Contributions of Hyperactive Mutations in Mpro from SARS-CoV-2 to Drug Resistance.

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.

Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease.

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.

Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses.

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 (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, 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 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.

Allosteric quinoxaline-based inhibitors of the flavivirus NS2B/NS3 protease.

Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.

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

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.

Post-Glycosylation Diversification (PGD): An Approach for Assembling Collections of Glycosylated Small Molecules.

Many small molecule natural products with antibiotic and antiproliferative activity are adorned with a carbohydrate residue as part of their molecular structure. The carbohydrate moiety can act to mediate key interactions with the target, attenuate physicochemical properties, or both. Facile incorporation of a carbohydrate group on de novo small molecules would enable these valuable properties to be leveraged in the evaluation of focused compound libraries. While there is no universal way to incorporate a sugar on small molecule libraries, techniques such as glycorandomization and neoglycorandomization have made signification headway toward this goal. Here, we report a new approach for the synthesis of glycosylated small molecule libraries. It puts the glycosylation early in the synthesis of library compounds. Functionalized aglycones subsequently participate in chemoselective diversification reactions distal to the carbohydrate. As a proof-of-concept, we prepared several desosaminyl glycosides from only a few starting glycosides, using click cycloadditions, acylations, and Suzuki couplings as diversification reactions. New compounds were then characterized for their inhibition of bacterial protein translation, bacterial growth, and in a T-cell activation assay.