biostudies-literatureJing LTaishan Scholar Program of Shandong ProvinceHercules Foundation (Research Foundation Flanders) and Rega Foundation, KU LeuvenNational Natural Science Foundation of ChinaNational Key Research and Development ProgramShandong Laboratory Program670https://www.ebi.ac.uk/biostudies/studies/S-EPMC11765572biostudies-literatureUnknown26(2)With the aim of developing novel anti-SARS-CoV-2 drugs to address the ongoing evolution and emergence of drug-resistant strains, the reported SARS-CoV-2 Mpro inhibitor WU-04 was selected as a lead to find novel, highly potent, and broad-spectrum inhibitors. Using a fragment-based multilevel virtual screening strategy, 15 hit compounds were identified and subsequently synthesized. Among them, A5 (IC50 = 1.05 μM), A6 (IC50 = 1.08 μM), and A9 (IC50 = 0.154 μM) demonstrated potent SARS-CoV-2 Mpro inhibition comparable to or slightly weaker than WU-04. Antiviral activity evaluations revealed that compound A9 exhibited the strongest antiviral activity with an EC50 value of 0.18 μM, quite cInternational journal of molecular sciencesOptimization of SARS-CoV-2 Mpro Inhibitors by a Structure-Based Multilevel Virtual Screening Method.PMC11765572the 'Caps-It' research infrastructure (project ZW13-02)82373727tsqn2024080552023YFE0206500SYS202205Zhan PLiu XMeng BNeyts JJing LKang DZheng LZhao FJochmans DGao SLaporte MDe Jonghe SfalseOptimization of SARS-CoV-2 Mpro Inhibitors by a Structure-Based Multilevel Virtual Screening Method.With the aim of developing novel anti-SARS-CoV-2 drugs to address the ongoing evolution and emergence of drug-resistant strains, the reported SARS-CoV-2 Mpro inhibitor WU-04 was selected as a lead to find novel, highly potent, and broad-spectrum inhibitors. Using a fragment-based multilevel virtual screening strategy, 15 hit compounds were identified and subsequently synthesized. Among them, A5 (IC50 = 1.05 μM), A6 (IC50 = 1.08 μM), and A9 (IC50 = 0.154 μM) demonstrated potent SARS-CoV-2 Mpro inhibition comparable to or slightly weaker than WU-04. Antiviral activity evaluations revealed that compound A9 exhibited the strongest antiviral activity with an EC50 value of 0.18 μM, quite c2025-01-01T00:00:00Z2025 Jan2026-05-27T08:02:44.765Z2025-04-04T22:06:48.412ZS-EPMC117655723985938210.3390/ijms26020670