Anticancer/Antiviral Agent Akt Inhibitor-IV Massively Accumulates in Mitochondria and Potently Disrupts Cellular Bioenergetics

[Image: see text] Inhibitors of the PI3-kinase/Akt (protein kinase B) pathway are under investigation as anticancer and antiviral agents. Akt inhibitor-IV (ChemBridge 5233705, CAS 681281-88-9, AKTIV), a small molecule reported to inhibit this pathway, exhibits potent anticancer and broad-spectrum antiviral activity. However, depending on concentration, this cationic benzimidazole derivative exhibits paradoxical positive or negative effects on the phosphorylation of Akt that are not well understood. To elucidate its mechanism of action, we investigated its spectroscopic properties. This compound proved to be sufficiently fluorescent (excitation λ(max) = 388 nm, emission λ(max) = 460 nm) to enable examination of its uptake and distribution in living mammalian cells. Despite a low quantum yield of 0.0016, imaging of HeLa cells treated with AKTIV (1 μM, 5 min) by confocal laser scanning microscopy, with excitation at 405 nm, revealed extensive accumulation in mitochondria. Treatment of Jurkat lymphocytes with 1 μM AKTIV for 15 min caused accumulation to over 250 μM in these organelles, whereas treatment with 5 μM AKTIV yielded concentrations of over 1 mM in mitochondria, as analyzed by flow cytometry. This massive loading resulted in swelling of these organelles, followed by their apparent disintegration. These effects were associated with profound disruption of cellular bioenergetics including mitochondrial depolarization, diminished mitochondrial respiration, and release of reactive oxygen species. Because mitochondria play key roles in both cancer proliferation and viral replication, we conclude that the anticancer and antiviral activities of AKTIV predominantly result from its direct and immediate effects on the structure and function of mitochondria.