Statine-based peptidomimetics as SARS-CoV-2 Papain-like protease inhibitors: in Silico and in vitro studies

Abstract

We investigated statin-based peptidomimetic compounds as inhibitors of SARS-CoV-2 papain-like protease (PLpro) by in silico methods, including molecular docking/dynamic simulations and ADMET prediction, as well as enzymatic in vitro assays. Five compounds (LQMed 426, 428, 430, 431, and 432) were identified as having promising interactions with the active site and an allosteric site of PLpro. The docking poses in the active site revealed that the compounds interacted with the catalytic triad (Cys111, His272, and Asp286). Compound 426 stood out for forming significant hydrophobic interactions and hydrogen bonds. In molecular dynamic (MD) simulations, there was a tendency for the compounds to migrate to a pocket in the ubiquitin-like (Ubl) domain region, indicating possible allosteric inhibition. Principal component analysis (PCA) and free energy landscape (FEL) plot showed significant differences in the conformational stability of protein-ligand complexes. Complex 432 had the highest stability (PC1: 24.9%, PC2: 10.5%) with restricted movements, while complex 430 had the lowest stability and the highest PC1, indicating broader movements. Complexes 426 and 428 had intermediate stability. FEL analysis confirmed that complex 432 had multiple minimum energy clusters, suggesting a greater likelihood of stable enzyme-ligand complex. In vitro assays demonstrated that all compounds inhibited PLpro activity by at least 50%, with IC₅₀ values ranging from 0.85 µM to 4.06 µM. Compounds 426 and 432 were the most promising, with IC₅₀ of 0.85 µM and 1.46 µM, respectively, values comparable to the GRL-0617 reference inhibitor. Binding free energy analysis indicated that compounds 430 and 431 showed significant affinity for the allosteric site (-27 kcal/mol and - 14 kcal/mol, respectively), while compound 428 showed reduced stability. In addition, in silico pharmacokinetics and toxicity (ADMET) prediction revealed that statine-based compounds are promising compared to GRL-0617 due to the low potential of inhibition of CYP's family and toxicity risk. Overall, the tested compounds, especially 426 and 432, showed potential as inhibitors of SARS-CoV-2 PLpro and may act by non-competitive or mixed mechanisms, making them promising candidates for developing antivirals against CoViD-19.

Keywords: CoViD-19; Inhibitors; Molecular Docking; Molecular dynamics; PLpro; Statine.

Fig. 1

Best pose by docking simulations of the statine-based peptidomimetics (LQMed compounds) on the active site of SARS-CoV-2 PLpro: (A) 426; (B) 428; (C) 430; (D) 431; and (E) 432. The residues involved in H-bond interactions (dashed yellow lines) with the ligands are represented in ball-and-stick models, and residues involved in hydrophobic interactions are in stick models. In the 2D structures, the atoms (or groups) of the ligands involved in H-bond interactions are circled in pink.

Fig. 2

RMSD graph during 500 ns of the MD simulations of the protein-ligand complexes based on the ligand’s atoms of compounds (A) 426; (B) 428; (C) 430; (D) 431; and (E) 432.

Fig. 3

RMSD graph during 500 ns of the MD simulations of the protein-ligand complexes based on the protein Cα-atoms from PLpro in the presence of compounds (A) 426, (B) 428, (C) 430, (D) 431, and (E) 432.

Fig. 4

Cα-RMSF graph relative to the MD simulations of the PLpro-LQMed complexes and holoenzyme (PLpro). Ligands 426 (red line), 428 (orange line), 430 (green line), 431 (blue line), and 432 (pink line); and holoenzyme (black line). Ribbon diagram of the PLpro enzyme: The cyan (zinc finger domain), red (BL2), yellow (active site), green (S2 helix), and pink (Ubl) arrows (and cartoons) indicate amino acid residues with the highest RMSF fluctuations.

Fig. 5

H-bond interactions by molecular dynamics simulations of the PLpro-LQMed complexes. Ligands: (A) 426; (B) 428; (C) 430; (D) 431; (E) 432. Donor and acceptor groups involved in the H-bonds and their lifetime (%). The 3D structure represents the ligands (blue balls and sticks) involved in H-bond interactions (dashed yellow lines) with residues (orange sticks).

Fig. 6

RMSD graph during 200 ns of the MD simulations of the protein-ligand complexes (starting from the frames with the compounds located in the allosteric site) based on ligands atoms of compounds: (A) 426; (B) 428; (C) 430; (D) 431; and (E) 432. The representative poses of each ligand during MD (yellow) and molecular docking (cyan) simulations are shown for the best comparison.

Fig. 7

RMSD graph during 200 ns of the MD simulations of the protein-ligand complexes (starting from the frames with the compounds located in the allosteric site) based on protein Cα-atoms in the presence of compounds: (A) 426; (B) 428; (C) 430; (D) 431; and (E) 432. (F) Cα-RMSF analysis was relative to the MDs of the PLpro-LQMed complexes.

Fig. 8

H-bond interactions from 200 ns of the MD simulations of the PLpro-LQMed complexes (starting from the frames with the compounds located in the allosteric site) for compounds: (A) 426, (B) 428, (C) 430, (D) 431, and (E) 432. The ligands (blue balls and sticks) that performed H-bond interactions (dashed yellow lines) with residues (orange sticks).