Abstract
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 Å 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.
Highlights
Many current drugs were originally discovered through observation of unexpected biological activities
The compound collection was dominated by kinase-inhibitor scaffolds, we identified a nonkinase target of hydroxy-PP, carbonyl reductase 1 (CBR1), an NADPH-dependent reductase
The wellcharacterized protein kinase inhibitors (K252a [4], SKB203580 [5], VK-1911 [6], and PP2 [7]) served as positive controls and ‘‘landmarks’’ for the phenotypes likely to be induced by the less-characterized compounds in the collection (Figure S1 contains a complete list of all compounds tested)
Summary
Many current drugs were originally discovered through observation of unexpected biological activities (e.g., penicillin, benzodiazepines, sildenafil [Viagra]). The search for drug-like hits by high-throughput approaches is dominated by in vitro single-enzyme activity– based screens and single-readout cell-based assays. These approaches measure very limited regions of biological space and do not reveal potent effects on pathways not being measured directly. In order to systematize the understanding of the full activity of new small molecules, we quantified dosedependent morphological changes induced in five cell types, thereby identifying ‘‘hit’’ compounds with unique activities. Cell-typespecific components are known to utilize distinct pathways and cellular programs to control fundamental processes affecting the features of the organelles and the overall cellular morphology.
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