*3.7. Molecular Docking and Molecular Dynamic Simulation (MDS)*

Molecular docking studies confirmed the experimental results for the BSA-ERL interaction. The site probe experiments with phenylbutazone suggest that the ERL interacted with BSA's subdomain IIA (Site I). However, an earlier study using molecular docking suggested that ERL binds to subdomain IB (Site III) and subdomain IIIA (site II) of BSA [53]. Furthermore, the site probe study with ibuprofen (Site II marker) did not suggest the interaction of ERL at Site II of BSA. Moreover, the quenching curves for the BSA with ERL in the presence or absence of ibuprofen did not change, suggesting no interaction of ERL with site II of BSA.

Molecular docking conformation for the interaction of ERL to Site I and Site III of BSA is given in Figure 5a,c. The binding energy for Site I <sup>−</sup>33.63 kJ·mol−<sup>1</sup> was lower than the binding energy <sup>−</sup>29.91 kJ·mol−<sup>1</sup> for Site III of BSA. The binding pocket of Site I of BSA (Figure 5b) was surrounded by Glu291, Leu218, Leu233, Arg198, Phe222, Arg256, Arg217, Ser286, Leu237, Leu259, His241, Ile289, Tyr149, Ile263, Ala290, Trp213, Gln195, Ser191, Ala260, Arg194, and the amino acids that surrounded the binding pocket at Site III were Lys 116, Pro117, Leu115, Ile181, Leu122, Glu125, Tyr137, Lys136, Arg185, Ile141, Tyr160, Phe133, Met184. Furthermore, the fluorophore residues Trp213, Tyr149, and Phe222 were found in the vicinity of the binding pocket. Thus, the interaction of ERL with BSA might influence these fluorophore residues. In addition, the hydrogen bond between Ligand C19 and Ser286, 3.41 Å, and pi hydrogen bond between 6-ring and Ala290, 3.79 Å were also observed in the BSA-ERL interaction. The BSA-ERL interaction was also investigated for any changes in the presence of QUR. In this study, three hydrogen bonds, Ligand C15 and Phe133, 2.95 Å, Ligand O3 and Phe133, 2.65 Å, Ligand O4, and Lys136, 3.08 Å, were found. In addition, two pi-H bonds 6-ring and Leu115, 4.16 Å, 6-ring and Leu122, 3.90 Å, were observed.

**Figure 5(a-d).** Two-dimensional molecular docking conformation for BSA ERL system at Site I (**a**) and Site III (**c**); three-dimensional docking conformation of BSA-ERL system Site I (**b**) and Site III (**d**). **Figure 5.** Two-dimensional molecular docking conformation for BSA ERL system at Site I (**a**) and Site III (**c**); three-dimensional docking conformation of BSA-ERL system Site I (**b**) and Site III (**d**).

At Site III (Figure 5d), the BSA and ERL formed three hydrogen bonds and one pi– hydrogen bond. The three hydrogen bonds formed were between Ligand C17 and Glu125, 3.51 Å , Ligand O4 and Lys136, 3.15 Å , Ligand N7, and PRO117, 3.15 Å , and the one pi-At Site III (Figure 5d), the BSA and ERL formed three hydrogen bonds and one pi–hydrogen bond. The three hydrogen bonds formed were between Ligand C17 and Glu125, 3.51 Å, Ligand O4 and Lys136, 3.15 Å, Ligand N7, and PRO117, 3.15 Å, and the one pi-hydrogen bond formed was between 6-ring and Leu122, 3.79 Å.

hydrogen bond formed was between 6-ring and Leu122, 3.79 Å . As reported by earlier studies, QUR also binds to site I of serum albumin [47], and thus competition for the same binding Site might occur. Therefore, the molecular docking and the experimental results concluded that the presence of QUR might influence the binding of ERL to BSA. As reported by earlier studies, QUR also binds to site I of serum albumin [47], and thus competition for the same binding Site might occur. Therefore, the molecular docking and the experimental results concluded that the presence of QUR might influence the binding of ERL to BSA.

The conformation stability for the BSA-ERL system was studied with MDS. The MDS study was conducted for Site I and Site III of BSA with ERL. The complex's stability was evaluated based on the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) studies. The RMSD studies for BSA and BSA Site I and Site III with ERL are provided in Figure 6 a,b, respectively, whereas the RMSF plots are given in Figure 6c and d, respectively. The most critical deviations from the crystal structure are found at the residue level in the most mobile parts of the protein, i.e., loops, terminal regions, and helix ends. In contrast, the transmembrane segments remain stable in all the simulations. The conformation stability for the BSA-ERL system was studied with MDS. The MDS study was conducted for Site I and Site III of BSA with ERL. The complex's stability was evaluated based on the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) studies. The RMSD studies for BSA and BSA Site I and Site III with ERL are provided in Figure 6a,b, respectively, whereas the RMSF plots are given in Figure 6c,d, respectively. The most critical deviations from the crystal structure are found at the residue level in the most mobile parts of the protein, i.e., loops, terminal regions, and helix ends. In contrast, the transmembrane segments remain stable in all the simulations.

on the N-terminus were observed.

**Figure 6.** Molecular dynamic simulation RMSD plot for Site I (**a**) and Site III (**b**) and RMSF plot for Site I (**c**) and Site III (**d**). **Figure 6.** Molecular dynamic simulation RMSD plot for Site I (**a**) and Site III (**b**) and RMSF plot for Site I (**c**) and Site III (**d**).

Figure 6a shows the RMSD per residue for all the simulations reported in the present work for Site I. Three major peaks in subdomain 1 (loops C2) Ala78, Pro110, and Cys168

Further, in the simulation of the complex and comparison to the native protein residue Cys168 and Ala78, the N-terminus seems to be responsible for the higher RMSD values from 14–20 ns. Additional minor peaks can also be observed in the remaining loops Figure 6a shows the RMSD per residue for all the simulations reported in the present work for Site I. Three major peaks in subdomain 1 (loops C2) Ala78, Pro110, and Cys168 on the N-terminus were observed.

299, 309, and 504 and the C-terminus. In the simulations between 2 to 8 ns, the resulting RMSD plots seem to be responsible for the higher RMSD values beyond residue Pro110 on the N-terminus for the native protein. No fluctuation was observed in the RMSD or RMSF plots of the BSA-ERL system at Further, in the simulation of the complex and comparison to the native protein residue Cys168 and Ala78, the N-terminus seems to be responsible for the higher RMSD values from 14–20 ns. Additional minor peaks can also be observed in the remaining loops 299, 309, and 504 and the C-terminus.

either of the binding sites, Site I or Site III. The RMSD averages were 1.98 and 1.82 Å, respectively, and the variation in the RMSD for the BSA-ERL system was between 0.258– 3.013 Å for Site I and 0.214–2.318 Å for Site III. Therefore, a stable complex between BSA and ERL is concluded as the RMSD values did not fluctuate too high. Further, the residual flexibility is interpreted from the RMSF studies. The RMSF plot for both the studied sites (Site I and Site III) of the BSA-ERL system suggests that the complex formed between BSA and ERL was stable with a fluctuation of less than 3 Å [54]. **4. Conclusions**  This study examined the influence of flavonoid QUR on the BSA-ERL interaction by In the simulations between 2 to 8 ns, the resulting RMSD plots seem to be responsible for the higher RMSD values beyond residue Pro110 on the N-terminus for the native protein. No fluctuation was observed in the RMSD or RMSF plots of the BSA-ERL system at either of the binding sites, Site I or Site III. The RMSD averages were 1.98 and 1.82 Å, respectively, and the variation in the RMSD for the BSA-ERL system was between 0.258–3.013 Å for Site I and 0.214–2.318 Å for Site III. Therefore, a stable complex between BSA and ERL is concluded as the RMSD values did not fluctuate too high. Further, the residual flexibility is interpreted from the RMSF studies. The RMSF plot for both the studied sites (Site I and Site III) of the BSA-ERL system suggests that the complex formed between BSA and ERL was stable with a fluctuation of less than 3 Å [54].

#### multispectroscopic and computational methods. Quercetin presence in the BSA-ERL sys-**4. Conclusions**

This study examined the influence of flavonoid QUR on the BSA-ERL interaction by multispectroscopic and computational methods. Quercetin presence in the BSA-ERL system reduced the binding constant of the BSA-ERL system to almost half of what was observed in its absence. Thus, it can be concluded that there will be a higher free drug fraction of ERL in the system in the presence of QUR. However, the use of ERL in a therapeutic regimen leads to several adverse events, which in turn are associated with ERL plasma concentrations. Thus, co-administration of QUR and ERL might influence the pharmacokinetics of ERL and needs to be investigated by in vivo studies. Further, QUR is highly metabolized in the human body, necessitating studying the effect of the QUR on co-administered drugs in future studies. Hence, the information gained from such studies can benefit from dose optimization where the two drugs are intended to be co-administered.

**Author Contributions:** Conceptualization: T.A.W., S.Z.; Methodology: T.A.W., S.Z.; Software: A.H.B.; Formal analysis: N.A.A., M.M.A.; Investigation: A.H.B., T.A.W., S.Z., O.M.A.; Resources: T.A.W.; Writing: S.Z.; T.A.W.; Review & Editing: A.A.K.; Project administration: T.A.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** Researchers Supporting Project number (RSP-2021/357), King Saud University, Riyadh, Saudi Arabia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data will be available on request to corresponding author.

**Acknowledgments:** The authors extend their appreciation to Researchers Supporting Project number (RSP-2021/357), King Saud University, Riyadh, Saudi Arabia, for funding this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples are to be prepared freshly for analysis.
