658 CHEM RES CHINESE UNIVERSITIES VoL 27 and ketanserin are shown in Fig 4, which indicates that ketan- serin mainly makes contacts with the residues in TM-3, TM-7 and EL-2. Closer inspection of this docking study reveals that ketanserin binds to 5-hT2a by three main interactions. The quinazolinedione fragment penetrates into the hydrophobic pocket formed by TM-6 and TM-7, it is in close contact with Y7. 43 and F6.51. The fluorine atom is halogen bonded to the hydroxyl group of S5.43. However, the most important and conserved interaction remains a salt bridge between the proto- nated nitrogen of the piperidine ring and the carboxy late group of D3. 32. Our docking result is in agreement with most site directed mutagenesis experiments/ 21 22), confirming the accura- Fig 5 Positively charged cluster detected by y of our model. The obtained antagonistic conformation of positive probe NI The red sphere represent cluster of positive probe NI, it forms electrostatic 5-HT2A will be used in the building of pharmacophore model. "sandwich"" interaction with the side chains of D3.32 and F6.51 S5.46. Two big hydrophobic clusters were detected by probe DRY pocket formed by residues F6.51 and F6.52 at the active site and the other is close to hydrophobic residues of 13.29 and Leu228. These hydro- phobic features are thus chosen since they can significantly stabilize the complex. A small negatively charged cluster was omitted due to two reasons: ) the interaction energy with the S5.43 residues at the active site of protein is relatively high, (ID) the binding pocket of the protein has a tremendous negative poten- 743 tial, it repulses the negatively charged ligand and prevents entrance. Finally, a pharmacophore model was generated( Fig. 6) It contains one positive ionizable group, two hydrophobic groups, and one HBd group. As shown in Fig. 6(B), the dis- tances from positive ionizable group, which locates in the cen- ter of the active site. to the hbd. hydri. and hydra are 0.560, 0.832 and 0.997 nm, respectively. They distribute dif- ferent sites at the active site. It shows that our model is quite Fig 4 Structure of ketanserin(A)and the interaction node between 5-HT2A receptor and its anta- gonist ketanserin(B) The key residues involved in the interactions are shown. The hydrogen bonds are shown by dashed lines, ketanserin is represented by sticks and TEOHHYDR2 the key residues if they are the active site of 5-HT2A are shown in stick 3.4 Pharmacophore Model generation HBD The program GRiD 22 was used to generate an interaction ap of the active site residues in the antagonistic conformation with five different probes(DRY, COo, NI,O and NI)ex- 46HBI plained in the methods section. It resulted in a total of 5 inte raction maps. For each probe used, the resulting combined map was overlaid on the active site of 5-HT2A. This helped to iden model, a strategy was used to select probes that would com- plement essential conserved groups at the active site The essential residues at the active site of 5-HT2A are Fig 6 Four-feature pharmacophore model with 6 ex- conserved S5.43 and S5.46. The D3. 32 is essential for the clusion volumes(A)and pharmacophore model binding of the protonated amino group of ligand, S5.43 and at the active site of 5-HT2A(B) S5. 46 may form the conserved interaction with the hydroxyl(A) The hydrophobic features are shown in cya group of the ligand A positively charged cluster was detected sizable feature in red color, the hydrogen bond donor feature in purple by positive probe NI, and it formed electrostatic"sandwich" color and the exclusion volumes in grey;(B)5-HT2A is shown in fanc interaction with the side chains of D3.32 and F6.51(Fig. 5). The cartoon representation. The hydrophobic features(HYDRI and HYDR2)are shown as cyan color meshes, the positive ionizable feature(POS)as a red HBd cluster was detected by o probe and it formed hydrogen mesh, and the HBD as purple mesh, and the key residues if they are at the ond interaction with the hydroxyl group of the side chain of active site of 5-HTzA are shown in stick. The unit of the distances is nm658 CHEM. RES. CHINESE UNIVERSITIES Vol.27 and ketanserin are shown in Fig.4, which indicates that ketan￾serin mainly makes contacts with the residues in TM-3, TM-7 and EL-2. Closer inspection of this docking study reveals that ketanserin binds to 5-HT2A by three main interactions. The quinazolinedione fragment penetrates into the hydrophobic pocket formed by TM-6 and TM-7, it is in close contact with Y7.43 and F6.51. The fluorine atom is halogen bonded to the hydroxyl group of S5.43. However, the most important and conserved interaction remains a salt bridge between the proto￾nated nitrogen of the piperidine ring and the carboxylate group of D3.32. Our docking result is in agreement with most site￾directed mutagenesis experiments[21,22], confirming the accura￾cy of our model. The obtained antagonistic conformation of 5-HT2A will be used in the building of pharmacophore model. Fig.4 Structure of ketanserin(A) and the interaction mode between 5-HT2A receptor and its anta￾gonist ketanserin(B) The key residues involved in the interactions are shown. The hydrogen bonds are shown by dashed lines, ketanserin is represented by sticks and the key residues if they are the active site of 5-HT2A are shown in stick. 3.4 Pharmacophore Model Generation The program GRID 22 was used to generate an interaction map of the active site residues in the antagonistic conformation with five different probes(DRY, COO– , N1+ , O and N1) ex￾plained in the methods section. It resulted in a total of 5 inte￾raction maps. For each probe used, the resulting combined map was overlaid on the active site of 5-HT2A. This helped to iden￾tify the conserved positions of each probe. In developing the model, a strategy was used to select probes that would com￾plement essential conserved groups at the active site. The essential residues at the active site of 5-HT2A are conserved S5.43 and S5.46. The D3.32 is essential for the binding of the protonated amino group of ligand, S5.43 and S5.46 may form the conserved interaction with the hydroxyl group of the ligand. A positively charged cluster was detected by positive probe N1+ , and it formed electrostatic “sandwich” interaction with the side chains of D3.32 and F6.51(Fig.5). The HBD cluster was detected by O probe and it formed hydrogen bond interaction with the hydroxyl group of the side chain of Fig.5 Positively charged cluster detected by positive probe N1+ The red sphere represent cluster of positive probe N1+ , it forms electrostatic “sandwich” interaction with the side chains of D3.32 and F6.51. S5.46. Two big hydrophobic clusters were detected by probe DRY. One is close to the hydrophobic pocket formed by residues F6.51 and F6.52 at the active site and the other is close to hydrophobic residues of I3.29 and Leu228. These hydro￾phobic features are thus chosen since they can significantly stabilize the complex. A small negatively charged cluster was omitted due to two reasons: (I) the interaction energy with the residues at the active site of protein is relatively high; (II) the binding pocket of the protein has a tremendous negative poten￾tial, it repulses the negatively charged ligand and prevents its entrance. Finally, a pharmacophore model was generated(Fig.6). It contains one positive ionizable group, two hydrophobic groups, and one HBD group. As shown in Fig.6(B), the dis￾tances from positive ionizable group, which locates in the cen￾ter of the active site, to the HBD, HYDR1, and HYDR2 are 0.560, 0.832 and 0.997 nm, respectively. They distribute dif￾ferent sites at the active site. It shows that our model is quite Fig.6 Four-feature pharmacophore model with 6 ex￾clusion volumes(A) and pharmacophore model at the active site of 5-HT2A(B) (A) The hydrophobic features are shown in cyan color, the positive(POS) ionizable feature in red color, the hydrogen bond donor feature in purple color and the exclusion volumes in grey; (B) 5-HT2A is shown in fancy cartoon representation. The hydrophobic features(HYDR1 and HYDR2) are shown as cyan color meshes, the positive ionizable feature(POS) as a red mesh, and the HBD as purple mesh, and the key residues if they are at the active site of 5-HT2A are shown in stick. The unit of the distances is nm