It has been more than four years since the first report of SARS-CoV-2, and humankind has experienced a pandemic with an unprecedented impact. Moreover, the new variants have made the situation even worse. Among viral enzymes, the SARS-CoV-2 main protease (M^pro) has been deemed a promising drug target vs. COVID-19. Indeed, M^pro is a pivotal enzyme for viral replication, and it is highly conserved within coronaviruses. It showed a high extent of conservation of the protease residues essential to the enzymatic activity, emphasizing its potential as a drug target to develop wide-spectrum antiviral agents effective not only vs. SARS-CoV-2 variants but also against other coronaviruses. Even though the FDA-approved drug nirmatrelvir, a M^pro inhibitor, has boosted the antiviral therapy for the treatment of COVID-19, the drug shows several drawbacks that hinder its clinical application. Herein, we report the synthesis of new thiazolidine-4-one derivatives endowed with inhibitory potencies in the micromolar range against SARS-CoV-2 M^pro. In silico studies shed light on the key structural requirements responsible for binding to highly conserved enzymatic residues, showing that the thiazolidinone core acts as a mimetic of the Gln amino acid of the natural substrate and the central role of the nitro-substituted aromatic portion in establishing π-π stacking interactions with the catalytic His-41 residue.

Since its emergence in late 2019, SARS-CoV-2, the causative agent of COVID-19, has continued to spread globally, with more than 7 million reported deaths as of March 2025. Among the viral nonstructural proteins, nsp12 serves as the RNA-dependent RNA polymerase (RdRp), mediating viral genome replication and transcription in concert with its cofactors nsp7 and nsp8. To date, only two nucleoside analogs specifically targeting SARS-CoV-2 nsp12, remdesivir and molnupiravir, have been authorized by the FDA for COVID-19 treatment. In response to the need for additional safe and effective antiviral agents, we screened two extensive in silico libraries of safe-in-man compounds (>9,000) and natural compounds (>249,000), against the SARS-CoV-2 nsp12/7/8 complex, targeting the orthosteric and two allosteric nsp12 sites, using the EXSCALATE (EXaSCale smArt pLatform Against paThogEns) platform. Compounds were then selected based on docking score significance, novelty for the target, and clinical safety profiles. The top 119 candidates were subsequently evaluated in a biochemical assay to assess their potential to inhibit SARS-CoV-2 nsp12/7/8 polymerase activity, identifying 42 compounds able to block it, among which four showed IC₅₀ and EC₅₀ values in the nanomolar or low micromolar range. When tested in cell-based assays to evaluate their efficacy on SARS-CoV-2 replication, they proved to inhibit it in the same concentration ranges. Mechanism of action studies revealed different modalities of inhibition. These results provide the basis for the development of novel antiviral compounds against SARS-CoV-2, targeting both the RdRp active site and an allosteric site, further suggesting that the Computer-Aided Drug Discovery (CADD) approach, together with experimental validation, can provide the basis for accelerated antiviral drug development.

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.

The endoribonuclease Nsp15 is essential for coronavirus pathogenesis and evasion of host defenses and is therefore a promising drug target. We determined optimal parameters for an endoribonuclease FRET biochemical assay and use it for a high-throughput drug screen. Measurement of Z'-factor confirmed robust assay performance (Z' = 0.7- 0.9). We screened a commercially available library (LOPAC 1280) and identified three molecules able to inhibit the catalytic endonuclease activity of Nsp15 in the low micromolar range. Among them, a promising hit compound, Reactive Blue 2 showed also anti-SARS-CoV-2 activity with EC₅₀ value of 2 μM, and low cytopathic effect in multiple cell types (CC₅₀ > 28 μM). More importantly, SARS-CoV-2 replication was inhibited in cells with an intact IFN system, but not in IFN-deficient cell lines.

Starting from the crystal structure of a Zika virus (ZIKV) NS2B-NS3 protease with bound allosteric inhibitor, we generated dynamic pharmacophore models derived from representative molecular dynamics (MD) conformations to capture key interaction patterns. These models guided a pharmacophore-based virtual screening of commercial libraries, including commercial compounds and approved drugs, enabling the rapid selection of virtual hit compounds. Biochemical evaluation identified several inhibitors, with the antiretroviral drug Nelfinavir emerging as the most promising compound. Nelfinavir displayed the strongest inhibition of ZIKV and West Nile virus (WNV) proteases and showed antiviral activity in cell-based assays, with EC50 values of 1.79 ± 0.01 µM on ZIKV and 3.01 ± 1.25 µM in WNV, supporting its relevance as a repositionable scaffold for modulation of ZIKV and WNV infections. The activity observed across flavivirus proteases highlights the value of targeting conserved allosteric regions. Overall, this integrated computer aided drug design (CADD) strategy demonstrates the power of dynamic, structure-based pharmacophores to uncover novel allosteric inhibitors and accelerate antiviral drug repurposing.