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 (M
pro
) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 M
pro
bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of M
pro
, 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 M
pro
inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.
With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have ...been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (M
) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 M
, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 M
. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.
Drugs that target the main protease (Mpro) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of ...resistance mutations. To understand resistance potential in Mpro, we performed comprehensive surveys of amino acid changes that can cause resistance to nirmatrelvir (Pfizer), and ensitrelvir (Xocova) in a yeast screen. We identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir, many of which have not been previously reported. Ninety-nine mutations caused apparent resistance to both inhibitors, suggesting likelihood for the evolution of cross-resistance. The mutation with the strongest drug resistance score against nirmatrelvir in our study (E166V) was the most impactful resistance mutation recently reported in multiple viral passaging studies. Many mutations that exhibited inhibitor-specific resistance were consistent with the distinct interactions of each inhibitor in the substrate binding site. In addition, mutants with strong drug resistance scores tended to have reduced function. Our results indicate that strong pressure from nirmatrelvir or ensitrelvir will select for multiple distinct-resistant lineages that will include both primary resistance mutations that weaken interactions with drug while decreasing enzyme function and compensatory mutations that increase enzyme activity. The comprehensive identification of resistance mutations enables the design of inhibitors with reduced potential of developing resistance and aids in the surveillance of drug resistance in circulating viral populations.
The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target ...enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here, we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but have not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a fluorescence resonance energy transfer (FRET)-based yeast readout. We identified hundreds of mutations that significantly increased the Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed 3 times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory-grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.
The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target ...enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (M
) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in M
that reduce binding to inhibitors; here, we investigate mutations that affect enzyme function. Hyperactive mutations that increase M
activity can contribute to drug resistance but have not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in M
affect enzyme function using a mutational scanning approach with a fluorescence resonance energy transfer (FRET)-based yeast readout. We identified hundreds of mutations that significantly increased the M
activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed 3 times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory-grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in M
and provide a comprehensive list for future surveillance efforts.
We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 ...(SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.
