Nathan Soper, PhD
New York University
Small molecule viral inhibitors disrupt RNA transcription-replication by a common mechanism
Small-molecule inhibitors targeting viral helicases or polymerases are typically characterized as isolated enzymes, yet their effects when associated within their respective replication complexes remain poorly defined. Here, we investigate how helicase- and polymerase-directed inhibitors disrupt the activity of the SARS-CoV-2 replication–transcription complex (RTC). Using a reconstituted mini-RTC (Nsp12/7/8 + Nsp13), we show that Nsp13 forms a stable complex with the holo-RdRp and induces ATPase-dependent stalling of primer extension in vitro. Catalytically inactive Nsp13 (E375A) fails to stall polymerization, suggesting that ATP hydrolysis is required for this regulatory effect. The previously characterized Nsp13 inhibitor IOWH-032 prevents Nsp13-mediated stalling, as it has been shown to disrupt helicase-RNA engagement as well as inhibiting ATPase activity. Unexpectedly, IOWH-032 also directly inhibits holo-RdRp polymerization activity, revealing it as a general RNA transcription-replication inhibitor. In a comparison of IOWH-023 and suramin (a well established competitive inhibtor of the polymerase), electrophoretic mobility shift assays and fluorescence polarization assays reveal that both IOWH-032 and suramin displace RNA from holo-RdRp, whereas strictly allosteric inhibitors (i.e. SSYA10-001) do not. These findings demonstrate that helicase-targeting compounds can exert unanticipated effects on polymerase function within the RTC and highlight the importance of evaluating small molecules in the context of intact viral replication machinery to improve our understanding of the polyprotein complexes.
Small-molecule inhibitors targeting viral helicases or polymerases are typically characterized as isolated enzymes, yet their effects when associated within their respective replication complexes remain poorly defined. Here, we investigate how helicase- and polymerase-directed inhibitors disrupt the activity of the SARS-CoV-2 replication–transcription complex (RTC). Using a reconstituted mini-RTC (Nsp12/7/8 + Nsp13), we show that Nsp13 forms a stable complex with the holo-RdRp and induces ATPase-dependent stalling of primer extension in vitro. Catalytically inactive Nsp13 (E375A) fails to stall polymerization, suggesting that ATP hydrolysis is required for this regulatory effect. The previously characterized Nsp13 inhibitor IOWH-032 prevents Nsp13-mediated stalling, as it has been shown to disrupt helicase-RNA engagement as well as inhibiting ATPase activity. Unexpectedly, IOWH-032 also directly inhibits holo-RdRp polymerization activity, revealing it as a general RNA transcription-replication inhibitor. In a comparison of IOWH-023 and suramin (a well established competitive inhibtor of the polymerase), electrophoretic mobility shift assays and fluorescence polarization assays reveal that both IOWH-032 and suramin displace RNA from holo-RdRp, whereas strictly allosteric inhibitors (i.e. SSYA10-001) do not. These findings demonstrate that helicase-targeting compounds can exert unanticipated effects on polymerase function within the RTC and highlight the importance of evaluating small molecules in the context of intact viral replication machinery to improve our understanding of the polyprotein complexes.
