The Role of Ribose Modifications on the Structural Stability of Nucleotide Analogues with α-Thiotriphosphate at the Active Site of SARS-CoV-2 RdRp

As a promising drug target, RNA-dependent RNA polymerase (RdRp) has attracted much attention recently due to its notable conserved active site, especially in the context of COVID-19 spreading. To inhibit the function of RdRp, a nucleotide analogue is a common choice for acting as a chain terminator or RNA corruptor. Although some nucleotide analogues have shown the ability to terminate the extension of a nascent strand of SARS-CoV-2, most of them are likely to be excised due to the proofreading of SARS-CoV-2 nsp14/nsp10. A previous experimental study found that introducing sulfur modification into analogues' phosphate moieties (α-thiotriphosphate; "thio" modification) can break the bottleneck. For instance, Sofosbuvir with α-thiotriphosphate modification successfully escaped the excision of nsp14/10. However, it is unknown how the α-thiotriphosphate affects the structural stability of nucleotide analogues with different ribose modifications at the active site of SARS-CoV-2 RdRp. Thus, in this study, we performed extensive molecular dynamics simulations on four nucleotide analogues with α-thiotriphosphate to elucidate what kind of ribose modification combined with "thio" modification would benefit the analogue's structural stability at the active site. We found that the "thio" modification led to the torsion of phosphate moieties, profoundly affecting the overall conformation of analogues and surrounding residues, determining the Watson-Crick base pairing and catalytic efficiency. Interestingly, chemical modification on the ribose, especially the 1' and 3'-ribose positions, increases the structural stability of analogues through hydrogen bond interactions. Our results revealed nucleotide analogues' structural and dynamical features with "thio" modification at the active site, which may contribute to future drug design or repurposing aimed at the SARS-CoV-2 RdRp.