Substrate and target selectivity of 4'-fluoroadenosine against viral and host polymerases

Developing safe and effective treatments against emerging RNA viruses is an important goal in pandemic preparedness efforts. 4'-fluorouridine (4'-FlU) is a broad-spectrum antiviral that was shown to inhibit viral RNA-dependent RNA polymerases (RdRps). Given its notable range of antiviral activity, this class of nucleoside analogs warrants further investigation. Here, we studied the antiviral activity and underlying mechanism of inhibition of 4'-fluoroadenosine (4'-FlA). Like 4'-FlU, 4'-FlA demonstrates a broad-spectrum of antiviral activity against eight prototypic viruses representing diverse families. Enzyme kinetics shows that the triphosphate (4'-fluoroadenosine triphosphate) is efficiently incorporated by viral RdRps. A cryo-EM structure of the RdRp of severe acute respiratory syndrome coronavirus 2 in complex with double-stranded RNA and the incorporated monophosphate (4'-fluoroadenosine monophosphate) characterizes interactions at the active site. The incorporated analog elicits heterogeneous inhibition patterns in primer extension reactions. In contrast, templates with embedded 4'-fluoroadenosine monophosphate inhibit incorporation of complementary uridine triphosphate (UTP) across the viral RdRps. However, incorporation of 4'-fluoroadenosine triphosphate is not limited to viral polymerases and likewise includes human mitochondrial RNA polymerase. These results demonstrate the general potential for 4'-fluorinated nucleotides as antiviral drugs and guide the development of more selective derivatives for medical use in appropriate settings.