A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase

The current COVID-19 pandemic has highlighted the necessity of more efficient antiviral compounds. The antiviral efficacy of adenosine-based analogs, the main repurposed drugs for SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition, is mainly assessed through in vitro or cell-free polymerization assays, under arbitrary conditions that do not reflect the physiological environment. We show that SARS-CoV-2 RdRp inhibition efficiency of remdesivir and cordycepin, two common adenosine analogs, is influenced by endogenous adenosine level, and that the current clinically approved concentrations for COVID-19 treatment are suboptimal for effective RdRp inhibition. Furthermore, we identified GTP as the rate-limiting nucleotide of SARS-CoV-2 replication. Our results demonstrate that nucleotide sensitivity of the RdRp complex and competition of nucleoside analog drugs against endogenous concentrations of nucleotides are crucial elements to be considered when designing new SARS-CoV-2 antiviral compounds.