Dual-Site Inhibition of SARS-CoV-2 RNA-Dependent RNA Polymerase by Small Molecules Able to Block Viral Replication Identified through a Computer-Aided Drug Discovery Approach

Malune et al., ACS Infectious Diseases, doi:0.1021/acsinfecdis.5c00517, Sep 2025

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In vitro and in silico study showing benefit with four small-molecule RdRp inhibitors.

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Malune et al., 26 Sep 2025, peer-reviewed, 12 authors.

In vitro studies are an important part of preclinical research, however results may be very different in vivo.

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Dual-Site Inhibition of SARS-CoV-2 RNA-Dependent RNA Polymerase by Small Molecules Able to Block Viral Replication Identified through a Computer-Aided Drug Discovery Approach

Paolo Malune, Daniela Iaconis, Candida Manelfi, Stefano Giunta, Roberta Emmolo, Filippo Lunghini, Annalaura Paulis, Carmine Talarico, Angela Corona, Andrea Rosario Beccari, Enzo Tramontano, Francesca Esposito

ACS Infectious Diseases, doi:10.1021/acsinfecdis.5c00517

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 RNAdependent 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 IC 50 and EC 50 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.

Authors Paolo Malune -Department of Life

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