## 3 Results and Discussion  
### Sequence Comparison and Homology Modeling  
The bioinformatics analysis of the protein sequences of the four adenosine receptor subtypes has already been presented and discussed in the literature [54; 55; 56]. The overall sequence identity/similarity between human adenosine receptors is relatively high. Interestingly, the adenosine A\({}_{3}\) receptor reveals higher sequence identity/similarity to the adenosine A\({}_{1}\) receptor (48/69%) than to the adenosine A\({}_{2A}\) (37/48%) receptor, as summarized in Tables 1 and 2. In homology modeling, the simplest template selection rule is to select the structure with the highest sequence similarity to the modeled sequence [57]. Moreover, when it is possible, a template bound to the same or similar ligands as the modeled sequence should generally be used. By the analysis of data shown in Table 2 and Figure 5, i.e., the SI% and SS% for all the receptor domains involved in the recognition of agonists and antagonists, the hA\({}_{1}\) AR can be considered a better possible template than the hA\({}_{2A}\) AR. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**A3/A1**} & \multicolumn{2}{c}{**A3/A2a**} \\ \cline{2-5}  & **SI(\%)** & **SS(\%)** & **SI(\%)** & **SS(\%)** \\ \hline
**TM1** & 48.1 & 66.7 & 51.9 & 63 \\
**IL1** & 33.3 & 66.7 & 66.7 & 83.3 \\
**TM2** & 65.5 & 89.7 & 51.7 & 62.1 \\
**EL1** & 20 & 40 & 60 & 80 \\
**TM3** & 64.7 & 85.3 & 47.1 & 76.5 \\
**IL2** & 50 & 70 & 20 & 30 \\
**TM4** & 58.3 & 79.2 & 50 & 66.7 \\
**EL2** & 25 & 40.6 & 12.5 & 25 \\
**TM5** & 52.9 & 79.4 & 44.1 & 67.6 \\
**IL3** & 23.1 & 58.3 & 13.3 & 20 \\
**TM6** & 69.4 & 80.6 & 50 & 66.7 \\
**EL3** & 14.3 & 28.6 & 12.5 & 12.5 \\
**TM7** & 52 & 76 & 48 & 80 \\ \hline overall & **48.2** & **68.7** & **31.6** & **47.8** \\ \hline max & _TM6_ & _TM2_ & _IL1_ & _IL1_ \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Sequence identity (SI) and sequence similarity (SS) percentages calculated for each alignment performed on structural domains of crystal structures SUEN (hA\({}_{1}\) AR) and 3 PWH (hA\({}_{2A}\)). Overall values are also reported along with domains showing the highest SI and SS percentages. As anticipated in the Introduction, the A\({}_{3}\) AR has not yet been crystallized. To appreciate the qualitative and the quantitative differences derived by using hA\({}_{1}\) AR as alternative templates of the conventional hA\({}_{2A}\) AR, we decided to retrospectively compare the performance of molecular docking in posing and scoring a selection of known potent and selective agonists and antagonists, summarized in Figures 2 and 3. Moreover, we have recently reported that the presence of the sodium ion and its first sphere of hydration improves the accuracy and precision of positioning of the antagonist, with respect to the crystallographic poses. This occurs through most of the docking programs and regardless of the chemical nature of the ligand [51]. These data are strongly consistent with the crystallographic evidence which would seem to indicate sodium and his first hydration sphere being an integral part of the orthosteric site that recognizes the structure of antagonist, even for those crystallographic structures in which these have not been solved. By logical extension, sodium and its first hydration sphere should be incorporated into all docking studies, taking into account the interaction between an antagonist and its orthosteric receptor cavity. On the contrary, the presence of sodium and its first hydration sphere, as observed crystallographically, are not an integral part of the orthosteric site that recognizes the structure of the agonist.