The efficiency of high-throughput screening (HTS) and in-silico data analysis during medical emergencies: Identification of effective antiviral 3CLpro inhibitors
Debora Zian, Daniela Iaconis, S. Nenci, Alessandra Crusco, Sanjeevani Tawde, Mariangela Sodano, Rocco Vitalone, Ameya Raje, Martina Palamini, Daniele Carettoni, Angela Molteni, Candida Manelfi, Valerio Tazzari, Andrea R. Beccari, Paolo Malune, Stefania Maloccu, Annalaura Paulis, Angela Corona, Salvatore Nieddu, Silvano Coletti · Antiviral Research · 2025
The COVID-19 pandemic highlighted the importance of accelerating the drug discovery process. The 3-chymotrypsin-like protease (3CLpro) is a critical enzyme in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral replication process and was quickly identified as a prime target for drug development. This study leverages High-Throughput Screening (HTS) to identify novel 3CLpro inhibitors. We screened a different library of 325,000 compounds, leading to the discovery of two new chemical scaffolds with selective inhibitory activity against 3CLpro. In-silico analysis and further experimental validation, elucidated the binding modes and mechanisms of action, revealing a covalent inhibitor targeting the catalytic pocket and two allosteric inhibitors affecting the monomer/dimer equilibrium of 3CLpro. The identified compounds demonstrated significant antiviral activity in vitro, reducing SARS-CoV-2 replication in VeroE6 and Calu-3 cell lines. This study highlights the potential of combining HTS and computational approaches to accelerate the discovery of effective antiviral agents, suggesting a workflow to support the research and the design of effective therapeutic strategies. • HTS approach allowed us to identify effective antiviral compounds, acting as inhibitor of 3CLpro. • We identified two stable novel chemical classes inhibiting 3CLpro activity. • The identified inhibitors are selective for coronavirus proteases. • In-silico analysis showed compounds’ binding modes and mechanisms of action, affecting 3CLpro activity and dimerization. • Allosteric inhibitors were identified by in-silico driven data analysis.