The next generation insect cell phenotyping technology “Cell Painting” effectively unravels the Modes of Action of V-ATPase and F-ATPase inhibitors

Cellular screening techniques have proven to be invaluable for modern drug discovery processes. With the introduction of Cell Painting, a next-generation cell phenotypic screening technology, multiple cellular processes can be assessed in parallel. Cell Painting uses high-content microscopy and a combination of fluorescent dyes to "paint" cells. This strategy allows to capture in parallel distinct information about different cellular organelles, cytoskeletal structures, and cellular components, allowing for the detection of subtle phenotypic changes in response to chemical treatments. By assessing these phenotypic fingerprints, Cell Painting enables diagnostics of mechanism of action and assessments of efficacy. However, it is currently almost exclusively applied in pharmaceutical research. Here we demonstrate the application of Cell Painting to insect cell lines and show its value in studying the efficacy and mechanism of action of insecticidal compounds. We showcase how Cell Painting can differentiate the phenotypic responses and functional roles of compounds that target V- and F-ATPases. Despite the shared structural features of these ATPases, the resulting phenotypic profiles revealed distinct inhibitor-induced phenotypic changes, enabling the assessment of the similarity and specificity of compound effects. We further quantify the ATPase-specific activities using biochemical assays and verify the specific inhibition of each ATPase. Notably, we were able to provide strong evidence that several public and proprietary V- and F-ATPase inhibitors, believed to be specific, act on both molecular targets. In essence we demonstrate how Cell Painting enables the identification of specific compounds ultimately enabling the design of specific, selective and safe next generation insecticides.