Uncovering Flexible Active Site Conformations of SARS-CoV-2 3CL Proteases through Protease Pharmacophore Clusters and COVID-19 Drug Repurposing

Abstract

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease–ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 “PPC consensus anchors” (a distinct set observed in each PPC) were observed, of which three “PPC core anchors” EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived “PPC scoring S” to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 μM and an antiviral activity with EC50 of 49.89 μM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 μM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 μM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.

A head-to-head comparison of the inhibitory activities of 15 peptidomimetic SARS-CoV-2 3CLpro inhibitors

“…1 ). Examples of electrophilic warheads include aldehydes, 19 , 20 , 21 ethyl propenoate, 19 hydroxymethylketone, 22 hydroxymethyl sulfonic acid, 16 , 23 ketoamides, 16 , 24 , 25 and ketobenzothiazoles. 26 , 27 This peptide-warhead strategy has led to the approval of Boceprevir and Telaprevir in 2011 ( Fig.…”

“…Intriguingly, HCV NS3 protease inhibitors Boceprevir and Telaprevir were also reported to inhibit SARS-CoV-2 3CLpro, suggesting that they can be repurposed for treating COVID-19. 16 , 25 , 29 …”

Dynamic Profiling of β-Coronavirus 3CL Mpro Protease Ligand-Binding Sites

“…It lacks a typical pocket shape; however, it can be a key site where hydrophilic side chains can interact with the solvent. 25 Moreover, in the homodimer structure of the M pro enzyme, the N-finger (residues 1–7) of one protomer is squeezed between domains I and II to shape the substrate-specificity pocket. This shows the importance of dimerization and N-finger orientation for substrate specificity and catalysis.…”

DRDOCK: A Drug Repurposing platform integrating automated docking, simulations and a log-odds-based drug ranking scheme

“… Plaque reduction percentage of two repurposed SARS-CoV2 nsp16 active site inhibitors tegaserod and fenoterol in comparison with an earlier reported SARS-CoV2 3CL protease inhibitor, Nelfinavir that serves as a positive control (Pathak et al, 2021) in this experiment. The inhibition percentage is calculated as [1-(V D /V C )]×100%, where V D and V C refer to the virus titer in the presence and absence of the inhibitors, respectively.…”