Identification of novel proteolytically inactive mutations in coronavirus 3C‐like protease using a combined approach

Abstract

Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C-like proteases (3CL pro s) from CoVs (also called main proteases) are essential for viral replication and are also involved in polyprotein cleavage and immune regulation, making them attractive and effective targets for the development of antiviral drugs. Herein, the 3CL pro from the porcine epidemic diarrhea virus, an enteropathogenic CoV, was used as a model to identify novel crucial residues for enzyme activity. First, we established a rapid, sensitive, and efficient luciferase-based biosensor to monitor the activity of PDEV 3CL pro in vivo. Using this luciferase biosensor, along with confirming the well-known catalytic residues (His41 and Cys144), we identified 4 novel proteolytically inactivated mutants of PDEV 3CL pro , which was also confirmed in mammalian cells by biochemical experiments. Our molecular dynamics (MD) simulations showed that the hydrogen bonding interactions occurring within and outside of the protease's active site and the dynamic fluctuations of the substrate, especially the van der Waals contacts, were drastically altered, a situation related to the loss of 3CL pro activity. These data suggest that changing the intermolecular dynamics in protein-substrate complexes eliminates the mechanism underlying the protease activity. The discovery of novel crucial residues for enzyme activity in the binding pocket could potentially provide more druggable sites for the design of protease inhibitors. In addition, our in-depth study of the dynamic substrate's envelope model using MD simulations is an approach that could augment the discovery of new inhibitors against 3CL pro in CoVs and other viral 3C proteases.

Drug repurposing for coronavirus (COVID-19): in silico screening of known drugs against coronavirus 3CL hydrolase and protease enzymes

“…The known coronavirus 3-chymotrypsin-like protease (3CLpro), also known as Mpro, is the main protease, which is required for proteolytic maturation of the corona virus. This Mpro have an essential role in the immune regulation and cleaving the polyproteins pp1a and pp1ab, which making them attractive and important targets for anti-COVID-19 drugs (Zhou et al, 2019). Functional proteins such as RNA polymerase, endoribonuclease and exoribonuclease are generated by cleavage of pp1a and pp1ab polyproteins by Mpro .…”

Screening of phytochemicals as potent inhibitor of 3-chymotrypsin and papain-like proteases of SARS-CoV2: an in silico approach to combat COVID-19

“…3CLpro and PLpro are encoded by ORF-1 of virus genome which plays a major role in the processing of inactive polyproteins (pp1a and pp1ab) into active non-structural proteins (nsps). These nsps finally helps in the replication, generation, as well as infection of the virus (Zhou et al., 2019 ). Inhibition of these proteases can be crucial for further replication and infection of SARS-CoV2 (Wu et al., 2020 ).…”

Pharmacological Therapeutics Targeting RNA-Dependent RNA Polymerase, Proteinase and Spike Protein: From Mechanistic Studies to Clinical Trials for COVID-19

“…Bananin (BN) effectively suppresses both SARS-CoV RNA/DNA helicase nucleic acid unwinding function and SARS-CoV RNA-viral replication in cell culture [72]. In addition, high-throughput screening (HTS) approaches were used to screen potent inhibitors of the SARS-CoV main proteinase [73,74]. Recent computational studies found that lopinavir, oseltamivir and ritonavir are able to bind with SARS-CoV-2 protease [75].…”