Synthetic host defense peptide inhibits SARS-CoV-2 replication in vitro

Although myriads of potential antiviral agents have been tested against SARS-CoV-2, only a handful have proven to be effective in clinical trials. During the COVID-19 pandemic, many known or novel peptides were evaluated for their ability to inhibit SARS-CoV-2 replication; however, testing of D-enantiomers that resist body and viral proteases has been limited. Here, we characterized the ability of D-3006, a D-enantiomeric synthetic host defense peptide, to inhibit SARS-CoV-2 replication in vitro. A battery of authentic SARS-CoV-2 variants (ancestral, Mu, Delta, and Omicron BA.1) and a comprehensive panel of β-coronavirus spike pseudotyped lentiviruses were used to demonstrate that D-3006 safely (CC₅₀value = 430 µg/mL) blocked spike-mediated entry (EC₅₀ values ranging from 1.57 to 5.37 µg/mL) and also had synergistic anti-SARS-CoV-2 activity in vitro when combined with the viral polymerase inhibitor remdesivir. We also showed that D-3006 inhibited influenza A virus (H1N1) replication in vitro, suggesting that this synthetic host defense peptide could have potential broad antiviral activity against multiple enveloped viruses. These data, together with negative-stain transmission electron microscopy analysis, suggest that the mechanism of action of D-3006 is associated with non-specific binding to the viral membrane, most likely causing virus aggregation and interfering with virus attachment and entry. The potential broad-spectrum antiviral activity of D-3006, its innate resistance to host proteases, as well as the possibility of being used in combination with other antiviral drugs suggest that this host synthetic peptide could be developed as a candidate for the treatment of SARS-CoV-2 and/or other respiratory viral infections.