A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M^pro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC₅₀ values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC₅₀ values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic M^pro inhibitors. A complex crystal structure of SARS-CoV-2 M^pro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M^pro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

X-ray crystal structures of SARS-CoV-2 M ^pro with dual inhibitors provide a new direction for the designing of COVID-19 antivirals.

The SARS-CoV-2 main protease (M^pro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in M^pro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring M^pro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k_cat/K_m < 10-fold change) while being resistant to nirmatrelvir (K_i > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that M^pro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in M^pro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.

Despite major advances in the beta-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the Class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the Class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including Class B and Class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a beta-lactam antibiotic for the treatment of a wide range of multidrug resistant gram-negative bacterial infections, by both intravenous and oral administration.

The papain-like protease (PL^pro) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PL^pro inhibitors including Jun9-72-2 and Jun9-75-4 with improved enzymatic inhibition and antiviral activity compared to GRL0617, which was reported as a SARS-CoV PL^pro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL^pro inhibitors in the BSL-2 setting. X-ray crystal structure of PL^pro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that Jun9-72-2 and Jun9-75-4 engaged in more extensive interactions than GRL0617. Overall, the PL^pro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PL^pro assay is a suitable surrogate for screening PL^pro inhibitors in the BSL-2 setting.