# Selective Control by _Pistacia vera_ L. and Its Carotenoid Zaxanthin on SARS-CoV-2 Virus
## 3 Discussion  
The present study explored the potential of pistachio extracts (NRRE and RURE) and zeaxanthin as inhibitors of SARS-CoV-2 entry, utilizing a luciferase-expressing pseudovirus system. NRRE and RURE extracts demonstrated significant inhibition of entry for both \(\alpha\)- and o-SARS-CoV-2 pseudoviruses, as shown in Figure 1. The natural product zeaxanthin, previously shown to inhibit HSV-1 entry, also acted as a SARS-CoV-2 entry inhibitor, although the targets and mechanisms differ [19]. Molecular modeling studies demonstrated that zeaxanthin strongly interacts with the RBD of the omicron variant's spike protein (Figures 3 and 4). These findings correlate  
Figure 7: In vitro inhibition of SARS-CoV-2 3 CLPro and PLPro. Purified recombinant SARS-CoV-2 3 CLPro and SARS-CoV-2 PLPro were combined with zeaxanthin (10 uM) and added to 3 CLPro substrate solution for 60 min at 37 °C. GC376 and Camostat were used as positive protease inhibitor controls. The reaction was stopped by adding Luciterin Detection Reagent, and the luminescence was recorded on a plate-reading luminometer. Results were expressed as percentages of RLU (**a,b**) and 3 CLPro and PLPro inhibitory activity (**c,d**). **\({}^{*}\)**_p_ \(<\) 0.01, ***\({}^{*}\)_p_ \(<\) 0.001, ***\({}^{*}\)_p_ \(<\) 0.0001.  
with the observed reduction in viral entry in in vitro studies (Figure 2), suggesting that zeaxanthin may play a role in inhibiting viral infection. The combination of molecular docking and MD simulations provides a robust computational framework for in silico analysis of drug-protein interactions, offering critical insights that complement and validate in vitro experimental results [22]. To evaluate the potential of zeaxanthin in modulating spike RBD-ACE2 interactions, we employed the Autodock4-GPU docking software (v. 4.2.6). Zeaxanthin was docked to the RBD of the SARS-CoV-2 spike protein of the alpha and omicron variants. While the binding energies were comparable (\(-6.90\) kcal/mol for the alpha variant and \(-7.10\) kcal/mol for the omicron variant), the 200 ns MD simulations revealed distinct behavioral differences between the two variants. The best docking poses of zeaxanthin with the RBD domain highlighted the different stability of the complexes. Specifically, the zeaxanthin/o-RBD complex exhibited greater stability compared to the alpha variant. In the case of the omicron variant, zeaxanthin remained stably positioned within the RBD region throughout the 200 ns simulation, as evidenced by minimal RMSD fluctuations. The proximity of zeaxanthin to the RBD/ACE2 interaction interface suggests that the compound may destabilize the protein complex, thereby hindering viral entry. This hypothesis is further supported by the free energy of binding analyses conducted using MM-PBSA calculations over the 200 ns simulation. According to YASARA analysis, more negative free energy of binding values indicates stronger binding affinity. The results showed average free energy of binding of \(-6.90\) kcal/mol for the \(\alpha\)-RBD complex and \(-8.77\) kcal/mol for the o-RBD complex. Additionally, the docking-based method used to calculate binding energy during the MD simulation indicated a higher affinity for the o-RBD complex (\(-4.86\) kcal/mol) compared to the \(\alpha\)-RBD complex (\(-4.34\) kcal/mol). These findings and the MD simulation results underscore the more favourable interactions in the omicron complex (Figure S9). The different inhibitory effects of zeaxanthin on SARS-CoV-2 variants likely stem from structural variations within the RBD of these variants. This hypothesis is supported by a previous study investigating the binding affinities of various carotenoids, including zeaxanthin, with SARS-CoV-2 spike protein mutants [22]. Although the omicron variant was not included in that study, the authors observed variant-dependent binding affinities, with zeaxanthin exhibiting stronger binding to the delta variant and weaker binding to the alpha variant.