Structural basis of SARS-CoV-2 polymerase inhibition by nonnucleoside inhibitor HeE1-2Tyr

Targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 with small molecules is a promising therapeutic strategy against COVID-19, but potent and safe inhibitors are lacking. HeE1-2Tyr, a nonnucleoside inhibitor of Dengue virus RdRp, was also shown to inhibit SARS-CoV-2 RdRp in vitro and to have antiviral activity in cells, but the underlying mechanism remains unclear. Here, we elucidate the molecular mechanism of HeE1-2Tyr-mediated SARS-CoV-2 RdRp inhibition. Biochemical assays confirm that HeE1-2Tyr inhibits RdRp with an IC₅₀ of 5 µM and show that it competes with RNA binding to RdRp in vitro. Structural analysis using cryo-EM reveals that a stack of three HeE1-2Tyr molecules binds to the RNA binding site of RdRp. The identification of the conserved HeE1-2Tyr binding site and its intriguing inhibition mechanism of three stacked molecules that outcompete RNA may facilitate further development of pan-corona nonnucleoside inhibitors.