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 EC₅₀ 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.