Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV‑2 Main Protease

Creating
small-molecule antivirals specific for severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle
coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease
inhibitors. We performed a structure–activity relationship
(SAR) study of noncovalent compounds that bind in the enzyme’s
substrate-binding subsites S1 and S2, revealing structural, electronic,
and electrostatic determinants of these sites. The study was guided
by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040
(compound 1), in which protonation states were directly
visualized. Virtual reality-assisted structure analysis and small-molecule
building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray
structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry
of an inhibitor’s P1 group is essential to preserve the hydrogen
bond with the protonated His163; (2) a positively charged linker is
preferred; and (3) subsite S2 prefers nonbulky modestly electronegative
groups.