Abstract
Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.
Highlights
Phenotypic screening has recently experienced a resurgence in drug discovery after many years of focus on target based approaches [1]
One of the main advantages of phenotypic screening lies in the potential of identifying functionally active chemical modulators without the need to know their precise mode of action (MoA)
In comparison to target-based drug discovery methods, phenotypic screening does not rely on the knowledge of a specific target per se and works as a tool to address complex relations of poorly understood diseases [5]
Summary
Phenotypic screening has recently experienced a resurgence in drug discovery after many years of focus on target based approaches [1] Methods such as cell painting [2,3,4] or phenomics are gaining interest due to their ability to detect disease relevant morphological and expression signatures. One way to circumvent these complications is the use of better annotated chemical libraries, consisting of highly target-specific chemical probes [6,7,8] or chemogenomics libraries which contain well-characterized inhibitors with narrow but not exclusive target selectivity [9,10] The latter have gained increasing interest as a new approach in drug discovery [11,12] as chemogenomic libraries may cover a large diversity of targets and a larger fraction of druggable proteins. The expansion of this CGC collection to cover the entire druggable proteome will be the goal of the Target 2035 [18]
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