Energetic vs. entropic stabilization between a Remdesivir analogue and cognate ATP upon binding and insertion into...

SARS-CoV-2 RNA dependent RNA polymerase (RdRp) serves as a highly promising antiviral drug target such as for a Remdesivir nucleotide analogue (RDV-TP or RTP). In this work, we mainly used alchemical all-atom simulations to characterize relative binding free energetics between the nucleotide analogue RTP and natural cognate substrate ATP upon initial binding and pre-catalytic insertion into the active site of SARS-CoV-2 RdRp. Natural non-cognate substrate dATP and mismatched GTP were also examined for computation control. We first identified significant differences in dynamical responses between nucleotide initial binding and subsequent insertion configurations to the open and closed active sites of the RdRp, respectively, though the RdRp protein conformational changes between the active site's open and closed states are subtle. Our alchemical simulations indicated that upon initial binding (active site open), RTP and ATP show similar binding free energies to the active sites while in the insertion state (active site closed), ATP is more stabilized (∼-2.4 kcal mol^-1) than RTP in free energetics. Additional analyses show, however, that RTP is more stabilized in binding energetics than ATP, in both the insertion and initial binding states, with RTP more stabilized due to the electrostatic energy in the insertion state and due to vdW energy in the initial binding state. Hence, it appears that natural cognate ATP still excels at association stability with the RdRp active site due to that ATP maintains sufficient flexibilities e.g., in base pairing with the template, which exemplifies an entropic contribution to the cognate substrate stabilization. These findings highlight the importance of substrate flexibilities in addition to energetic stabilization in antiviral nucleotide analogue design.