Ginkgolic Acid as a fusion inhibitor: Targeting RBD-ACE2 interaction with in silico, in vitro evidence of Allosteric modulation and potent virucidal effect

This study investigates Ginkgolic acid, a naturally occurring phenolic compound from Ginkgo biloba, for its potential to inhibit SARS-CoV-2 by targeting the RBD-ACE2 interface through a combination of computational and in vitro methodologies. In silico molecular docking and 500 ns molecular dynamics (MD) simulations were employed to examine the binding conformation and stability of Ginkgolic acid at the RBD-ACE2 interface. In vitro studies included cytotoxicity profiling, plaque reduction assays, qRT-PCR, luciferase-based viral entry quantification, and membrane fusion inhibition in Vero E6 and ACE2-expressing HEK293T cells. Ginkgolic acid demonstrated dose-dependent antiviral activity in Vero E6 cells with half-maximal inhibitory concentration (IC50) values ranging from 0.025 to 0.102 µM. MD simulations revealed its strong binding affinity at the RBD-ACE2 interface, particularly through interactions with conserved residues His34 and Asp38, leading to increased inter-residue distances and destabilization of the complex. In vitro assays confirmed significant virucidal activity, reduced viral entry, and inhibition of spike-mediated membrane fusion. Despite limited intracellular uptake, its antiviral efficacy appears to be predominantly extracellular. Ginkgolic acid acts as an allosteric modulator and fusion inhibitor by targeting conserved RBD-ACE2 interface residues, disrupting viral attachment and fusion. Its dual mechanism-direct virucidal effect and host-cell entry inhibition-presents a promising scaffold for future antiviral development. These findings warrant further preclinical validation to assess its efficacy across SARS-CoV-2 variants.