(+)-Usnic acid and its salts, inhibitors of SARS-CoV-2, identified by using in silico methods and in vitro assay

Abstract

The pandemic caused by severe acute respiratory Coronavirus-2 (SARS-CoV-2) has been ongoing for over two years, and treatment for COVID-19, other than monoclonal antibodies, is urgently required. Accordingly, we have investigated the inhibitors of SARS-CoV-2 protein targets by high-throughput virtual screening using a marine natural products database. Considering the calculated molecular properties and availability of the compounds, (+)-usnic acid was selected as a suitable hit. In the in vitro antiviral assay of (+)-usnic acid by the immunofluorescence method, IC₅₀ was 7.99 μM, which is similar to that of remdesivir used as a positive control. The generalized Born and surface area continuum solvation (MM/GBSA) method was performed to find the potent target of (+)-usnic acid, and the Mpro protein showed the most prominent value, -52.05 kcal/mol, among other SARS-CoV-2 protein targets. Thereafter, RMSD and protein-ligand interactions were profiled using molecular dynamics (MD) simulations. Sodium usnate (NaU) improved in vitro assay results with an IC₅₀ of 5.33 μM and a selectivity index (SI) of 9.38. Additionally, when (+)-usnic acid was assayed against SARS-CoV-2 variants, it showed enhanced efficacy toward beta variants with an IC₅₀ of 2.92 μM and SI of 11.1. We report the in vitro anti-SARS-CoV-2 efficacy of (+)-usnic acid in this study and propose that it has the potential to be developed as a COVID-19 treatment if its in vivo efficacy has been confirmed.

Figure 1

The anti-SARS-CoV-2 efficacy of (+)-usnic acid and controls; (a) The structure of (+)-usnic acid on the left and dose response curve of (+)-usnic acid against SARS-CoV-2 on the right; (b) The dose response curve of controls (chloroquine, lopinavir and remdesivir); the inhibition of infection and cell number to mock is shown using blue and red lines, respectively.

Figure 2

Predicted binding-free energies (kcal/mol) of 8 SARS-CoV-2 target proteins bound with (+)-usnic acid, calculated by Prime MM-GBSA.

Figure 3

The MD simulation of (+)-usnic acid bound to the SARS-CoV-2 Mpro protein; (a) RMSD of the alpha-carbon of Mpro; (b) The ligand RMSD of (+)-usnic acid bound to Mpro, simulated for 100 ns; (c) The Mpro amino acid interaction with (+)-usnic acid showing H-bonds, hydrophobic interactions and water bridges; (d) The 2D % interaction illustration of (+)-usnic acid in tautomeric form; (e) The 3D illustration of (+)-usnic acid bound to Mpro showing interacting amino acids and hydrogen bonds.