Discovery and Optimization of Inhibitors of SARS-CoV-2 Polymerase and Exonuclease

Summary: Discovery and Optimization of Inhibitors of SARS-CoV-2 Polymerase and Exonuclease SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) (Nsp12/7/8) and exonuclease (ExoN) (Nsp14/10) play critical roles in viral genome replication and transcription. These enzymes are highly conserved among the coronaviruses and have no counterparts in human host cells, serving as unique inhibitor targets. We discovered that SARS-CoV-2 ExoN, acting as a proofreader, removes nucleotide inhibitors, such as Remdesivir, that are incorporated by RdRp into viral RNA during its synthesis. This mechanism might be responsible for the relatively low efficacy of these drugs for treating COVID-19. We reasoned that combinations of inhibitors of the viral RdRp and ExoN could overcome this deficiency. We showed that inhibitors of the RdRp and ExoN act synergistically to overcome deficiencies of using RdRp inhibitors alone to block SARS-CoV-2 replication in vitro. We have developed high resolution molecular assays to assess SARS-CoV-2 RdRp and ExoN activities to screen inhibitors of these enzymes. We identified and validated a set of bona fide inhibitors for both enzymes. We demonstrated that the oral drug Pibrentasvir, an HCV NS5A inhibitor, also inhibits SARS-CoV-2 ExoN, and in the presence of Pibrentasvir, RNAs terminated with the RdRp inhibitors Remdesivir, Favipiravir, Molnupiravir, and AT-527 were largely protected from ExoN excision. These results indicate that all the nucleoside/nucleotide- based oral drug candidates currently in COVID-19 clinical trials will potentially benefit from this RdRp/ExoN inhibitor combination approach. In this project, we will focus on the discovery and optimization of new inhibitors of these two enzymes. Their ability to stop SARS-CoV-2 replication will be systematically tested. We will perform molecular, structural, and in vitro/in vivo studies to optimize this synergistic strategy to develop new molecules as effective COVID-19 drugs. In close collaboration with all the Cores of the MAVDA Center, first, we’ll screen and characterize SARS-CoV-2 RdRp and ExoN inhibitors using optimized molecular assays. Second, we’ll perform structural characterization of SARS-CoV-2 RdRp and ExoN complexed with the identified inhibitors. Third, we’ll design and synthesize new SARS-CoV-2 RdRp and ExoN inhibitors based on the molecular scaffolds of the above compounds that have demonstrated viral inhibitor activity. Finally, we’ll test SARS-CoV-2 RdRp and ExoN inhibitors for their potential synergistic effects using in vitro and in vivo assays. This four-pronged approach will be performed iteratively for the development of oral COVID-19 therapeutics. We expect the newly developed SARS-CoV-2 RdRp inhibitors will inhibit RNA polymerases in flaviviruses (ZIKV, DENV) and alphaviruses (CHIKV, EEEV), and we will assess their ability to inhibit these viruses as well.