Cell painting in activated cells illuminates phenotypic dark space and uncovers novel drug mechanisms of action
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Abstract
As drug and natural product libraries expand, assays for assessing mechanisms of action (MoA) are increasingly critical. Performing cytological profiling using the Cell Painting (CP) assay enables image-based profiling of cellular states upon treatment, yet many bioactive compounds remain uncharacterized due to undetectable cellular effects under standard conditions. To address this, we combined drug dosing with cell activation using the protein kinase C (PKC) agonist phorbol myristate acetate (PMA). Profiling A549 lung cancer cells treated with 8,387 compounds at two concentrations (1 and 10 µM) in both resting and PMA-activated states allowed us to detect phenotypic effects for up to 40% of all screened compounds, effectively illuminating new phenotypic “dark space”. Over 1,000 compounds exhibited phenotypes exclusively under PMA activation, establishing its advantage for MoA studies. We introduce novel quality control measures for CP screens and demonstrate that integrating phenotypic signatures enhances MoA discovery. Notably, 2-methoxycinnamaldehyde clustered with glucocorticoid receptor modulators and induced nuclear translocation, emphasizing the power of this approach in uncovering novel drug mechanisms and, therefore, aiding in improving therapeutic strategies.
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In this study, we demonstrate how cell activation with91PMA reveals previously undetectable phenotypes and enables the functional annotation of92compounds that would otherwise remain in the phenotypic dark space
This is a cool way to "induce" more phenotypic variation from cells in response to drugs. Are there other pathways that could be stimulated and be orthogonal to PKC for even more possibility of finding phenotypes?
How physiologically relevant is this activation of cells? Is this perturbation just a way to stress the cells or does it actually place them in a more clinically-relevant state?
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The decision to screen compounds at both 1 μM and 10 μM concentrations provided514complementary insights into compound activities. While the higher concentration revealed515more dramatic phenotypic changes and enabled the detection of weakly active compounds, the516lower concentration helped identify more specific and potentially physiologically relevant517effects while minimizing cytotoxicity.
Is it possible to distinguish drug-induced cytotoxicity from a drug-induced phenotype?
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After drug treatment, the cells were activated.
Is there a specific reason why activation should happen after drug treatment versus before? Does the phenotype from activation start to reverse after some amount of time?
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