Design and implementation of high-content imaging platforms: lessons learned from end user-developer collaboration.

Automated high-content screening and analysis (HCS/HCA) technology solutions have become indispensable in expediting the pace of drug discovery. Because of the complexity involved in designing, building, and validating HCS/HCA platforms, it is important to design, build, and validate a HCS/HCA platform before it is actually needed. Managed properly, collaboration between technology providers and end users in research is essential in accelerating development of the hardware and software of new HCS/HCA platforms before they become commercially available. Such a collaboration results in the cost effective creation of new technologies that meet specific and customized industrial requirements. This review outlines the history of, and considerations relevant to, the development of the Cytometrix Profiling System by Cytokinetics, Inc. and the "Complete Imaging Solution" for high-content screening, developed by Molecular Devices Corporation (MDC) (now MDS Analytical Technologies), from original conception and testing of various components, to multiple development cycles from 1998 to the present, and finally to market consolidation.

Compound classification using image-based cellular phenotypes.

Compounds with similar target specificities and modes of inhibition cause similar cellular phenotypes. Based on this observation, we hypothesized that we could quantitatively classify compounds with diverse mechanisms of action using cellular phenotypes and identify compounds with unintended cellular activities within a chemical series. We have developed Cytometrix technologies, a highly automated image-based system capable of quantifying, clustering, and classifying changes in cellular phenotypes for this purpose. Using this system, 45 out of 51 known compounds were accurately classified into 12 distinct mechanisms of action. We also demonstrate microtubule-binding activity in one of seven related cytochalasin actin poisons. This technology can be used for a variety of drug discovery applications, including high-throughput primary screening of chemical and siRNA libraries and as a secondary assay to detect unintended activities and toxicities.

An unbiased cell morphology-based screen for new, biologically active small molecules.

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.