The continuous spread of SARS-CoV-2 remains a serious threat to human life and health. Compared to vaccines and antibody drugs, small molecule antivirals have the advantages of broad-spectrum and sustained anti-coronavirus potential. The main protease (M^pro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Based on the in-depth understanding of the cocrystal structure between the lead compound (MCULE-5948770040) and M^pro, comprehensively employing computer-aided drug design, a multi-site binding and privileged structure assembly strategy, we report on the discovery of a range of non-covalent and covalent M^pro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. The most promising non-covalent inhibitor GC-14 (IC₅₀ = 0.40 μM, EC₅₀ = 1.0 μM) and covalent inhibitor GD-9 (IC₅₀ = 0.18 μM, EC₅₀ = 2.27 μM) showed remarkably increased activity compared with MCULE-5948770040 (IC₅₀ = 5.10 μM, EC₅₀ > 100 μM). Meanwhile, both of them presented favorable target selectivity for SARS-CoV-2 M^pro versus human cysteine proteases (IC₅₀> 50 μM and IC₅₀> 30 μM for cathepsins B/F/K/L and caspase 3, respectively). Notably, GC-14 exhibits extremely low cytotoxicity (CC₅₀ > 100 μM). Structural biology studies have confirmed GC-14 occupied each cavities of the M^pro active site with non-covalent interactions. A unique ‘sandwich’ conjugate effect, which was stacked by thiophene, dichlorophenyl and His41 imidazole ring, enhanced the target affinity. For GD-9, a covalent bond with Cys145 at the active site was formed. Taken together, two types of non-peptide M^pro inhibitors showed excellent anti-SARS-CoV-2 activity and high target selectivity, which provides a structural basis for further development of anti-COVID-19 drugs.

Shenghua Gao

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