Reference compounds for characterizing cellular injury in high-content cellular morphology assays

Abstract

Robust, generalizable approaches to identify compounds efficiently with undesirable mechanisms of action in complex cellular assays remain elusive. Such a process would be useful for hit triage during high-throughput screening and, ultimately, predictive toxicology during drug development. We generated cell painting and cellular health profiles for 218 prototypical cytotoxic and nuisance compounds in U-2 OS cells in a concentration-response format. A diversity of compounds causing cellular damage produced bioactive cell painting morphologies, including cytoskeletal poisons, genotoxins, nonspecific electrophiles, and redox-active compounds. Further, we show that lower quality lysine acetyltransferase inhibitors and nonspecific electrophiles can be distinguished from more selective counterparts. We propose that the purposeful inclusion of cytotoxic and nuisance reference compounds such as those profiled in this Resource will help with assay optimization and compound prioritization in complex cellular assays like cell painting.

A Decade in a Systematic Review: The Evolution and Impact of Cell Painting

“…30 One challenge with a target agnostic assay, such as Cell Painting, is that compounds active in the assay can act by on- and/or off-target effects, which in turn results in the difficult interpretation of a given bioactivity. 31 Hence, one practical solution is to include known reference compounds. Most recently, Chandrasekaran et al and Jamali et al introduced various sets of recommended control and landmark perturbations — including two compound plates and ORF and CRISPR perturbations plates.…”

“…30 One challenge with a target agnostic assay, such as Cell Painting, is that compounds active in the assay can act by on-and/or off-target effects, which in turn results in the difficult interpretation of a given bioactivity. 31 31 'Nuisance' compounds, in this context, are substances that frequently show up as hits in screening assays but are ultimately considered undesirable because their effects are often nonspecific, artifactual, or due to properties that interfere with the assay rather than a specific biological activity of interest. The exploration and analysis of this dataset highlighted relationships between different types of cellular injury with Cell Painting activity.…”

“…This dataset can be used to characterize cellular injuries. 31 Cell Painting is an in vitro assay that captures changes in cell morphology, but several studies have explored its ability to predict in vivo compound perturbation effects. In 2020, Nyffeler et al performed an in-vitro-to-in-vivo extrapolation (IVIVE) of in vitro potency estimates obtained through Cell Painting.…”

Detection of a Mitochondrial Stress Phenotype using the Cell Painting Assay

“…CPA has been employed to study cell and mitochondrial toxicity (Dahlin et al, 2023;Garcia de Lomana et al, 2023;Herman et al, 2023;Seal et al, 2022;Trapotsi et al, 2022). By using CPA profiles, gene expression signatures, chemical structural information and mitochondrial toxicity data, Seal et al could distinguish between mitochondrial toxicants and non-toxicants based on the morphological profiles (Seal et al, 2022).…”

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“…Given that the mouse brain size is comparable to our FOV, we maintain the physical size of the data by rescaling the images to match our effective pixel size. Additionally, we have collected 2,000 2D cell images of varying types, morphologies, and densities [5][6][7]. Unlike the 3D data, these cell images are generally smaller than our FOV and are thus not rescaled, preserving their original size.…”