Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity

Abstract

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (M pro ) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide M pro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits M pro with high potency (IC 50 = 0.40 μM) and displays excellent antiviral activity (EC 50 = 1.1 μM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC 50 > 100 μM) and excellent target selectivity for SARS-CoV-2 M pro (IC 50 > 50 μM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Discovery and Crystallographic Studies of Nonpeptidic Piperazine Derivatives as Covalent SARS-CoV-2 Main Protease Inhibitors

“…Of note, some warheads that had been highly active when they were attached to Ugi reaction-generated scaffolds, , turned out to be ineffective when fused to the piperazine ring of the present core structure. Two plausible explanations are conceivable: (i) co-crystal structures show that the N 4-substitution on the piperazine ring projects toward the protein surface near the oxyanion loop, and thus may give rise to steric collision and thereby block covalent warheads from reaching Cys145; (ii) covalent inhibitors reported here lack groups occupying the S1 cavity and interacting with the critical residue His163, which leads to weaker noncovalent interactions and reduces overall affinity. , …”

“…Main protease inhibition activities were measured for all target compounds in this study using a well-established fluorescence resonance energy transfer (FRET) assay . At a preliminary screening concentration of 10 μM, several compounds displayed significantly improved enzyme inhibition compared to the lead compound MCULE-5948770040 .…”

“… a Antiviral effect; nirmatrelvir and remdesivir were used as positive controls ( n = 3 biological replicates). b Error bars represent SD values of three independent experiments. c Cytotoxicity values were measured in Vero E6 cells with the MTS-based method …”

“…In the quest for novel piperazine-based noncovalent M pro inhibitors, GA-17S was discovered through rational drug design (Figure A). To increase permeability and in vivo antiviral activity, GC-14 with a replaced S1-binding group was designed, as recently reported …”

“…Development of piperazine-based M pro inhibitors. (A) Structures and activities of GA-17S and GC-14 , reported in our previous work . (B) Distance between the piperazine-N4 and the Cys145 thiol group (PDB ID: 8ACD), and general formula of the new GD series of compounds presented in this study.…”