Remdesivir triphosphate is a valid substrate to initiate synthesis of DNA primers by human PrimPol

Abstract

Remdesivir is a broad-spectrum antiviral drug which has been approved to treat COVID-19. Remdesivir is in fact a prodrug, which is metabolized in vivo into the active form remdesivir triphosphate (RTP), an analogue of adenosine triphosphate (ATP) with a cyano group substitution in the carbon 1’ of the ribose (1’-CN). RTP is a substrate for RNA synthesis and can be easily incorporated by viral RNA-dependent RNA polymerases (RdRp). Importantly, once remdesivir is incorporated (now monophosphate), it will act as a delayed chain terminator, thus blocking viral RNA synthesis. It has been reported that mitochondrial Polγ is also blocked in vitro by RTP, but the low impact in vivo on mitochondrial DNA replication stalling is likely due to repriming by the human DNA-directed DNA Primase/Polymerase (HsPrimPol), which also operates in mitochondria. In this work, we have tested if RTP is a valid substrate for both DNA primase and DNA polymerase activities of HsPrimPol, and its impact in the production of mature DNA primers. RTP resulted to be an invalid substrate for elongation, but it can be used to initiate primers at the 5´site, competing with ATP. Nevertheless, RTP-initiated primers are abortive, ocassionally reaching a maximal length of 4–5 nucleotides, and do not support elongation mediated by primer/template distortions. However, considering that the concentration of ATP, the natural substrate, is much higher than the intracellular concentration of RTP, it is unlikely that HsPrimPol would use RTP for primer synthesis during a remdesivir treatment in real patients.