## 3 Results and Discussion
### \"Antagonist-driven\" hA\({}_{3}\) AR Models  
In contrast to the construction of the \"agonist-driven\" hA\({}_{3}\) AR models, in the case of \"antagonist-driven\" hA\({}_{3}\) AR, it has been decided to build up three different homology models, with two of these models using as templates the two hA\({}_{2A}\) AR structures in complex with the antagonist ZM241385 (PDB codes: 3 PWH and 5 NM2, respectively), and the third one using as a template the hA\({}_{1}\) AR structure in complex with the antagonist DU1 (PDB code: 5 UEN). As already anticipated, all \"antagonist-driven\" hA\({}_{3}\) AR models have been built up considering the sodium cation and its first hydration sphere as an integral part of the orthostatic site. Figure 8: Per residue energy histograms and selected docking poses of MRS7110 (panel **A**) and MRS5644 (panel **B**) on either the hA\({}_{2A}\) based model (**a**) or the hA\({}_{1}\) based models (**b**,**c**). All other antagonists, as listed in Figure 3, have been docked in any of the three hA\({}_{3}\) ARs and the best docking poses have been summarized in Video S2 (available as Supplementary Information). As for the comparison of agonists-driven docking results, even for antagonists the correlation matrix, shown in Figure 9, summarizes the energetic contributions (electrostatic and van der Waals) assigned to the hA\({}_{1}\)-based and the hA\({}_{2\Delta}\) 5 NM2 model compared to the hA\({}_{2\Delta}\) 3 PWH model. From the analysis of the correlation matrix, in this case, it is deducible that not all homology models equally stabilize the accommodation of the different antagonists in the orthosteric binding site of hA\({}_{3}\) AR. In particular, the energetic contributions for antagonist-receptor interaction (electrostatic and/or van der Waals) would seem to show a much higher stabilization in the hA\({}_{1}\)-based model, compared to models based on both the A\({}_{2\Delta}\) structures, for several antagonists such as the highly selective 1,4-dihydropyridine derivative MRS-1334 (especially the 5- enantiomers) or the isoquinoline VUF-5455. As an example, the highly selective 1,4-dihydropyridine derivative MRS-1334 exists in two possible enantiomers, (4\(R\)) and (4\(S\)). It has been already demonstrated that the (\(S\))-enantiomer is the main responsible for selectivity for this class of compounds [47]. The (4\(S\))-enantiomer orients the \(p\)-nitrobenzyl substituent in the same direction of the aryl-ethynyl one, by intramolecular aromatic stacking interaction, conversely, the (4\(R\))-enantiomer does to the top between TMs 1 and 7 (Figure 10). Enantiomers' poses differ mainly in the orientation of the ethyl ester substituent, up (4\(S\)) and down (4\(R\)), leading to different interactions as well. The (4\(R\))-enantiomer shows very unfavorable interactions of the 2-phenyl moiety with the EL2 residue Val169. Conversely, the (4\(S\))-enantiomer ethyl ester substituent contracts favorable interactions with Val169 and strong hydrophobic interactions with Phe168 (EL2). Hydrophobic interactions are established also with Val65 and Leu90. In general, the latter gives the main contribution to both enantiomers of MRS-1344 poses. These data are largely in favor of the (4\(S\))-enantiomer, consistently with enantioselectivity observed. Asn250 (6.55) contribution is not determinant in this case, suggesting that excluding inevitable limitations of homology modeling, the _atypical_ dihydropyridine scaffold in its \(S\)-configuration could be unable to engage hydrogen bonds  
Figure 9: EM correlation matrix of energy contributions (electrostatic and van der Waals) for antagonists poses (\(y\)-axis), obtained from the hA\({}_{2\Delta}\)-based model (5 NM2) and the hA\({}_{1}\)-based model (5 UEN), in comparison with the reference model (3 PWH). with Asn250 (6.55), except if we consider a rotation of the amide side chain, i.e. the driving component of the final state may be mainly hydrophobic.