Defining the Substrate Envelope of SARS-CoV-2 Main Protease to Predict and Avoid Drug Resistance

Abstract

Coronaviruses, as exemplified by SARS-CoV-2, can evolve and spread rapidly to cause severe disease morbidity and mortality. Direct acting antivirals (DAAs) are highly effective in decreasing disease burden especially when they target essential viral enzymes, such as proteases and polymerases, as demonstrated in HIV-1 and HCV and most recently SARS-CoV-2. Optimization of these DAAs through iterative structure-based drug design has been shown to be critical. Particularly, the evolutionarily conserved molecular mechanisms underlying viral replication can be leveraged to develop robust antivirals against rapidly evolving viral targets. The main protease (Mpro) of SARS-CoV-2, which is evolutionarily constrained to recognize and cleave 11 specific sites to promote viral maturation, exemplifies one such target. In this study we define the substrate envelope of Mpro by determining the molecular basis of substrate recognition, through nine high-resolution cocrystal structures of SARS-CoV-2 Mpro with the viral cleavage sites. These structures enable identification of evolutionarily vulnerable sites beyond the substrate envelope that may be susceptible to drug resistance and compromise binding of the newly developed Mpro inhibitors.

Potential Resistance of SARS-CoV-2 Main Protease (Mpro) against Protease Inhibitors: Lessons Learned from HIV-1 Protease

“…The N142 is also prone to mutations (in total, 29 of the known variants: N142S, N142L, and N142D) (Figures 4b and 5b). Both M49 and N142 residues were found to be located at highly mobile sites and can adopt various conformations; hence, both residues were considered to be potential sites of resistance [86,93]. Different variants of G15 (G15S, G15C), P108 (P108S, P108L), and P132 (P132S, P132L) residues are among the most frequent mutations, indicating higher variability of these residues (Figure 4a).…”

“…Based on analysis of the crystal structures of enzyme-substrate complexes, the substrate envelope of SARS-CoV-2 Mpro was also defined [ 86 ]. Nirmatrelvir—binding to the S4-S1 sites of the enzyme—was found to fit the substrate envelope, and the sites where the inhibitor protrudes the envelope and which may be most vulnerable to resistance were also mapped.…”

“…Nirmatrelvir—binding to the S4-S1 sites of the enzyme—was found to fit the substrate envelope, and the sites where the inhibitor protrudes the envelope and which may be most vulnerable to resistance were also mapped. The enzyme-inhibitor interactions were found to be even beyond the substrate envelope (formed with, e.g., M49, N142, E166, and Q189 residues) [ 86 ]. Some of these positions are more variable (e.g., M49) and thus may be correlated to resistance development, while E166 is conserved due to its importance in the dimerization of the Mpro [ 87 ].…”

“…The N142 is also prone to mutations (in total, 29 of the known variants: N142S, N142L, and N142D) ( Figure 4 b and Figure 5 b). Both M49 and N142 residues were found to be located at highly mobile sites and can adopt various conformations; hence, both residues were considered to be potential sites of resistance [ 86 , 93 ].…”

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors

“…However, there is still significant room for discovering additional Mpro inhibitor scaffolds that may be tractable for drug discovery and development efforts, leading to even more efficacious treatments. Additionally, there may be the emergence of resistance against Paxlovid once widely deployed in the clinic, necessitating additional inhibitors 5,6 .…”