Cell Painting reveals polypharmacological activity on the V-ATPase by the F-ATPase inhibitor Cyhexatin

Polypharmacology, a single drug affecting multiple targets, poses a significant challenge in drug discovery. There is a particular potential for polypharmacological effects for structurally similar proteins that have different functions. Examples of such proteins are the vacuolar V₀V₁-ATPases (V-ATPases) and mitochondrial F₁F₀-type ATPases (F-ATPases). They share structural homology yet perform opposing cellular roles in different subcellular compartments. V-ATPases are involved in ATP-hydrolysis-driven proton transport for organelle acidification, while F-ATPases work in reverse-mode utilizing a proton gradient to generate ATP in mitochondria. In this study, we applied Cell Painting, a high-content phenotypic screening technique, to distinguish the differential effects of compounds targeting V- and F-ATPases. By capturing detailed cellular morphologies, Cell Painting enabled the generation of phenotypic signatures that differentiated the functional roles and compound responses of these two ATPases. Despite their shared structural features, the resulting profiles revealed distinct inhibitor-induced phenotypic changes relating to their physiological mode of action (MoA). To corroborate these findings, we used biochemical assays to quantify ATPase-specific activities and further verify the selective inhibition of each ATPase. Additionally, in vivo validation was performed through compound injections into Spodoptera frugiperda larvae, assessing the physiological impact on the whole animal. These complementary approaches provide strong evidence for Cyhexatin, a registered miticide, to not only target F-ATPase but to also have a polypharmacological effect on the V-ATPase, another established insecticidal target. This integrative strategy has the potential to unravel the specificity of compounds, identify those with minimal off-target effects, and ultimately design specific, selective and safe next generation insecticides.