Kobophenol A Inhibits Binding of Host ACE2 Receptor with Spike RBD Domain of SARS-CoV-2, a Lead Compound for Blocking COVID-19

Keywords: Kobophenol A, SARS-CoV-2, ACE2, Spike protein, S1-RBD, COVID-19, natural products, antiviral, docking, molecular dynamics

Abstract
In the search for inhibitors of COVID-19, we targeted the interaction between the human angiotensin-converting enzyme 2 (ACE2) receptor and the spike receptor binding domain (S1-RBD) of SARS-CoV-2. Virtual screening of a library of natural compounds identified Kobophenol A as a potential inhibitor. Kobophenol A was found to block the interaction between ACE2 and S1-RBD in vitro with an IC₅₀ of 1.81 ± 0.04 μM and inhibit SARS-CoV-2 viral infection in cells with an EC₅₀ of 71.6 μM. Blind docking calculations identified two potential binding sites, and molecular dynamics simulations predicted binding free energies of −19.0 ± 4.3 and −24.9 ± 6.9 kcal/mol for Kobophenol A at the spike/ACE2 interface and the ACE2 hydrophobic pocket, respectively. In summary, Kobophenol A, identified through docking studies, is the first compound that inhibits SARS-CoV-2 binding to cells through blocking S1-RBD to the host ACE2 receptor and may serve as a good lead compound against COVID-19.

Introduction
The new coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), rapidly spread as the COVID-19 global pandemic. As of December 2020, there were over 68 million active cases and over 1.57 million deaths worldwide. The absence of specific treatments and the high death toll have prompted a global effort to identify inhibitors of key viral processes. Natural products, with their structural diversity, continue to inspire new drug discovery, especially for antiviral drug development.

While much research has focused on inhibiting the SARS-CoV-2 main protease (MPro/3CLPro), this study focused on the spike proteins, which are critical for viral entry into host cells. The spike protein consists of two subunits: S1, which contains the receptor-binding domain (RBD) that interacts with the ACE2 receptor on host cells, and S2, which facilitates membrane fusion. Understanding the conformational states of the spike protein, particularly the “down” (receptor-inaccessible) and “up” (receptor-accessible) states, has been crucial for rational drug and vaccine design.

Blocking the interaction between the S1-RBD and ACE2 is a validated target for preventing viral entry. Previous studies have attempted to identify small molecule inhibitors of this interaction, but none had demonstrated potent in vitro inhibition prior to this work.

Methods
A virtual screening approach was used, targeting the ACE2/S1-RBD interface based on the X-ray crystal structure (PDB ID: 6M0J). A library of 25 natural compounds, including metabolites, was evaluated using AutoDock 4.2 for initial binding. Top candidates were further studied with molecular dynamics (MD) simulations to predict binding stability and free energies.

Results
Docking and Binding Sites
Kobophenol A was identified as the top candidate, effectively binding to two regions:

ACE2/spike interface: Kobophenol A formed a hydrogen bond with residue Gln325, with a docking energy of −11.15 kcal/mol.

ACE2 hydrophobic pocket: Kobophenol A formed hydrogen bonds with Glu375 and Thr347, with a docking energy of −9.98 kcal/mol.

Three metabolites of Kobophenol A (M1, M2, M3) also showed favorable docking energies at these sites.

In Vitro Inhibition
Kobophenol A inhibited ACE2 binding to S1-RBD in an ELISA assay with an IC₅₀ of 1.81 ± 0.04 μM. In a cell-based antiviral assay using VeroE6-EGFP cells, Kobophenol A inhibited SARS-CoV-2 infection with an EC₅₀ of 71.6 μM, comparable to FDA-approved drugs such as Indinavir and Favipiravir. Importantly, Kobophenol A showed no cytotoxicity to uninfected cells up to 100 μM.

Molecular Dynamics Simulations
MD simulations examined the interactions and conformational changes upon Kobophenol A binding at both the ACE2/spike interface and the ACE2 hydrophobic pocket. Root-mean-square deviation (RMSD) analysis showed that the S1-RBD region equilibrated quickly, while the ACE2 receptor required longer to stabilize, especially when Kobophenol A was bound in the hydrophobic pocket. Root-mean-square fluctuation (RMSF) analysis indicated greater fluctuations in the S1-RBD region when Kobophenol A was at the interface, particularly within residues 435–460 and 475–515, which form the receptor binding motif (RBM).

Hydrogen Bonding and Binding Free Energy
Hydrogen bonding analysis revealed that many of the interactions present in the crystal structure were lost during MD simulations, with only a subset remaining intact regardless of Kobophenol A binding. Notably, the Y495-K353 hydrogen bond, present in the Apo system, was eliminated when Kobophenol A was bound, suggesting a possible mechanism for inhibition.

Binding free energies calculated using the MM/PBSA method were −19.0 ± 4.3 kcal/mol for the ACE2/spike interface and −24.9 ± 6.9 kcal/mol for the ACE2 hydrophobic pocket, indicating strong binding in both locations.

Discussion
Kobophenol A is a natural oligomeric stilbenoid, a tetramer of resveratrol, isolated from the Caragana genus. It has previously demonstrated antiviral, neuroprotective, anti-inflammatory, cardioprotective, and skin-whitening activities, and is non-toxic in traditional medicinal use. Its additional anti-inflammatory, bronchodilator, cardioprotective, and antioxidant properties may provide added benefits for COVID-19 patients, potentially reducing mortality associated with cytokine storm and organ inflammation.

Pharmacokinetic studies in rats showed a half-life of 0.68 h (i.v.) and 5.78 h (oral). Kobophenol A’s metabolites (M1, M2, M3) also demonstrated good binding energies in silico.

Conclusion
This study demonstrates that Kobophenol A effectively inhibits the interaction between the ACE2 receptor and the S1-RBD domain of SARS-CoV-2, both in silico and in vitro. It is the first compound shown to block SARS-CoV-2 binding to host cells by targeting this critical interface, making it a promising lead compound for anti-COVID-19 drug development. Its favorable safety profile and additional health benefits further support its potential as a therapeutic agent or Compound 19 inhibitor supplement in COVID-19 treatment.