## 3 Results and Discussion
### Molecular Dynamics of SARS-CoV-1 S Protein
The analysis of ligands' RMSD values in respect to the protein coordinates and trajectories revealed a substantial difference between anthraquinone compared with emodin and promazine (Figure 4B,D). In particular, anthraquinone showed a marked increase of RMSD compared to the other two ligands reflecting a poorly stable interaction at the identified binding site. Indeed, an early detachment from the surface pocket with a clear one-way outward trajectory was observed. Emodin and promazine were, instead, able to remain at the surface pocket of S protein over time. Notably, emodin and promazine migrated closer to the region identified as a PPI hotspot for S protein-ACE2 interaction (see above). Conversely, anthraquinone, moves out of the pocket at an early stage and this is consistent with its low inhibitory potency towards S protein-ACE2 complex formation. In this respect, ligands diffusion on protein surface likely foreshadow ligand binding since it has been already observed in molecular dynamic studies [39] and it has been already demonstrated as the causal link for a relevant surface protein-ligand interaction [40]. Hence, these results are in line with our initial hypothesis and further corroborate the causal link between the diverse capacity of these compounds to persist at the protein-protein contact surface and the different experimental inhibitory potencies. The authors note that emodin and promazine did not record appreciable geometrical differences in terms of surface occupancy in the timeframe considered, in spite of the diverse activity found experimentally (the former being less potent than the latter) [7]. Therefore, the different activities reported could not be explained on the basis of their persistence at the surface pocket. However, assuming the duration of the interaction as an important aspect to determine the inhibitory potential, a more lasting interaction can be expected for promazine, which showed a stronger inhibitory activity in vitro [7] compared to emodin. Considering that emodin is the focus of this work, its capacity to disrupt the geometry of SARS-CoV-1 S protein-ACE2 complex was also assessed. The geometrical stability of the unbound SARS-CoV-1 S protein-ACE2 complex was compared to that of the complex bound to emodin. The RMSD values of the unbound complex described a rather stable geometry either for the protein-protein complex or for the S protein (Figure 5). In contrast, the geometry was less stable and associated with higher RSMD values for the complex in the bound state with emodin (Figure 5A,B). In addition, the radius of gyration (Rg) was calculated for the SARS-CoV-1 S protein-ACE2 complex for both its unbound and bound state with emodin. Rg reflects protein compactness and a large body of evidence is available describing that an increase of Rg compared to a stable folded state is typically associated with unfolding processes and complex dissociation [41; 42; 43]. The complex in the bound state with emodin showed higher Rg values compared to the unbound complex, particularly at the level of the S protein (Figure 5C,D). Taken together, these data highlight the capacity of emodin to destabilize SARS-CoV-1 S protein-ACE2 complex formation with a particular action on the S protein geometry. Moreover, both the analysis of hydrogen bonds and long-lasting short-range contacts between the S protein and ACE2 were performed to investigate the potential impact of emodin in weakening the network of interaction at the protein-protein interface. The analysis of the number of hydrogen bonds over time revealed a slightly higher number of contacts for the unbound complex toward the end of the simulation compared to the complex bound to emodin (Figure 5E). These results were in agreement with the analysis of the number of long-lasting short-range contacts namely, 46 and 43 for the unbound and bound complex respectively.