Kobophenol A Inhibits Binding of Host ACE2 Receptor with Spike RBD Domain of SARS-CoV-2, a Lead Compound for Blocking COVID-19
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Product name: Kobophenol A
Product Numbers: CFN92530
Journal of Physical Chemistry Letters: (IF 2021: 6.795)
Copyright: American Chemical Society
In the search for inhibitors of COVID-19, we have 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 then found to block the interaction between the ACE2 receptor 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 to 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 thus may serve as a good lead compound against COVID-19.
Kobophenol A was purchased from ChemFaces, China with a purity of ≥98% (catalog no. CFN92530). We also acquired the following: SARS-COV-2 S1-RBD (Novateinbio. USA, catalog no. PR-nCOV-2), ACE2 receptor protein (Novateinbio, catalog no. PR-nCOV-4), and goat anti-human IgG-Fc-HRP conjugate. Kobophenol A was dissolved in DMSO and stored at −20 °C. Molecular docking studies were carried out by using AutoDock ver. 4.2 on Windows. All the molecular dynamics simulations were carried out using the GPU-enabled Amber18 pmemd engine, and cpptraj was utilized for analyzing the trajectory.
The crystal structure of SARS-CoV-2 was retrieved from rcsb.org (PDB ID: 6M0J)(6) and used to generate initial 3D coordinates of the spike S1-RBD-ACE2 complex. Cocrystallized water molecules were deleted. Polar hydrogens were added, and Gasteiger charges were computed. The structures of selected natural compounds were superimposed against the predocked ligand in the PDB, and the latter was then removed to generate initial conformation of natural compound at the active site of SARS-CoV-2. As the natural compounds were not available in the X-ray crystal structure of S1-RBD bound with ACE2, a grid box was generated by considering the whole protein and blind docking was performed (PDB ID: 6M0J).(6) Finally, both Autogrid and AutoDock were run with the default parameters as described in following references: refs (45−47). Top scoring molecules were evaluated for their interactions.
In Vitro Spike S1-RBD and ACE2 Inhibitory Activity of SARS-CoV-2 by Enzyme-Linked Immune Sorbent Assay (ELISA). To test whether Kobophenol A, inhibits the interaction between ACE2 and S1-RBD of SARS- CoV-2, a 96-well plate was coated with recombinant 2019-nCoV S1-RBD (catalog no. PR- nCOV-2, Novatein Biosciences, USA) at 0.1–0.4 μg/mL overnight. Plates were washed 3× with PBS pH 7.2 (without Ca2+ and Mg2+) with 0.05% Tween-20 and blocked with 1% BSA in PBS. ACE2 receptor protein (catalog no. PR-nCOV-4), 0.1–0.2 μg/mL, was added in the presence or absence of Kobophenol A at various concentrations. For data sheets of human ACE2, spike RBD, and Kobophenol A, see Supporting Information Figure S5. Samples were incubated for 1–2 h in the binding buffer (0.1% BSA in PBS, pH 7.2). Plates were washed, and anti-human Fc-antibody-HRP 1:20,000 in binding buffer was added. After three washes 3,3′,5,5′-tetramethylbenzidine (TMB) was added for a signal; after stopping the reaction with an acidic solution, the plates were read at 450 nm. IC50 values were calculated by using GraphPad. The ELISA assay was performed twice by Novatein Biosciences, USA.