## 3 Results and Discussion
### Docking Study
#### 3.4.3 Docking Analysis  
The RMSD (root mean square deviation) measures the average distance between the atoms of two superimposed molecules. It is commonly used to quantify the structural similarity between two molecules in space. In the context of molecular docking, RMSD describes how far the predicted pose of a ligand differs from the native pose. Lower RMSD values indicate higher accuracy in pose prediction. The RMSD, given in A, was 0.67 and 0.83 for 1 HWK and 6 RZ4, respectively. This means that the redocking procedure was successful [66]. The molecular docking data revealed that the newly acquired derivatives exhibited affinity for the investigated protein domains. In Table 5, their affinity for the protein domains is comparable to the binding energies of the reference ligands. The conducted studies indicate that the analyzed ligands exhibited affinity profiles comparable to or lower than the atorvastatinatin as a reference ligand and higher than the caffeine as a reference ligand, thus indicating higher strength of binding to the 6 RZ4 protein  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Compound** & **Molecular Weight** & **HB** & **HB** & **Polar Surface** & **aLogP** & **Rotatable** \\  & **[g/mol]** & **Acceptors** & **Donors** & **Area [Å\({}^{2}\)]** & **Bonds** \\ \hline Atorvastatinatin & 559 & 5 & 4 & 111.79 & 6.31 & 12 \\
**1** & 706 & 4 & 1 & 59.91 & 11.61 & 11 \\
**3** & 653 & 6 & 0 & 88.12 & 7.02 & 9 \\
**4  
domain and lower affinities to 1 HWK. The best poses for the 1 HWK protein domain were 6 and 3, respectively, for compounds **4** and **8**. The poses with the lowest energy have binding energies of \(-9.3\) and \(-9.6\) kcal/mol, respectively [66]. The interactions between the ligands and the 1 HWK protein domain can be found in Figures 3-7, whereas the 6 RZ4 can be found in Figures 8-10. Figure 8: **(a)** The depiction of possible hydrogen bond formation between biocconjugate **4** and 6 KZ4 protein domain. **(b)** The 2D depiction of interactions between biocconjugate **4** and 6 KZ4 protein domain. Cyan lines with big dots—pi–pi interactions, green solid lines—hydrophobic contacts. Figure 6: **(a)** The depiction of possible hydrogen bond formation between biocconjugate **8** and 1 HWK protein domain. **(b)** The 2D depiction of interactions between biocconjugate **8** and 1 HWK protein domain. Blue dashed lines—hydrogen bonds, green solid lines—hydrophobic contacts. Figure 7: Native ligand (atorvastatin, grey-colored) and new ligands: biocconjugates **1** (pink-colored), **3** (bittersweet-colored), **4** (cyan-colored), and 8 (green-colored) at once in the binding site of the 1 HWK protein domain. The new ligands occupy the same space as the native ligands. All the predicted possible hydrogen bonds formed between the protein domains and the ligands are gathered in Table 6. This table contains only the highest probable hydrogen bonds if there are a few possible between certain atoms of a ligand and a protein domain. The potential hydrogen bonds whose length is higher than 3.5 A are not listed, as their probability of formation is very low [67]. A potential hydrogen bond between compound **4** and SER 155 A residue of the 6 RZ4 protein domain (Figure 8) can indeed be formed as the hydrogens are added automatically and can be placed at different angles, shortening the distance. The same applies to the potential hydrogen bond between compound **8** and THR 154 A residue of the 6 RZ4 protein domain (Figure 9). Analyzing bond distances in molecular docking studies is important for understanding how stable and biologically relevant ligand-protein interactions are. Generally, shorter bond distances suggest stronger interactions, which help make the ligand-protein complex more stable.