## 3 Results and Discussion
### Binding Interaction Analysis with PLIP
#### 3.5.1 Interactions with Estrogen Receptor
Some hydrophobic interactions seen for Pep were maintained, including those with Glu423D, Thr460B, and Tyr537B, while new interactions were formed with Val534A and Tyr537A of ER-\(\alpha\). Two \(\pi\)-stacking interactions occurred between Tyr residues of ER and Mut2. For Mut3, (Figure 2d and Supplementary Information Table S2d), eighteen H-bonds and eleven hydrophobic interactions occurred. New H-bonds formed with residues, including Thr371B, Arg412D, Glu419D, Met528A, Lys531A, Val533A, indicating a slight change in position of the Mut3 peptide within the receptor compared to Pep. Certain hydrophobic interactions were maintained, including those with Glu423D, Ser463B, and Tyr537B, while new hydrophobic interactions formed with several residues including Val422D, Asp426D, Leu462B, Tyr526A, Tyr537A, and Leu541A. A new \(\pi\)-cation interaction was observed between Tyr4 of Mut3 and Arg412D of ER-\(\alpha\) and the \(\pi\)-stacking interaction between Tyr537A of ER-\(\alpha\) and Tyr6 of the peptide was conserved and is likely responsible for the higher binding compared to Mut2. For Mut4 (Figure 2e and Supplementary Information Table S2e), eleven hydrophobic interactions and fourteen hydrogen bonds were formed between Mut4 and ER. Hydrophobic interactions were all formed with different residues compared to Pep. A H-bond was observed with Asp538A, as was seen for Pep, while all other H-bonds formed with new residues of ER-\(\alpha\). \(\pi\)-stacking was observed between Tyr537B of ER and Tyr4 of the Mut4 peptide and a \(\pi\)-cation interaction occurred between Lys520C of ER and the N-terminal Tyr of Mut4. Three salt bridges were formed between Arg515A, His516C, and Lys520C of ER and the C-terminal Asp of Mut4. These results indicate that the mutated peptides showed comparable binding interactions with ER-\(\alpha\), though for some there was a slight reduction (Mut1 and Mut2) compared  
Figure 2: PLIP results with ER-\(\alpha\) for (**a**) Pep; (**b**) Mut1 (**c**) Mut2 (**d**) Mut3 (**e**) Mut4. to Pep. Mut3 showed similar binding affinity to Pep, while Mut4 showed a very slight reduction. Further, new interactions were formed between some of the mutated peptides and ER-\(\alpha\), which could potentially enhance the peptides' selectivity and stability in the receptor binding site. After examining the binding interactions of the peptides, we then examined the binding interactions of the polyphenol-peptide conjugates. The interactions are shown in Supplementary Information Table S3 and Figure 3.  
In a recent study, comparative molecular binding studies of ER-\(\alpha\) models with their native ligand estradiol, the drug genistein, and homoisoflavones demonstrated that homoisoflavones exhibited comparable binding affinity for ER-\(\alpha\) as that of estradiol and genistein, indicating their potential applications as ER-\(\alpha\) ligands. Several residues, including Leu339, Leu343, Phe356, Phe475, Glu305, and Ile373, were particularly implicated in binding interactions [58]. Various polyphenols have also been shown to act as ER-\(\alpha\) ligands and this property is implicated in their anti-tumorigenic properties in ER positive cancers [59]. Thus, we hypothesized that the designed polyphenol-peptide conjugates may also display favorable binding interactions with ER\(\alpha\). Figure 3: PLIP results for polyphenol peptide conjugates bound to ER\(\alpha\) (**a**) CGA-Pep; (**b**) CSA-Pep (**c**) GLA-Pep (**d**) RMA-Pep (**e**) Mut1-CGA; (**f**) Mut1-CSA; (**g**) Mut1-GLA; (**h**) Mut1-RMA; (**i**) Mut2-CGA (**j**) Mut2-CGA; (**k**) Mut2-GLA; (**l**) Mut2-RMA; (**m**) Mut3-CGA; (**n**) Mut3-CSA; (**o**) Mut3-GLA; (**p**) Mut3-RMA; (**q**) Mut4-CGA; (**r**) Mut4-CSA; (**s**) Mut4-GLA; (**t**) Mut4-RMA. The results obtained after conjugation with polyphenols are shown in Figure 3. Results for CGAPep are shown in Supplementary Information Table S3a and Figure 3a. Extensive hydrophobic interactions (ten) and hydrogen bonds (twenty) occurred.