捕获大多数已知类别的药理剂的表型读数，对小分子诱导的形态学效应进行无偏分析，并且有可能读出知之甚少的信号通路。用包含四种不同蛋白激酶抑制剂支架的107种小分子处理四种人类癌细胞系和一种非癌原代细胞类型。通过对细胞核、微管和高尔基体区室的形态、染色强度和空间属性进行多变量统计分析，对每种化合物诱导的细胞表型进行量化。主成分分析用于鉴定已知蛋白激酶抑制剂未靶向的细胞成分的抑制剂。在此，我们重点关注已知的Src家族激酶抑制剂PP2的羟基取代类似物（羟基-PP），因为它诱导了与该集合中所有已知激酶抑制剂不同的细胞特异性形态特征。我们使用亲和纯化来鉴定羟基-PP的靶点——羰基还原酶1（CBR1），一种短链脱氢酶-还原酶。我们解析了CBR1/羟基-PP复合物的X射线晶体结构，分辨率达到1.24 Å。基于结构设计更有效和选择性更高的CBR1抑制剂，为分析CBR1在A549细胞中的生物学功能提供了探针。这些研究揭示了CBR1在血清饥饿诱导的细胞凋亡中以前未知的功能。进一步的研究表明，CBR1抑制剂可能会增强抗癌蒽环类药物的疗效。对多种癌细胞类型进行基于形态学的筛选，为发现用于细胞生物学研究的强效新小分子探针和抗癌药物候选物提供了一种方法。
