Researchers at the University of California, Santa Cruz, developed a method that pairs cell activation with cytological profiling to identify drug effects that standard assays fail to detect, according to a report in Nature Communications. The technique combines compound dosing with cellular activation using the protein kinase C agonist phorbol myristate acetate, known as PMA.
The scientists tested A549 lung cancer cells against 8,387 compounds at concentrations of 1 and 10 micromolar. Profiling the cells across both resting and PMA-activated states revealed phenotypic responses for up to 40 percent of the screened compounds.
Combining the image-based Cell Painting fingerprints from both activated and resting states uncovered phenotypic activity for 565 compounds that showed no effect in resting cells alone. The team also established quality control measures for Cell Painting screens to assist in determining compound mechanisms of action.
Testing validated specific biological interactions within the library. The compound 2-methoxycinnamaldehyde clustered with glucocorticoid receptor modulators and induced nuclear translocation. To assess this mechanism, the authors used a glucocorticoid receptor nuclear translocation immunofluorescence protocol provided by Karolien De Bosscher and Laura Van Moortel at the VIB Center for Medical Biotechnology at Ghent University.
Authors Akshar Lohith and Derfel Terciano contributed equally to the work alongside Matylda A. Zietek, Beverley M. Rabbitts, Aswad Khadilkar, John B. MacMillan, and R. Scott Lokey. The project used randomized plate maps provided by Benjamin David and Assistant Professor Paul Jensen from the University of Michigan. Research instruments and compound libraries were supplied through the UCSC Chemical Screening Center, funded in part by National Institutes of Health grants.
