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

Abstract

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 (M pro ) 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 M pro 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 M pro 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.

Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease

“…Nonetheless, the Glu166 carboxylate rotates by about 90° from its observed position in the inhibitor-free M pro structure. It forms a new hydrogen bond with the neutral His172, as was observed in our other inhibitor-bound M pro structures 34 , 43 , 53 . P2, P3, and P4 groups of BBH-1 are hydrophobic and do not form hydrogen bonds with M pro .…”

“…The carbonyl oxygen makes a short 1.7 Å hydrogen bond with the protonated imidazolium side chain of His163. A similar hydrogen bond was observed between the P1 uracil of a noncovalent inhibitor Mcule-5948770040 and His163 34 . However, unlike the uracil group, the γ-lactam doesn’t make a C-H…O interaction with His172.…”

“…In both M pro /BBH-1 and inhibitor-free M pro XN structures, Nε2 of His164 donates its D in a hydrogen bond with the Thr175 hydroxyl, which forms a hydrogen bond with the main-chain carbonyl of Asp176. This hydrogen bond is conserved in all XN structures we have determined 34 , 43 , 53 . The neutral protonation state of His172 is also maintained because it is secured by the hydrogen bond with main-chain amide N of Gly138.…”

“…A P1 group must have a hydrogen bonding acceptor capability to form a strong hydrogen bond with His163 in the S1 substrate binding subsite. Recent neutron crystallographic structures of M pro revealed that His163 adapts from the Nδ1-D tautomer to the protonated and positively charged state in inhibitor complexes 34 , 53 . We therefore designed and synthesized the hybrid reversible covalent inhibitors BBH-1, BBH-2, and NBH-2 (Fig.…”

“…For example, several research groups have focused their attention on structure-based drug design of noncovalent M pro inhibitors starting from the anti-epileptic drug perampanel 28 , 29 , or from ML188 and ML300 previously designed to inhibit SARS-CoV-1 M pro 30 – 33 . We have recently performed a structure-activity relationship study 34 on a noncovalent compound newly discovered by high-throughput virtual screening 35 . Others have designed and evaluated a series of peptidomimetic reversible or irreversible covalent inhibitors 36 – 38 , or nonpeptidic small molecules with a novel chemical scaffold 39 .…”