Integrated computational protocol for sampling molecular databases towards allosteric inhibition of SARS-CoV-2 spike...

The opening of the receptor-binding domain of the SARS-CoV-2 spike (S) glycoprotein is a key conformational change required for viral entry. In this work, we use an integrated computational approach to evaluate how small molecules may allosterically alter this process. From an initial library of ∼ 60,000 ZINC compounds screened using physicochemical, absorption, and spatial criteria, 739 candidates were identified and clustered to select nine representatives to study their allosteric effect on the protein activation (opening). Classical molecular dynamics simulations revealed distinct differences in ligand stability, while steered (non-equilibrium) simulations quantified how each compound affected the opening motion of the receptor-binding domain. Free-energy profiles constructed from umbrella sampling, together with conformational population density maps derived from principal component analysis, showed that small ligands provide limited inhibition, medium-sized ligands produce the most consistent stabilizing effects, and two of the heaviest ligands substantially reshape the activation pathway by stabilizing closed or intermediate conformations. Overall, the presented approach provides a practical screening framework for analyzing the effect of small molecules on large conformational changes in viral fusion proteins allowing the assessment of reaction barriers in nanosecond timescale.