The SARS-CoV-2 main protease (M pro ) is a crucial enzyme for viral replication and has been considered an attractive drug target for the treatment of COVID-19. In this study, virtual screening techniques and in vitro assays were combined to identify novel M pro inhibitors starting from around 8000 FDA-approved drugs. The docking analysis highlighted 17 promising best hits, biologically characterized in terms of their M pro inhibitory activity. Among them, 7 cephalosporins and the oral anticoagulant betrixaban were able to block the enzyme activity in the micromolar range with no cytotoxic effect at the highest concentration tested. After the evaluation of the degree of conservation of M pro residues involved in the binding with the studied ligands, the ligands' activity on SARS-CoV-2 replication was assessed. The ability of betrixaban to affect SARS-CoV-2 replication associated to its antithrombotic effect could pave the way for its possible use in the treatment of hospitalized COVID-19 patients.

BackgroundThe SARS-CoV-2 pandemic stimulated an outstanding global sequencing effort, which allowed to monitor viral circulation and evolution. Nuoro province (Sardinia, Italy), characterized by a relatively isolated geographical location and a low population density, was severely hit and displayed a high incidence of infection.MethodsAmplicon approach Next Generation Sequencing and subsequent variant calling in 92 respiratory samples from SARS-CoV-2 infected patients involved in infection clusters from March 2020 to May 2021.ResultsPhylogenetic analysis displayed a coherent distribution of sequences in terms of lineage and temporal evolution of pandemic. Circulating lineage/clade characterization highlighted a growing diversity over time, with an increasingly growing number of mutations and variability of spike and nucleocapsid proteins, while viral RdRp appeared to be more conserved. A total of 384 different mutations were detected, of which 196 were missense and 147 synonymous ones. Mapping mutations along the viral genome showed an irregular distribution in key genes. S gene was the most mutated gene with missense and synonymous variants frequencies of 58.8 and 23.5%, respectively. Mutation rates were similar for the S and N genes with one mutation every ∼788 nucleotides and every ∼712 nucleotides, respectively. Nsp12 gene appeared to be more conserved, with one mutation every ∼1,270 nucleotides. The frequency of variant Y144F in the spike protein deviated from global values with higher prevalence of this mutation in the island.ConclusionThe analysis of the 92 viral genome highlighted evolution over time and identified which mutations are more widespread than others. The high number of sequences also permits the identification of subclusters that are characterized by subtle differences, not only in terms of lineage, which may be used to reconstruct transmission clusters. The disclosure of viral genetic diversity and timely identification of new variants is a useful tool to guide public health intervention measures.

The current SARS-CoV-2 pandemic and the likelihood that new coronavirus strains will emerge in the immediate future point out the urgent need to identify new pancoronavirus inhibitors. Strigolactones (SLs) are a class of plant hormones with multifaceted activities whose roles in plant-related fields have been extensively explored. Recently, we proved that SLs also exert antiviral activity toward herpesviruses, such as human cytomegalovirus (HCMV). Here we show that the synthetic SLs TH-EGO and EDOT-EGO impair β-coronavirus replication including SARS-CoV-2 and the common cold human coronavirus HCoV-OC43. Interestingly, in silico simulations suggest the binding of SLs in the SARS-CoV-2 main protease (M pro ) active site, and this was further confirmed by an in vitro activity assay. Overall, our results highlight the potential efficacy of SLs as broad-spectrum antivirals against β-coronaviruses, which may provide the rationale for repurposing this class of hormones for the treatment of COVID-19 patients.