Structural insights into an aptainhibitor of the SARS-CoV-2 E protein using molecular docking, molecular dynamics, and biophysical analyses.

The envelope (E) protein is a highly conserved structural protein that plays key roles in viral assembly, membrane curvature, and pathogenesis. The E-protein transmembrane (TM) domain oligomerizes into a pentameric ion channel, which contributes to the activation of the NLRP3 inflammasome that is directly linked to acute respiratory distress syndrome (ARDS). Therefore, targeted disruption of E-protein oligomerization represents a powerful therapeutic strategy to attenuate viral virulence. In this study, we report the first DNA aptainhibitors to block E-protein oligomerization. The lead aptainhibitor, Apt-E-36-3, a 30-mer derivative, was characterized using long-timescale molecular dynamics (MD) simulations and surface plasmon resonance (SPR) assays. MD identified Apt-E-36-3 contacts near conserved TM residues on monomeric E protein, suggesting that TM engagement may interfere with membrane insertion. SPR analysis of an E-protein construct consisting of residues 39-75 revealed direct binding to the C-terminal domain (CTD). Apt-E-36-3 demonstrated affinities of 1.97 and 11.8 nM for the full-length E protein and the CTD construct, respectively. The aptamer demonstrated potent functional inhibition, achieving 50% blockade of E-E self-association in the presence of the aptamer. Our findings provide an atomic-level structural rationale for designing variant-resistant therapeutics that specifically inhibit conserved viral proteins.