Cell Painting in activated cells illuminates phenotypic dark space and drug mechanisms of action.

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 combine 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 enables detection of phenotypic effects for up to 40% of all screened compounds, effectively illuminating more phenotypic 'dark space.' The combined cellular CP fingerprints from PMA-activated and resting cells reveal phenotypes for 565 compounds that are inactive under the resting condition alone, supporting its use in MoA studies. We introduce quality control measures for CP screens and demonstrate that integrating phenotypic signatures supports MoA discovery. Notably, 2-methoxycinnamaldehyde clusters with glucocorticoid receptor modulators and induces nuclear translocation, emphasizing the utility of this approach in identifying drug mechanisms and, therefore, aiding in improving therapeutic strategies.