The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC₅₀) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC₅₀) of 91.65 µg/mL, a CC₅₀ of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors.

Macrophomina phaseolina, a necrotrophic fungus with global distribution, has emerged as a source of bioactive natural products. This study evaluated the antiviral potential of metabolites from M. phaseolina against herpes simplex virus 1 (HSV-1). A crude extract of M. phaseolina was obtained from the culture medium, and the secreted metabolites were purified using chromatographic techniques. Major metabolites were identified using NMR and HRMS techniques. The antiviral activity of these metabolites was assessed using Vero cells and virus quantitation through qPCR. In addition, a viral gene expression and intracellular genome quantification was performed using qPCR. Six metabolites were isolated: (−)-botryodiplodin, mellein, hydroxymellein, acetylphomalactone, asperlin, and isoasperlin. Their structures were elucidated through NMR and HRMS. Most metabolites showed limited antiviral efficacy; however, (−)-botryodiplodin demonstrated potent activity, with an EC₅₀ of 2.44 µg/mL and a selectivity index of 4.79. In addition, acyclovir was included as a control, demonstrating >97% antiviral inhibition. Mechanistic studies showed that (−)-botryodiplodin inhibited HSV-1 production, reduced viral genome levels, and suppressed immediate–early, early, and late viral gene expressions. These findings demonstrate that (−)-botryodiplodin acts as an early-stage inhibitor of HSV-1 prior to viral transcription, establishing this natural metabolite as a promising chemical scaffold for the rational design of novel antiviral agents.