MasitinibL shows promise as a drug-like analog of masitinib that elicits comparable SARS-Cov-2 3CLpro inhibition with low kinase preference

Abstract

SARS-CoV-2 infection has led to several million deaths worldwide and ravaged the economies of many countries. Hence, developing therapeutics against SARS-CoV-2 remains a core priority in the fight against COVID-19. Most of the drugs that have received emergency use authorization for treating SARS-CoV-2 infection exhibit a number of limitations, including side effects and questionable efficacy. This challenge is further compounded by reinfection after vaccination and the high likelihood of mutations, as well as the emergence of viral escape mutants that render SARS-CoV-2 spike glycoprotein-targeting vaccines ineffective. Employing de novo drug synthesis or repurposing to discover broad-spectrum antivirals that target highly conserved pathways within the viral machinery is a focus of current research. In a recent drug repurposing study, masitinib, a clinically safe drug against the human coronavirus OC43 (HCoV-OC43), was identified as an antiviral agent with effective inhibitory activity against the SARS-CoV-2 3CLpro. Masitinib is currently under clinical trial in combination with isoquercetin in hospitalized patients (NCT04622865). Nevertheless, masitinib has kinase-related side effects; hence, the development of masitinib analogs with lower anti-tyrosine kinase activity becomes necessary. In this study, in an attempt to address this limitation, we executed a comprehensive virtual workflow in silico to discover drug-like compounds matching selected pharmacophore features in the SARS-CoV-2 3CLpro-bound state of masitinib. We identified a novel lead compound, "masitinibL", a drug-like analog of masitinib that demonstrated strong inhibitory properties against the SARS-CoV-2 3CLpro. In addition, masitinibL further displayed low selectivity for tyrosine kinases, which strongly suggests that masitinibL is a highly promising therapeutic that is preferable to masitinib.

Figure 1

Pharmacophore modelling and virtual screening workflow. (A) The 3CLpro-bound pose of masitinib with selected pharmacophore features for chemical database screening. (B) The four selected pharmacophore points with individual distances. The single hydrogen bond donor is shown in a white color mesh, while the aromatic and hydrophobic features (mapped on each other) are shown in purple and green color meshes, respectively. (C) A workflow for the virtual screening protocol, starting from the pharmacophore generation stage to the final MM-GBSA ranking. (D) Zoomed-in superimposition of the docked masitinib 3D structure (represented in yellow stick format) against the 3CLpro co-crystallized masitinib structure (represented in cyan stick format) for the reliability test of the virtual screening protocol.

Figure 2

Lead compound identification through protein–ligand interaction fingerprinting. (A) AuPosSOM-calculated dendrogram for the top 10 hits of the MM-GBSA binding energy ranking and masitinib. The image was generated with the Dendroscope software. (B) The binding of masitinib, CHEMBL3642843, and CHEMBL2058939 to the SARS-CoV-2 3CLpro active site. (C) A zoomed-in view of the binding pose similarity between masitinib, CHEMBL3642843, and CHEMBL2058939 based on the protein–ligand interaction fingerprinting grouping. Masitinib is shown in cyan, while CHEMBL3642843 and CHEMBL2058939 are displayed in purple and yellow, respectively.

Figure 3

Protein–ligand interaction profile prediction of masitinib and the selected lead compounds. (A) The predicted interaction profile of masitinib in complex with the SARS-CoV-2 3CLpro. Masitinib is displayed in cyan-colored stick while interacting active site residues of the 3CLpro are displayed in green-colored sticks. (B, C) The predicted interaction profiles of CHEMBL2058939 (yellow stick) and CHEMBL3642843 (purple stick) respectively, after docking against the 3CLpro active site. All interacting active site residues are shown in green sticks.

Figure 4

Root mean square deviation plots from molecular dynamics simulation. (A) represent the RMSD plot for the 3CLpro C-alpha backbone. Trajectories for the Apo, and complexes with masitinib, CHEMBL3642843 and CHEMBL2058939 are shown in black, red, green, and blue colors respectively. (B) The ligand RMSD plot. Trajectories for masitinib, CHEMBL3642843 and CHEMBL2058939 are shown in black, red, and green colors, respectively.

Figure 5

Root mean square fluctuation and radius of gyration plots. (A) The RMSF plot for the 3CLpro structural backbone. The 3CLpro apo trajectory and trajectories of its complexes with masitinib, CHEMBL3642843 and CHEMBL2058939 are displayed in black, red, green, and blue colors respectively. (B) The radius of gyration plot from the 300 ns molecular dynamics simulation run. Trajectories of the apo protein and its complexes with masitinib, CHEMBL3642843 and CHEMBL2058939 are displayed in black, red, green, and blue colors respectively.

Figure 6

Hydrogen bond plots from molecular dynamics simulation. (A) The intra-protein H-BOND analysis plot of the SARS-CoV-2 3CLpro. The apo protein H-BOND plot and the plot of its complexes with masitinib, CHEMBL3642843 and CHEMBL2058939 are shown in black, red, green, and blue, respectively. (B) The protein–ligand H-BOND plot over 300 ns of simulation time. Trajectories for masitinib, CHEMBL3642843 and CHEMBL2058939 are shown in black, red, and green, respectively.

Figure 7

Solvent accessible surface area and principal component analysis plots. (A) represents the plot of solvent accessibility for the apo protein and its complexes with masitinib, CHEMBL3642843 and CHEMBL2058939. Their individual trajectories are shown in black, red, green, and blue, respectively. (B) A 2-dimensional projection of the principal components of each system. The collective motion trajectory of the apo protein and its complex with masitinib, CHEMBL3642843 and CHEMBL2058939 are displayed in black, red, green, and blue, respectively.

Figure 8

Free energy landscape (FEL) plots of the first two principal components. (A) Apo protein FEL plot, (B) FEL plot of the 3CLpro-masitinib complex, (C) FEL plot of the 3CLpro-CHEMBL3642843 complex, (D) FEL plot of the 3CLpro-CHEMBL2058939 complex.

Figure 9

Pie chart showing the top 15 predicted target classes for each compound. (A) The predicted target class for masitinib. (B) The predicted classes of target for CHEMBL3642843. (C) The predicted classes of target for CHEMBL2058939.

Figure 10

2D representation of the dynamic protein–ligand interaction profile of CHEMBL in complex with the SARS-CoV-2 3CLpro. All observed forms of interaction include hydrophobic interaction, hydrogen bond, pi stacking interaction, pi cation interaction, and halogen bond.