Abstact

Epidemic and pandemic outbreaks of respiratory illness caused by three different coronaviruses over the past two decades have underscored the importance of pharmaceutical agents that could offer broad-spectrum activity across this family of pathogens. Two coronavirus inhibitors characterized by broad in vitro potency were synthesized and studied with X-ray crystallography. Their high-resolution structures in complex with six α-, β-, and γ-coronaviruses delineate the requirements for pan-coronavirus inhibition by drug-like molecules targeting the S1-S4 subsites of the viral 3CL-protease, which performs a critical function during coronavirus polyprotein processing. Anchoring by polar contacts in S1, utilization of hydrophobic packing in S2, compact substitutions in S3, and mid-sized hydrophobic modifications in S4 are all factors contributing to inhibitor activity. Interactions in S2 are modulated by the amino acid identity of three key residues, and in S4, where sequence conservation is the lowest, pan-coronavirus coverage is facilitated by solvent exposure of the diverging side chains.