The library, as shown NT 157 before [47]. However, the overall performance of this screen is in line with previous docking INCB-039110 chemical information studies that identified numerous and potent GPCR ligands [9,45?9]. As was the case here, most of these campaigns targeted a Class A GPCR that binds small organic molecules. Such receptors tend to have rather narrow, well-defined binding sites ?in contrast to the CXCR4 receptor, the only peptide-bound GPCR structure elucidated so far [50]. Smaller binding pockets make for narrower physical search spaces which is likely one of the reasons behind the tractability of these GPCRs by docking and similar approaches.Third, for receptors with high degrees 12926553 of similarity, such as the ARs, selective compounds cannot be predicted solely by docking to one receptor subtype. Most of the ligands identified as A1AR hits also bound to one of the other AR subtypes, and vice versa. In fact, the screen directed toward the A1AR worked even better against the A3AR, with a hit rate of 36 and the most potent compound inhibiting with a Ki of 36 nM. This is an advantage if it is desired to discover ligands for other related GPCR subtypes within a single screening process. However, there is one compound, 8, which appears selective for the A1AR by the criteria used in this screen. In addition, some of the ligands were also moderately selective in binding to the A3AR, which may be due to the fact that the binding pocket of the A3AR is the most divergent one when comparing the three AR subtypes (Table S3), suggesting the relative ease of achieving A3AR selectivity. This tendency to cross over to other subtypes in the screening process can be expected from the similarity of the binding sites. It is difficult to estimate, however, to what degree the use of homology models affected the selectivity of the compounds. Bias stemming from the template used (the A2AAR) cannot be ruled out, but cannot be the only factor as evidenced by the many compounds binding to A3AR. Very likely, even computational screens employing X-ray structures result in 23727046 similarly nonsubtypeselective hit compounds. However, because biochemical testing is limited to the targeted subtype in most studies, this does not become apparent. As a further example of this observation, in the A2AAR screen by Carlsson et al. [10], which is based on a crystal structure, several ligands were found that had mixed selectivity for the A2A and A3ARs. Docking will undoubtedly continue to play a significant role in the quest for novel GPCR ligands, as it has been able to consistently identify potent and chemically novel ligands for a variety of receptors. The targeted identification of selective compounds by combining multiple approaches to model the same 
receptor and closely related members of the same protein family will be the topic of future investigations. Furthermore, the most promising hits from this study, such as a mixed A1/A2AAR ligand, i.e. the 2H-chromen-2-imine derivative 17, or a moderately potent and slightly selective A3AR ligand, i.e. 1,3,5-triazine derivative 24, could now be optimized structurally for AR affinity and selectivity.Supporting InformationTable 
S1 Ligands that were tested and replaced less than 50 of radioligand at 10 mM in all targets. **n = 2. (PDF) Table S2 Compounds in ChEMBL most similar to the ligands identified in this study. (PDF) Table S3 Comparison of binding site residues between A1AR, A2AAR and A3AR. asuperscripts give the Ballesteros-Weinstein numbers. (PDF)Ackno.The library, as shown before [47]. However, the overall performance of this screen is in line with previous docking studies that identified numerous and potent GPCR ligands [9,45?9]. As was the case here, most of these campaigns targeted a Class A GPCR that binds small organic molecules. Such receptors tend to have rather narrow, well-defined binding sites ?in contrast to the CXCR4 receptor, the only peptide-bound GPCR structure elucidated so far [50]. Smaller binding pockets make for narrower physical search spaces which is likely one of the reasons behind the tractability of these GPCRs by docking and similar approaches.Third, for receptors with high degrees 12926553 of similarity, such as the ARs, selective compounds cannot be predicted solely by docking to one receptor subtype. Most of the ligands identified as A1AR hits also bound to one of the other AR subtypes, and vice versa. In fact, the screen directed toward the A1AR worked even better against the A3AR, with a hit rate of 36 and the most potent compound inhibiting with a Ki of 36 nM. This is an advantage if it is desired to discover ligands for other related GPCR subtypes within a single screening process. However, there is one compound, 8, which appears selective for the A1AR by the criteria used in this screen. In addition, some of the ligands were also moderately selective in binding to the A3AR, which may be due to the fact that the binding pocket of the A3AR is the most divergent one when comparing the three AR subtypes (Table S3), suggesting the relative ease of achieving A3AR selectivity. This tendency to cross over to other subtypes in the screening process can be expected from the similarity of the binding sites. It is difficult to estimate, however, to what degree the use of homology models affected the selectivity of the compounds. Bias stemming from the template used (the A2AAR) cannot be ruled out, but cannot be the only factor as evidenced by the many compounds binding to A3AR. Very likely, even computational screens employing X-ray structures result in 23727046 similarly nonsubtypeselective hit compounds. However, because biochemical testing is limited to the targeted subtype in most studies, this does not become apparent. As a further example of this observation, in the A2AAR screen by Carlsson et al. [10], which is based on a crystal structure, several ligands were found that had mixed selectivity for the A2A and A3ARs. Docking will undoubtedly continue to play a significant role in the quest for novel GPCR ligands, as it has been able to consistently identify potent and chemically novel ligands for a variety of receptors. The targeted identification of selective compounds by combining multiple approaches to model the same receptor and closely related members of the same protein family will be the topic of future investigations. Furthermore, the most promising hits from this study, such as a mixed A1/A2AAR ligand, i.e. the 2H-chromen-2-imine derivative 17, or a moderately potent and slightly selective A3AR ligand, i.e. 1,3,5-triazine derivative 24, could now be optimized structurally for AR affinity and selectivity.Supporting InformationTable S1 Ligands that were tested and replaced less than 50 of radioligand at 10 mM in all targets. **n = 2. (PDF) Table S2 Compounds in ChEMBL most similar to the ligands identified in this study. (PDF) Table S3 Comparison of binding site residues between A1AR, A2AAR and A3AR. asuperscripts give the Ballesteros-Weinstein numbers. (PDF)Ackno.
