Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance

Abstract

Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 Mpro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of Mpro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed Mpro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir

“…Although the results reassure that the efficacy of nirmatrelvir is not compromised by the current dominant SARS-CoV-2 variants, drug resistance to nirmatrelvir is anticipated given the experience from the clinic use of HIV and HCV protease inhibitors (11,12). Several studies have been conducted to evolve or predict the nirmatrelvir resistant M pro mutants (13)(14)(15)(16).…”

Predicting antiviral resistance mutations in SARS-CoV-2 main protease with computational and experimental screening

“…To select these residues, we first analyzed the substrate and inhibitor bound SARS-CoV-2 M pro structures (PDB: 7TE0 and 7N89, respectively). [38][39][40] Set 1 includes the sub-pockets S4-S1 and S1'-S3' at the active site, which contribute to both substrate and inhibitor non-covalent interactions with the residues P4-P1 and P1'-P3' respectively, in addition to other residues within van der Waals, salt bridge or hydrogen bonding distance to substrate or inhibitor. The specific interactions between enzyme and its various peptide recognition sites have recently been mapped, and are consistent with this initial set (T25, T26, L27, H41, S46, M49, Y54, F140, L141, N142, G143, S144, H163, H164, M165, E166, L167, P168, F185, D187, Q189, T190, A191, and Q192) (Figure 1, Supporting Information Figure 1).…”

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors

“…Other inhibitors show much promise 4,[8][9][10][11][12][13][14][15][16] , including a non-covalent MPro inhibitor from the international Covid-19 Moonshot consortium that may be characterized as an advanced pre-clinical candidate [17][18][19] , and more experimental molecules that are relatively potent but have not proceeded far from hit to lead 20 . Notwithstanding these successes, both the resistance that may be expected to emerge 21,22 , and the inevitable liabilities of the early drugs support the discovery of new scaffolds. Accordingly, we targeted the structure of MPro for large library docking, seeking new starting points for lead discovery.…”

“…Notwithstanding these successes, both the resistance that may be expected to emerge 21, 22 , and the inevitable liabilities of the early drugs support the discovery of new scaffolds. Accordingly, we targeted the structure of MPro for large library docking, seeking new starting points for lead discovery.…”