AMPK-specific autophagy activator based on 1,3-diaza-2-oxophenoxazine

The dynamic equilibrium between synthesis and degradation of biomolecules is maintained by cells, however, with aging, this balance is disrupted, resulting in the onset of diseases, including diabetes and neurodegenerative diseases. A decrease in autophagy, a key cellular process that is involved in lysosome-mediated degradation of damaged or dysfunctional cellular components, may contribute to this imbalance. Autophagy is strictly regulated within the cell through multiple signaling pathways, e.g., through the AMPK-dependent pathway, which functions as a key sensor of cellular energy limitation. In this study, we assessed the autophagy/mitophagy activation ability of a small set of 1,3-diaza-2-oxophenoxazine derivatives and analogs using a fluorescent reporter assay and immunoblot analysis. The two lead compounds, AR493 and AR900, which exhibited the highest autophagy induction levels, were demonstrated to activate the AMPK-dependent pathway. The introduction of a 2′-hydroxyl group into AR493 had almost no influence on its activity, while subsequent attachment of a metabolizable masked phosphate group resulted in a notable increase in activity, although accompanied by substantial toxicity. When analyzing the specificity of the lead compounds to AMPK and its main upstream regulator SIRT1 on the corresponding knockout cell lines, AR493 demonstrated the greatest specificity of action to AMPK. Molecular docking revealed that AR493 binds to Site 2 of the AMPK γ-subunit, which may promote AMPK activation by two possible mechanisms: by preventing ATP binding to Site 3, thus favoring AMP binding; and by directly engaging the αRIM2 motif to stabilize its interaction with the γ-subunit. © 2025 Elsevier B.V., All rights reserved.