Discovery and optimization of phenazopyridine hydrochloride as novel SARS-CoV-2 RdRp inhibitors

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen of coronavirus disease (COVID-19) causing a pandemic with growing global transmission. The viral RNA-dependent RNA polymerase (RdRp) is conserved especially for variants of concern (VOCs), making it as an effective antivirals target. Due to the proofreading activity of coronavirus nsp14/nsp10, limited the efficacy of nucleoside analogs in vivo. Herein, we identified that Phenazopyridine hydrochloride (PAP) inhibits SARS-CoV-2 with EC₅₀ of 5.37 μmol/L. Furthermore, PAP can effectively inhibit SARS-CoV-2 RdRp with EC₅₀ value of 7.37 μmol/L, after further optimization, compound PAP-22 exhibits the most potential inhibition, with EC₅₀ of 1.11 μmol/L. PAP and its derivatives can bind directly to SARS-CoV-2 RdRp, fully resistance to the exoribonuclease (ExoN) and exhibit broad spectrum anti-CoV activities. Combined with the current data available on the safe and pharmacokinetics of PAP as an approved drug in clinical use, these results provide a path for the urgently needed antivirals to combat SARS-CoV-2.