Tiliroside From Pavonia malacophylla (Link & Otto) Garcke as an Anti-SARS-CoV-2 Cell Entry Agent

Flavonoids are recognized for interfering with stages of SARS-CoV-2 life cycle. Then, tiliroside (kaempferol 3-O-glucoside-6''-E-coumaroyl), an O-glycosylated flavonoid isolated from Pavonia malacophylla (Link & Otto) Garcke, was investigated for its ability to block SARS-CoV-2 viral entry. Tiliroside was chemically characterized and tested for inhibition of the SARS-CoV-2 Spike receptor-binding domain (RBD)-human ACE2 interaction using a bioluminescent immunoassay. At 10 µM, it inhibited 63% of RBD:ACE2 binding. Flow cytometry and pseudovirus assays assessed Spike internalization in endothelial cells and Spike-pseudotyped infection, respectively. Flow cytometry confirmed reduced internalization after tiliroside treatment, while neutralization assays showed a dose-dependent inhibition of pseudovirus infection, with an EC₅₀ of 18.5 µM. Molecular docking revealed strong binding of tiliroside to the Spike RBD, involving Lys417, Gly496, Tyr495, Tyr453, and Tyr505, with a predicted higher affinity than that of remdesivir and nafamostat. These results provide the first in vitro evidence that tiliroside may be a SARS-CoV-2 entry inhibitor, highlighting O-glycosylated flavonoids as antiviral scaffolds.