## 3 Results and Discussion
### \"Agnostic-driven\" hA\({}_{3}\) AR Models
From an interpretative point of view, if this value is more negative it means that this energetic contribution is much more favorable for the binding of the ligand versus the hA\({}_{1}\)-based model compared to the hA\({}_{2A}\)-based model, whereas when this value is more positive, the energetic contribution is much more favorable for the binding of the ligand versus the hA\({}_{2A}\)-based model and compared to the hA\({}_{1}\)-based model. From the analysis of the correlation matrix is deducible that all homology models, independently from the original template, are able to properly accommodate all selected agonists inside the orthosteric binding site of the hA\({}_{3}\) AR. However, in general, the energetic contributions for agonist-receptor interaction (electrostatic and/or van der Waals) indicates a higher stabilization in the hA\({}_{1}\)-based model compared to a model based on A\({}_{2A}\) structure, in particular for the agonists MRS7110 and MRS5644. Interestingly, when docked to the hA\({}_{1}\)-based model, MRS-7110 achieves a very suitable accommodation for the 2-(4-aryl-1,2,3-triazoleyl) substituent, in the proximity to both EL2 and TM2 (Figure 8). The 2-aryl-triazoleyl derivative shows the conserved binding pattern of agonists and a ribosyl C2\({}^{\prime}\)-endo conformation. Conversely, MRS-5644 is partially displaced from the binding site. MRS-5644 presents a huge planar 2-arylethylynyl substituent, which is thought to be responsible for selectivity towards the hA\({}_{3}\) AR. A favorable as possible accommodation of the substituent (without prohibitive sterical hindrance) must be determinant for hA\({}_{3}\) selectivity. None of the built models is able to predict the most suitable pose for this ligand, but the hA\({}_{1}\)-based model can accommodate its analog MRS-7710, which shares a geometrically similar substituent in C2. This seems to be consistent with the evidence that the different conformation of the ELs and the TMs' shift observed for the hA\({}_{1}\) AR, as compared with the hA\({}_{2A}\) subtype, leads to a different shape of the binding cavity. It is reasonable to assume that peculiar residues of the hA\({}_{3}\) AR sequence could further enhance this aspect, making the binding cavity more prone to locate even rigid bulky substituents, especially towards the TM2 and EL2. In order to explore this hypothesis, we built up another structure, based on the already obtained 6D9H-A\({}_{3}\) model, exploiting the MOE Induced Fit functionality in presence of the MRS-7110 ligand pose, whose coordinates had been previously superposed to the MRS-5644 pose (indeed, these ligands differ only for the respective functionalities in C2). Figure 7: EM correlation matrix of energy contributions (electrostatic and van der Waals) for agonist poses (\(y\)-axis) obtained from the hA\({}_{1}\)-based model with induce fit (6D9H-in) and without induce fit (6D9H), as compared to the reference model (2 YDO). Docking results on the 6D9H-in A\({}_{3}\) AR model are visibly better: 5 out 5 poses are in line with the canonical binding interaction geometry of AR ligands, as shown in Video S2. Furthermore, interaction with Thr94 (3.36) and Asn250 (6.55) is significantly more stable for each ligand. Importantly, as hypothesized, MRS-5644 reaches a reliable orientation (Figure 8), with the 2-aryl-ethynyl substituent directed towards the TM2, on the side of EL2. Indeed, the model, as compared to the template 6D9H, shows a slight outward shift of the TMs 2, 3 and 7 upper regions, while an inward shift of the EL2 is observed (the measured C\(\alpha\)-RMSD is about 0.28 A). These results indicate that, ideally, the orthosteric site of the hA\({}_{3}\) AR active state may accommodate the peculiar substituents of selective agonists in the proximity of EL2 and TMs 2-3 through some expansion of the cavity, favored by the conserved and not conserved residues located in these regions, as Leu90 (3.32) [58], Gln167 (EL2) [59] and Thr94 (3.36).