The Potential of Usnic-Acid-Based Thiazolo-Thiophenes as Inhibitors of the Main Protease of SARS-CoV-2 Viruses

Abstract

Although the COVID-19 pandemic caused by SARS-CoV-2 viruses is officially over, the search for new effective agents with activity against a wide range of coronaviruses is still an important task for medical chemists and virologists. We synthesized a series of thiazolo-thiophenes based on (+)- and (-)-usnic acid and studied their ability to inhibit the main protease of SARS-CoV-2. Substances containing unsubstituted thiophene groups or methyl- or bromo-substituted thiophene moieties showed moderate activity. Derivatives containing nitro substituents in the thiophene heterocycle-just as pure (+)- and (-)-usnic acids-showed no anti-3CL^pro activity. Kinetic parameters of the most active compound, (+)-3e, were investigated, and molecular modeling of the possible interaction of the new thiazolo-thiophenes with the active site of the main protease was carried out. We evaluated the binding energies of the ligand and protein in a ligand-protein complex. Active compound (+)-3e was found to bind with minimum free energy; the binding of inactive compound (+)-3g is characterized by higher values of minimum free energy; the positioning of pure (+)-usnic acid proved to be unstable and is accompanied by the formation of intermolecular contacts with many amino acids of the catalytic binding site. Thus, the molecular dynamics results were consistent with the experimental data. In an in vitro antiviral assay against six strains (Wuhan, Delta, and four Omicron sublineages) of SARS-CoV-2, (+)-3e demonstrated pronounced antiviral activity against all the strains.

Keywords: 3CLpro; SARS-CoV-2; main protease; molecular modeling; usnic acid.

Figure 1

Structure of (+)-and (−)-usnic acid and their natural sources. The images are taken from [46] with permission.

Scheme 1

Synthesis of compounds 3a–g.

Figure 2

The curves of residual protease activity versus inhibitory concentration of compound (+)-3e.

Figure 3

The dependence of the initial rate of substrate cleavage by SARS-CoV-2 3CL^pro without (A) or with a potential inhibitor (+)-3e (B). Each data point are an average of at least three independent experiments; the values are presented as the mean ± SE.

Figure 4

Contacts between atoms of the ligand and surrounding amino acid residues, as recorded throughout the entire simulation of the 3CL^pro–(+)-3e complex. The amino acids of the catalytic dyad are highlighted with a red square.

Figure 5

Contacts between atoms of the ligand and surrounding amino acid residues, as recorded throughout the entire simulation of the 3CL^pro–(+)-3g complex. The amino acids of the catalytic dyad are highlighted with a red square.

Figure 6

A summary of contacts recorded during molecular dynamics simulations. For complexes 3CL^pro–(+)-3e and 3CL^pro–(+)-3g, contacts are shown that were detectable for more than 20% of the simulation time for the 3CL^pro–(+)-1 complex, they were present for more than 10% of the simulation time. Various substituents are highlighted in red.

Figure 7

Molecular modelling study: Positioning of the studied ligands (+)-3e (A), (+)-3g (B) and (+)-UA (C), respectively, in the active binding site of subunit 3CLpro of the main protease. Letter designations S1–S3 correspond to significant cavities in the catalytic binding site.