Structural basis for inhibition of the SARS‐CoV‐2 nsp16 by substrate‐based dual site inhibitors

Coronaviruses, including SARS‐CoV‐2, possess an mRNA 5' capping apparatus capable of mimicking the natural eukaryotic capping signature. Two SAM‐dependent methylating enzymes play important roles in this process: nsp14 methylates the N7 of the guanosine cap, and nsp16‐nsp10 methylates the 2'‐O‐ of subsequent nucleotides of viral mRNA. The 2'‐O‐methylation performed by nsp16‐nsp10 is crucial for the escape of the viral RNA from innate immunity. Inhibition of this enzymatic activity has been proposed as a way to combat coronaviruses. In this study, we employed X‐ray crystallography to analyze the binding of the SAM analogues to the active site of nsp16‐nsp10. We obtained eleven 3D crystal structures of the nsp16‐nsp10 complexes with SAM‐derived inhibitors, demonstrated different conformations of the methionine substituting part of the molecules, and confirmed that simultaneous dual‐site targeting of both SAM and RNA sites correlates with higher inhibitory potential.