Allosteric Interactions of Flavonoids with ACE2 and Potential Implications for COVID-19: Computational Insights, Metabolic and Mitochondrial Effects of Rutin in A549 Cells

Abstract

There has been a compelling need to identify new therapeutic targets and agents owing to the increasing complexity of disease mechanisms. Angiotensin-converting enzyme 2 (ACE2), the primary host receptor for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, plays a crucial role in the pathophysiology of COVID-19, making ACE2 a potential biological target. Identification of small molecules that can interact with ACE2 may offer insights into a host-targeted approach relevant to viral entry and ACE2-associated cellular pathways. The study evaluated allosteric interactions of selected flavonoids with ACE2, the cytotoxicity and metabolic effects of the promising candidate in A549 cells through computational and in vitro methods. Molecular docking identified flavonoid binding to ACE2, followed by a 120 ns molecular dynamics (MD) simulation to characterize the stability and dynamic behaviour of ACE2-flavonoid complexes. MM/GBSA binding free energy calculations and per residue energy decomposition were performed to identify key interactions stabilizing ligand binding. The in silico analyses showed ARG255, PRO328, GLU357, GLU384, GLU388, and ARG500 as the key allosteric residues contributing to ACE2-flavonoid complex stability. Rutin exhibited the most favourable binding affinity, followed by isoquercetin. Based on computational findings, rutin was selected for evaluation in A549 cells to assess cytotoxicity, metabolic and mitochondrial effects using MTT assay, ATP quantification, mitochondrial membrane potential (MMP), CYP3A4 activity and LDH release for membrane integrity. In vitro analyses showed that rutin is non-cytotoxic up to 500µM, enhances ATP production without adversely affecting MMP and membrane integrity. These findings indicate that rutin formed stable interactions with ACE2, with the potential to influence cellular energy metabolism without inducing cytotoxicity and therapeutic benefits in conditions linked to mitochondrial dysfunction and metabolic stress. While direct ACE2 enzymatic modulation was not confirmed experimentally, these findings provide a mechanistic basis for further studies on ACE2-flavonoid interactions and their potential biological implications in ACE2-associated diseases.