Design of SARS-CoV-2 protease inhibitors with improved affinity and reduced sensitivity to mutations

Inhibitors of the SARS-CoV-2 main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function on these mutants are thus urgently needed. We hypothesized that the covalent HCV protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more tightly than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to NTV. A crucial feature of ML2006a4 is a novel derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Finally, ML2006a4 is less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory drug design can preemptively address potential resistance mechanisms.