In silico analysis of SARS-CoV-2 papain-like protease potential inhibitors

Abstract

The emergent outbreak caused by severe acute respiratory syndrome coronavirus 2 continues spreading and causing huge social and economic disruption. Papain-like protease (PLpro) has a crucial role in the cleavage of viral polyproteins, and disruption of host responses. PLpro is considered an important goal for the development of SARS-CoV-2 inhibitors. ZINC101291108 (lead 1) and ZINC16449029 (lead 2) were identified as potent SARS-CoV-2 PLpro inhibitors with IC₅₀ values of 0.085 μM and 0.063 μM, respectively. Molecular dynamics simulations (MD) were carried out for lead 1, 2 and several reported SARS-CoV-2 inhibitors. Analysis results of the simulations confirmed the stability of both compounds and showed that they adopted two confirmations along the simulation period. The per-residue decomposition results revealed that the key residues involved in inhibitor binding were E167, P247, P248, Y264, Y268 and Q269. H-bond analyses showed H-bonds with G266 and N267 and salt bridges with G209 and Y273, which are essential for strengthening the substrate-binding pocket. Both inhibitors showed hydrophobic interactions with the S4 site and BL2 loop residues. The RMSD of the BL2 loop with the two inhibitors was investigated, and the results showed that the Y268 and Q269 BL2 loop residues moved outward to accommodate the large size of lead 2. The van der Waals interaction was the main energy contribution that stabilized lead 2, while van der Waals and electrostatic interactions were the main energy contributions stabilizing lead 1. Rational design strategies were suggested to replace the 2-(2-hydroxybenzylidene) hydrazine moiety with naphthalene or nitrobenzene at the P4 position of lead 2 and introduce polar substituents as aniline and benzoate groups at position P1 to enhance hydrophobic interactions and H-bonds, respectively.

Fig. 1. Representation of SARS-CoV-2 PLpro in the cartoon representation (PDB: 7JRN). The protein contains the ubiquitin-like (UBL) (purple), thumb (blue), palm (orange red), and finger (green) domains. In the putative site, BL2 loop (Salmon) with key residues Y268 and Q269. The GRL0617 (sick, violet). The bound Zn²⁺ ion in the finger domain is shown as a blue sphere coordinated with C192, C224 and C226.

Fig. 2. Structure of SARS-CoV PLpro inhibitors.

Fig. 3. Structure of SARS-CoV_2 PLpro inhibitors.

Fig. 4. The RMSD values; (A) lead1-protein complex (red), lead 2–protein complex (yellow), GRL0617–protein complex (green) protein (black). (B) RMSD values of compound 2-protein complex (red), compound 3–protein complex (green), compound 6–protein complex (blue), simeprevir (SIM)–protein complex (yellow), vaniprevir (VAN)–protein-complex (violet).

Fig. 5. The RMSF values; (A) lead 1–protein complex (red), GRL0617–protein complex (green), protein (black). (B) lead 2–protein complex (red), GRL0617–protein complex (green) protein (black), (C) RMSF value; compound 2–protein complex (red), compound 3–protein complex (green), compound 6–protein complex (blue), simeprevir (SIM)–protein complex (yellow), vaniprevir (VAN)–protein-complex.

Fig. 6. (A) Radius of gyration (R_g) of lead 1 (blue), lead 2 (red), GRL0617 (green) complexed with the protein and the nonligand protein (black). (B) R_g of CP2 (red), CP3 (green), CP6 (blue), simeprevir (SIM) (yellow) and vaniprevir (VAN) (violet) complexed with the protein. (C) SASA of lead 1 (blue), lead 2 (red), GRL0617 (green) complexed with the protein and the nonligand protein (black) (D) SASA of CP2, CP3, CP6, simeprevir (SIM) and vaniprevir (VAN) complexed with the protein.

Fig. 7. (A) number of H-bonds of GRL0617 with SARS-CoV-2 PLpro putative pocket. (B) Percentage of H-bonds, salt bridge and hydrophobic occupations of the SARS-CoV-2 PLpro residues contributed to GRL0617.

Fig. 8. Number of H-bonds with SARS-CoV-2 PLpro putative pocket; (A) lead 1. (B) Lead 2. Percentage of H-bonds, salt bridge and hydrophobic occupations of the SARS-CoV-2 PLpro residues; (C) lead1. (D) Lead 2.

Fig. 9. Representative structures of lead1 within PLpro putative pocket. (A) Structure of lead 1 (spheres, carbon atoms violet) at the beginning of MD simulation. (B) Interaction of lead 1 with the key residues. (C) Lead 1 after 30 ns (sphere, carbon atoms yellow), (D) the interaction of lead 1 at 30 ns and it is observed that 5-amino-2-methoxyphenol moved upward and showed hydrophobic interactions with P247 and A246 and 6-hydroxy-pyrmidine-dione formed H-bond with Y268. (E) Lead 1 at the end of the simulation (sphere, carbon atoms blue), (F) the interaction of lead 1 at the end of the MD.

Fig. 10. Representative structures of lead 2 within PLpro putative pocket. (A) Structure of lead 2 (spheres, carbon atoms blue) at starting of MD simulation. (B) interaction of lead 2 with the key residues (C) lead 2 after 30 ns (sphere, carbon atoms violet), (D) the interaction of lead 2 at 30 ns and it is observed that N-(2-hydroxy benzylidene-amino)- oxadiazole showed hydrophobic interactions with A249 and P299 and NH moiety formed H-bond with G266. (E) Lead 2 at the end of the simulation (sphere, carbon atoms yellow), (F) the interaction of lead 2 at the end of the MD.

Fig. 11. Per-residue free energy decomposition: (A) lead 1, (B) lead 2.

Fig. 12. Rational drug design of the SARS-CoV-2 PLpro inhibitors.2D structure of lead 2 with possible modified fragments.