## 3 Results and Discussion  
Emodin, promazine (Figure 1B), and to a minor extent the emodin congener anthraquinone (Figure 1C), were shown to inhibit the interaction of SARS-CoV-1 S protein with ACE2 [7]. The SARS-CoV-1 S protein-ACE2 complex is stabilized via protein-protein interaction (PPI) at the complex interface involving the RBD of the S protein's S1 subunit, as described in crystallographic studies (Figure 2) [20]. Of note, anthraquinones may efficiently prevent protein-protein complex formation disrupting the interaction hotspots of PPI that are mediated by alpha helixes [35; 36; 37]. Therefore, it was proposed that such a mechanism may contribute to emodin's action. Hence, the interaction of emodin at the SARS-CoV-1 S protein-ACE2 complex interface was studied to provide a plausible detailed mechanistic assessment of its previously documented antiviral action.  
Specifically, the interaction interface of S protein's RBD-ACE2 complex was analysed to: (i) describe potential PPI hotspots at the S protein-ACE2 complex interface; (ii) describe a possible site of emodin interaction at the S protein-ACE2 complex interface that may inhibit PPI.  
The analysis of interaction hotspots and the pocket search was followed by docking studies to assess the ability of emodin to fit the pocket at the complex interface. Subsequently, molecular dynamics were performed to investigate the emodin's ability to remain at the S protein contact surface over the time. Then, molecular dynamic simulations were used to assess whether emodin' activity may interact and interfere with the SARS-CoV-1 S protein-target receptor complex. Finally, the analysis was extended also to the SARS-CoV-2 S protein to check a possible functional conservation of emodin on the two orthologous S protein.  
Analysis of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1) S Protein-Angiotensin I Converting Enzyme 2 (ACE2) Complex  
The visual inspection of SARS-CoV-1 S protein-ACE2 complex highlighted the prominence of an ACE2 alpha helix (residues 20-53) at the contact interface (Figure 2). Interaction hotspots (i.e., residues with a key role to stabilize PPI) were analysed with a previously validated computational approach through the implementation of the so called \"computational alanine scanning\" [38] using the Robetta algorithm [24]. Tyr41, Gln42, Gln325 and Lys353 were identified as the amino acid positions of ACE2 with the most marked effect on the PPI, while Tyr442, Tyr475, Tyr484 and Tyr491 were the key residues of S protein (Figure 2; Table S2 Supplementary Materials). Therefore, two different regions with a marked calculated energetic contribution to the PPI were identified at the protein-protein contact surface (Figure 2). One was formed by six out of eight of the key residues identified and such a contact point, enriched in interaction hotspots, was hypothesised to have a prominent role in promoting the PPI. Subsequently, the protein-protein contact interface was visually inspected and a surface pocket that potentially allows hosting molecules with anthraquinone scaffold was identified (Figure 2). This potential site of interaction was contiguous to the protein-protein contact point that was found enriched in interaction hotspots. This result was in line with the aforementioned hypothesis. Indeed, emodin, promazine and to a minor extent anthraquinone, might prevent SARS-CoV-1 S protein-ACE2 complex by docking such a pocket, disrupting a focal contact point and eventually destabilizing the complex. The binding capacity of emodin, promazine and anthraquinone within such surface pocket was assessed by means of docking simulations and molecular dynamics.