Journal of chemical Information and modeling Article Chart 1. Representative S-HTIAR Agonists Buspirone F13640 Y7 X. Y-80A Figure 2. Side view of the R-8-OH-DPAT-S-HTIAR complex embedded in a hydrated POPC lipid bilayer(left). s-HTIAR is shown in cartoon representation with R-8-OH-DPAT in the upper center. Water molecules and lipid molecules are shown in sticks; and choline N(blue),P atom (orange)and Nat(gray), CL(green) ions are represented in spheres. The detail of the binding mode of R-8-OH-DPAT with S-HTIAR is amplified right).S-HTIAR is shown in gray ribbons and R-8-OH-DPAT in green ball and sticks. Hydrogen bonds are depicted in dotted lines. RESULTS AND DISCUSSION structure of P2 aR bound to the partial inverse agonist carazolol 3D Structure of the 5-HT1AR Model. Sequence alignment (PDB Code: 2RH1) F88), indicated that the sequence identity in the transmembrane our model was built taking the active state conformation of the region is 45% between S-HTIAR and B,AR. The most P2AR-Gs complex as template and represents the active recently disclosed crystal structure of the P,AR-Gs complex in conformation binding with agonists. Thus it is suitable f active state(PDB Code: 3SN6)was the optimal template to me investigation of discovery of 5-HTIAR agonists. construct the S-HTIAR model. Therefore, it was expected tha 8-OH-DPAT(Chart 1), a prototypical 5-HTIAR agonist, has een extensively used for pharmacological studies. R-8-OH- our model represents the active state of the receptor. Figure SI DPaT displays higher efficacy than its S-counterpart and acts as shows the final sequence alignment of 5-HT1ar to the a full S-HTIAR agonist, thus it was chosen herein to induce template. The PROCHECK statistics showed that 99.2% of active receptor conformations. The predicted binding mode the residues in the 5-HT,AR model (Figure la) were either in the most favored or in the additionally allowed regions of the ( Figure 2, right)is quite consistent with all theavailable experimental studies: the protonated nitrogen forms a salt Ramachandran plot; only one residue in the loop is located in negatively charged D3. 32, and the hydroxyl the disallowed region( Figure 1b), suggesting that the overall group is involved in the hydrogen bonds with Ser5. 42 and main chain and side chain conformations are reasonable Lys191(ECL2). The importance of conserved D3. 32 and S5.42 Besides, several structural features present in the 5-HT1aR for 5-HT binding to S-HTIAR was supported by a site-directed model are characteristic of active conformations of family a mutagenesis study. Additionally, F6.51 and F6.52are GPCRs: the broken Arg3.50-Glu630 ionic lock due to a large participated in the aromatic stacking with R-8-OH-DPAT separation of TM3-TM6 in the cytoplasmic ends and Tyr7.53 The two phenylalanine residues in TM6 contribute to of the NPxxY motif (so-called"Tyrosine Toggle Switch") hydrophobic interaction and were proved to be crucial for extending into the protein interior. Compared to previously ligand binding in the case of the dopamine D2 receptor. The eported S-HTIAR models which were built based on the crystal di-n-propyl substituent was found stretched in the hydrophobic dxdoLor/10. 1021/c400481p. Chem. Inf Model. 2013, 53, 3202-3211■ RESULTS AND DISCUSSION 3D Structure of the 5-HT1AR Model. Sequence alignment indicated that the sequence identity in the transmembrane region is ∼45% between 5-HT1AR and β2AR. The most recently disclosed crystal structure of the β2AR-Gs complex in active state (PDB Code: 3SN6) was the optimal template to construct the 5-HT1AR model. Therefore, it was expected that our model represents the active state of the receptor. Figure S1 shows the final sequence alignment of 5-HT1AR to the template. The PROCHECK statistics showed that 99.2% of the residues in the 5-HT1AR model (Figure 1a) were either in the most favored or in the additionally allowed regions of the Ramachandran plot; only one residue in the loop is located in the disallowed region (Figure 1b), suggesting that the overall main chain and side chain conformations are reasonable. Besides, several structural features present in the 5-HT1AR model are characteristic of active conformations of family A GPCRs:39 the broken Arg3.50-Glu6.30 ionic lock due to a large separation of TM3-TM6 in the cytoplasmic ends and Tyr7.53 of the NPxxY motif (so-called “Tyrosine Toggle Switch”) extending into the protein interior. Compared to previously reported 5-HT1AR models which were built based on the crystal structure of β2AR bound to the partial inverse agonist carazolol (PDB Code: 2RH1) or bovine rhodopsin (PDB Code: 1F88), our model was built taking the active state conformation of the β2AR-Gs complex as template and represents the active conformation binding with agonists. Thus it is suitable for the investigation of discovery of 5-HT1AR agonists. 8-OH-DPAT (Chart 1), a prototypical 5-HT1AR agonist, has been extensively used for pharmacological studies.40 R-8-OH￾DPAT displays higher efficacy than its S-counterpart and acts as a full 5-HT1AR agonist, thus it was chosen herein to induce active receptor conformations. The predicted binding mode (Figure 2, right) is quite consistent with all the available experimental studies: the protonated nitrogen forms a salt bridge with the negatively charged D3.32, and the hydroxyl group is involved in the hydrogen bonds with Ser5.42 and Lys191 (ECL2). The importance of conserved D3.32 and S5.42 for 5-HT binding to 5-HT1AR was supported by a site-directed mutagenesis study.41 Additionally, F6.51 and F6.52 are participated in the aromatic stacking with R-8-OH-DPAT. The two phenylalanine residues in TM6 contribute to hydrophobic interaction and were proved to be crucial for ligand binding in the case of the dopamine D2 receptor.42 The di-n-propyl substituent was found stretched in the hydrophobic Chart 1. Representative 5-HT1AR Agonists Figure 2. Side view of the R-8-OH-DPAT-5-HT1AR complex embedded in a hydrated POPC lipid bilayer (left). 5-HT1AR is shown in cartoon representation with R-8-OH-DPAT in the upper center. Water molecules and lipid molecules are shown in sticks; and choline N (blue), P atoms (orange) and Na+ (gray), CL− (green) ions are represented in spheres. The detail of the binding mode of R-8-OH-DPAT with 5-HT1AR is amplified (right). 5-HT1AR is shown in gray ribbons and R-8-OH-DPAT in green ball and sticks. Hydrogen bonds are depicted in dotted lines. Journal of Chemical Information and Modeling Article 3205 dx.doi.org/10.1021/ci400481p | J. Chem. Inf. Model. 2013, 53, 3202−3211