## 3 Results and Discussion
### \"Agnostic-driven\" hA\({}_{3}\) AR Models  
The first comparative validation was performed using the structure of adenosine, its endogenous agonist. We have compared the adenosine binding motif in both hA\({}_{1}\) AR and hA\({}_{2A}\) AR crystallographic structures with those obtained through our docking procedure on the hA\({}_{3}\) AR models derived from the use of the two different crystallographic templates, hA\({}_{1}\) AR and hA\({}_{2A}\) AR respectively. As shown in Figure 6, the best docking poses of adenosine in both the hA\({}_{3}\) ARs are geometrically similar to the crystallographic poses in hA\({}_{1}\) AR and hA\({}_{2A}\) AR. In particular, in each model, adenosine engages  
Figure 5: Sequence comparison calculations on each topological position and overall alignment. The percentage reported on the \(y\)-axis indicates how much the difference in identity (\(\Delta\)SI, cyan), or in similarity (\(\Delta\)SS, orange), favors alignment towards the hA1 or hA2a AR. its canonical stabilizing bidentate H-bond of N7 and N\({}^{6}\) H with Asn250 (6.55) and the \(\pi\)-\(\pi\) stacking interaction with Phe168 (EL2). However, in the case of the hA\({}_{2A}\) AR model, the ribose ring is not anchored to Thr94 (3.36) by hydrogen bond, conversely, in the hA\({}_{1}\) AR model, it is, by a pretty network. This is also confirmed by corresponding energy histograms, in which the hA\({}_{2A}\) AR model is shown to be highly disfavored not only because of Thr94 (3.36) but also of critical residues such as Val169 (EL2). Moreover, the \(\pi\)-\(\pi\) stacking interaction with Phe168 (EL2) is not verified, as no hydrophobic interaction is detected. It is evident that an increased stabilization of adenosine occurred in the hA\({}_{1}\) AR-based model due to some crucial amino acids, i.e. Thr94 (3.36), Phe168 (EL2) and the not conserved residues Val169 (EL2) and Leu90 (3.32). As for adenosine, all other agonists have been docked to any of the hA\({}_{3}\) ARs, and the best docking poses have been summarized in Video S1 (available as Supplementary Information). Summarizing the docking results, all analyzed agonists were preferably fitted in the orthosteric binding site of the hA\({}_{1}\)-based models of hA\({}_{3}\), as graphically shown in the correlation matrix of Figure 7, where the energetic contributions (electrostatic and van der Waals) assigned to the hA\({}_{1}\)-based model are compared with the hA\({}_{2A}\)-based one. The chromatic scale must be interpreted as follows: (a) the white color indicates favorable energetic contributions of the specific ligand (reported in the \(y\)-axis) for both hA\({}_{1}\)-based model and hA\({}_{2A}\)-based models (as for example calculated for ADO and MRS5127); (b) the orange color indicates unfavorable energetic contributions of the specific ligand for both hA\({}_{1}\)-based model and hA\({}_{2A}\)-based models; (c) the blue color indicates favorable energetic contributions of the specific ligand (reported in the \(y\)-axis) for the hA\({}_{1}\)-based model and unfavorable for the hA\({}_{2A}\)-based model (as for example calculated for MRS7110 and MRS5644); and, finally, (d) the red color indicates unfavorable energetic contributions of the specific ligand for the hA\({}_{1}\)-based model and favorable for the hA\({}_{2A}\)-based model. Moreover, the numerical value inside each cell describes, in logarithmic form, the ratio of the specific energy contribution calculated between the hA\({}_{1}\)-based model and the  
Figure 6: Per residue energy histograms and selected docking pose of adenosine on either the hA\({}_{2A}\) based model (**a**) or the induced fit hA\({}_{1}\)-based model (**b**). hA\({}_{2A}\)-based reference model (see Equation (2), \(x\) and \(y\) respectively).