Investigating the Binding Mode of a Naphthol‐Based Inhibitor Targeting SARS‐CoV‐2 Main Protease

Regardless of the massive global efforts to combat the virus causing SARS‐CoV‐2 syndrome, the infection remains a substantial health challenge worldwide in the years following 2019. To this direction, targeting the main viral protease M pro has been proposed as a tractable and particularly promising approach toward developing effective and safe COVID‐19 antivirals. By applying an integrated workflow combining a previously developed in silico consensus ranking protocol with two orthogonal in vitro methods, the NCI/DTP repository is screened and the discovery of an original naphthol scaffold with M pro inhibitory properties is reported. The hit is characterized in terms of structure and binding thermodynamics by combining X‐ray crystallography and isothermal titration calorimetry where a binding affinity constant of 1.55 μM is determined. The compound is further evaluated against virus‐infected cells, where an EC 50 value of 7.23 μM and comparable toxicity with nirmatrelvir are measured. Chemical synthesis is additionally employed to facilitate optimal exploration of the structure–activity relationship landscape regarding the new hit. By integrating computational, biophysical, and enzymatic methods, the suggested approach allows the combination of a structural hypothesis with functional evidence and shows its capacity toward identifying and rationally optimizing structurally original noncovalent M pro inhibitors.