In vitro activity: Oligomycin A is an inhibitor of ATP synthase, inhibits oxidative phosphorylation and all the ATP-dependent processes occurring on the coupling membrane of mitochondria.Oligomycin A inhibits ATP synthase by blocking its proton channel (Fo subunit), which is necessary for oxidative phosphorylation of ADP to ATP. The inhibition of ATP synthesis by oligomycin A will significantly reduce electron flow through the electron transport chain; however, electron flow is not stopped completely due to a process known as proton leak or mitochondrial uncoupling. In a group of cancer cells, Oligomycin at 100 ng/ml completely inhibits oxidative phosphorylation activity in 1 h and induces various levels of glycolysis gains by 6 h. Oligomycin, inhibitor of the F0 part of H+-ATP-synthase, suppresses the TNF-induced apoptosis. Oligomycin inhibits mitochondrial respiration in melanoma, sensitizes melanoma cells to therapy, blocks the emergence of the slow-cycling, long-term tumor-maintaining melanoma cells.

Cell Assay: ATP and Oligomycin Dose-Response Growth Measurement. Cellular ATP changes are measured by CellTiter-Glo reagent. To measure oligomycin dose-response curves, the cells are plated in 96-well plates at about 400–500 cells/well in 100 μl of culture, dosed the next day, and grown for 4 additional days followed by assaying with Cell-Titer-Glo reagent. The dose-response curves are plotted with nonlinear regression analysis of GraphPad Prism.