## 3 Results and Discussion
### Molecular Dynamics of SARS-CoV-1 S Protein  
Inhibitory activity towards the formation of the SARS-CoV-1 S protein-ACE2 complex was further assessed using molecular dynamics simulations. The best scored docking pose of each compound was used as input coordinates in molecular dynamic simulations to check their capability to persist at the proposed site of interaction. The S protein not bound to ACE2 was used first assuming that complex-disrupting compounds may also interact at the site of interaction prior to the complex formation. The root mean square deviation (RMSD) of protein C-alpha and ligands' atomic coordinates were analyzed along with ligand trajectories to measure the geometrical stability of complexes, which is inherently linked to ligands' activity and protein functions [18]. The overall geometrical stability of the S protein was more pronounced (lower RMSD values) when complexed with anthraquinone compared to that with emodin and promazine (Figure 4A). Figure 3: Binding poses of emodin, anthraquinone and promazine. Ligands and surrounding residues are represented in sticks. Yellow dotted lines indicate polar contacts. SARS-CoV-1 S protein is colored in yellow, while ACE2 is colored in white. Cyan, red and blue spheres indicate regions sterically and energetically able to receive hydrophobic, hydrogen bond acceptors and hydrogen bond donor groups respectively. (**A**) Binding pose of emodin. (**B**) Binding pose of anthraquinone. (**C**) Binding pose of promazine. (**D**) Binding pose of anthraquinone and the pharmacophoric fingerprint of pocket. Grey boxes indicate the pharmacophoric matches. (**E**) Binding pose of emodin and pharmacophoric fingerprint of pocket. Grey boxes indicate the additional pharmacophoric matches compared to anthraquinone. (**F**) Binding pose of promazine and the pharmacophoric fingerprint of pocket. Grey box indicates the additional pharmacophoric matches due to the branched amino portion. While assuming that the organization of the S protein is an important facet to recognize its target receptor, the ligand-dependent destabilization of the S protein has a likely role in determining the extent of ligand-dependent inhibition. Specifically, the low capability of anthraquinone to prevent the interaction of SARS-CoV-1 S protein with ACE2 could be partially explained by its lowest capacity to change S protein organization. Conversely, the more potent capacity of emotion and promazine  
Figure 4: Molecular dynamic of emodin, anthraquinone or promazine in complex with SARS-CoV-1 S protein. (**A**) Root mean square deviation (RMSD) plot of protein C\(\alpha\). (**B**) RSMD plot of ligands. (**C**) Time-step representation of ligands trajectory of localised protein motion (residues 470–490). The switch from red-to-blue color indicates the stepwise changes of protein coordinates along the molecular dynamics (MD) simulation. (**D**) Time-step representation of ligands trajectory. The from-red-to-blue color switch indicates the stepwise changes of ligand coordinates along the MD simulation. The white dotted rings indicate the localization of the hotspot of interaction enriched in protein-protein contacts. The white arrow indicates the outward trajectory of anthraquinone, while the dashed box highlights its late interaction site. to prevent SARS-CoV-1 S protein-ACE2 interaction compared to anthraquinone may be due to their higher capacity to change S protein organization. A close inspection revealed that the most marked effect was at the level of residues 470-490 (Figure 4C), which form one of the two PPI hotspots identified (see above). Specifically, that region was more stable and compact when the protein was complexed with anthraquinone compared to the complex with emodin or promazine. This localised difference in protein stability could explain in part the experimental differences in the reported activities of the compounds.