## 3 Results and Discussion
### Binding Interaction Analysis with PLIP
#### 3.5.1 Interactions with Estrogen Receptor
PLIP studies were next performed with Mut2 conjugated with all four polyphenols. The CGAMut2 conjugate (Supplementary Information Table S3i and Figure 3i) exhibited marked improvement in binding affinity. In total, seventeen H-bonds and eleven hydrophobic interactions, as well as the formation of a \(\pi\)-stacking interaction, a \(\pi\)-cationinteraction, and a salt bridge was observed. New hydrogen bonds were observed with Glu423C, Asp426C, Thr460A, Phe461B, His516C, Asn519A, Lys520D, Tyr526B, Ser536A, and Asp538A. New hydrophobic interactions were seen with Glu380B, Asp426C, Asn519A, Lys520C, Tyr526A, and Tyr526B. For CSAMut2, the results are shown in Figure 3j and Supplementary Information Table S3j. Six hydrophobic interactions and sixteen H-bonds were observed. New hydrophobic interactions were seen with Tyr526A, while new H-bonds occurred with Trp383A, Arg412C, Glu423C, Thr460A, Lys520C, Lys531B, Ser536B, and Leu539B. A salt bridge was also observed with Arg412C. The results obtained for GLAMut2 are shown in Supplementary Information Table S3k and Figure 3k. Additional hydrophobic interactions (a total of eleven) and H-bonding (a total of nineteen) compared to neat Mut2 were seen. A \(\pi\)-stacking interaction with Tyr537A was also seen. New hydrophobic interactions include those with Glu380A, Val422D, Tyr526A, Lys531A, Asn532A, and Leu539A, while new H-bonds included those with Phe461B, Ser464B, His516C, Lys520C, Tyr526A, Met528A, and Ser536A. For RMAMut2 (Figure 3l and Supplementary Information Table S3l), nineteen hydrogen bonds and five hydrophobic interactions occurred; a salt bridge was observed with His377A. New H-bonds included those with Cys381B, Tyr459B, Phe461B, Leu462A, Leu462B, Ser463B, and His516D. New hydrophobic interactions occurred with Asp426D, Tyr526B, and Asp538A. CGAMut3 showed a slight decrease in H-bonding (fifteen total); fewer hydrophobic interactions (six total) were observed compared to neat Mut3, while \(\pi\)-stacking occurred with Tyr526A and Tyr526B in addition to a salt bridge with His377B (Supplementary Information Table S3m and Figure 3m). Compared to Mut3, hydrogen bonds with Thr371B, Glu419D, Met528A, Val533A, Val534A, Tyr537A, Leu539A, and Glu542A were eliminated, while several other new H-bonds were observed. New hydrophobic interactions were also seen. In the case of CSAMut3, twelve hydrogen bonds and thirteen hydrophobic interactions occurred and \(\pi\)-stacking and \(\pi\)-cation interactions were not maintained, while a salt bridge was formed with Arg412D, as shown in Figure 3n and Supplementary Information Table S3n. Several hydrogen bonds seen for neat Mut3 were not observed, including those with Thr371B, Glu419D, Leu462B, Met528A, Lys531A, and Glu542A. New hydrophobic interactions formed included those with His377A, Val534A, and Asp538A. For GLAMut3 (Figure 3o and **Supplementary Information Table S3o**), four hydrophobic interactions and sixteen H-bonds occurred, while \(\pi\)-stacking and a salt bridge were observed. Hydrophobic interactions were seen with Tyr537A, Tyr537B, and Leu539B. Several hydrogen bonds disappeared, including those with Thr371B, Glu419D, Asp426D, Leu462B, Met528A, Lys531A, Ser536A, and Leu539A, while new H-bonds were formed with several other residues. The RMAMut3 conjugate showed four hydrophobic interactions and nineteen H-bonds, in addition to \(\pi\)-stacking interactions and a salt bridge (Supplementary Information Table S3p and Figure 3p). Hydrophobic interactions were observed with Glu380A, Val534A, Tyr537A, and Tyr537B. New H-bonds were formed with His373A, His373B, Glu380B, Arg412C, Val422C, Glu423C, Thr460B, His516C, and Lys520C, among other residues. The significant changes in the positions of receptor-ligand interactions indicate a change in the position of RMAMut3 within the binding pocket compared with Mut3. PLIP studies were then carried out with Mut4-conjugated polyphenols.