# Experimental and Simulation Identification of Xanthohumol as an Inhibitor and Substrate of ABCB1
## 3 Results
### XN Bound to the Central Transmembrane Domain (TMD) Site, Overlapping with the DOX Binding Site  
To probe the interactions between the ligands (DOX and XN) and ABCB1, two ABCB1-ligand-ATP complexes and 1000-ns MD simulations for each system were constructed. Our RMSD analysis indicated the convergence of the MD simulations in the last 400 ns for both systems (Figure S2). The detailed results of the protein secondary structure, protein-ligand contacts, and ligand torsion profiles are provided in the supporting material (Figures S3-S6). Here, we highlight some key observations. First, DOX and XN bound to similar central TMD sites. Figure 5A-C shows the superimposed structures containing the ligands from the last frames of the two MD simulations. It is noteworthy that the ligands DOX (red) and XN (blue) overlapped in the central TMD site. Figure 5D,E show the binding of DOX and XN to nearby residues in the central TMD site, respectively. DOX bound close to the residues Ile 340, Phe 983, Asn 839, Gln 990, Tyr 307, Gln 725, and Phe 343 (Figure 5D and Table S1). XN bound close to residues Phe 336, Phe 983, Ala 980, Phe 728, Asn 842, Tyr 953, and Phe 732 (Figure 5E and Table S1). The binding sites of XN overlapped with that of DOX, which suggested XN could block DOX and interact to ABCB1 competitively. Second, MM-GBSA binding energy data (Table 2) showed the binding of XN (\(-\)88.0 \(\pm\) 6.3 kcal/mol) to the transporter at the central TMD site was stronger than that of DOX (\(-\)86.1 \(\pm\) 8.6 kcal/mol) by \(-\)1.9 kcal/mol. The energy decomposition indicated that the hydrophobic interaction (\(\Delta\Delta\)Lip = \(-\)10.7 kcal/mol) was the major contributor to the energy difference (\(-\)1.9 kcal/mol) between the two ligands. This hydrophobic interaction could be reduced by the VDW interaction (\(\Delta\Delta\)VDW = 6 kcal/mol), and electrostatic interaction (\(\Delta\Delta\)GBELE = 2.9 kcal/mol). Third, both the protein (Figure 6) and ligand RMSF analyses (Figure 7) showed that the DOX-protein system had slightly higher position fluctuation than the XN-protein system. The average protein RMSF for the DOX complex was 4.51 A, whereas the protein RMSF for the XN complex was 2.89 A. Similarly, the average ligand RMSF for DOX was 2.58 A, whereas the average ligand RMSF for XN was 1.85 A. The XN complex experienced less fluctuation overall, compared to the DOX complex. This could indicate a more stable ligand-protein complex, providing evidence that XN bound to ABCB1 and acted as a competitive inhibitor of DOX. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**XB1/Inhibitor Complex** & **\(\Delta\)VDW** & **AGBELE** & **\(\Delta\)LIPO** & **\(\Delta\)TOT** & \(\Delta\)\(\Delta\)**TOT** \\ \hline XN Complex & \(-\)51.6 \(\pm\) 3.2 & 14.3 \(\pm\) 3.2 & \(-\)50.7 \(\pm\) 3.1 & \(-\)88.0 \(\pm\) 6.3 & \(-\)1.9 \\ DOX Complex & \(-\)57.5 \(\pm\) 3.4 & 11.4 \(\pm\) 6.5 & \(-\)39.9 \(\pm\) 3.0 & \(-\)86.1 \(\pm\) 8.6 & 0.0 \\ \hline \hline \end{tabular} \(\Delta\)VDW = Change in VDW energy in gas phase upon complex formation (Units: kcal/mol); \(\Delta\)GBELE = Change in GB reaction field energy + gas phase Elec. energy upon complex formation (Units: kcal/mol); \(\Delta\)LIPO = Change in lipophilic term (lipophilic energy) upon complex formation (Units: kcal/mol); \(\Delta\)TOT = \(\Delta\)VDW + \(\Delta\)LiPO + \(\Delta\)GBELE Change in potential energy in water upon complex formation (Units: kcal/mol).  
\end{table} 
Table 2: MM-GBSA binding energies. Figure 5: Structural representation of the predicted interaction between ABCB1 and its ligands. **(A)** Superimposed ABCB1 models from the last snapshot of the MD simulations. The model with DOX and ATP ligands is represented using red ligands in VDW and pink-colored ribbons. The model with XN and ATP ligands is represented using blue ligands in VDW and yellow-colored ribbons; **(B)** Close-up view of the superimposed DOX and XN complexes at the ABCB1 central TMD from the top.