CHEM. RES CHINESE UNIVERSITIES VoL 27 05245768 differs with ketanserin in that its hydroxyl tail forms molecular docking, it guided the optimization of the ligand by a hydrogen bond with $5.46 instead of the aforemen tioned enhancing the lipophilicity of its aromatic ring. More com- halogen bond between the fluorine atom and $5.43. This result pounds as designed will be purchased or synthesized for fur- is consistent well with previous studies 24,251. From the docking ther biological test to discover more effective inhibitors of result, we can predict that in order to keep or increase the acti- 5-HT2A receptor. In this study, we developed a pharmaco- vity, the protonated nitrogen atom of the ligand is necessary for phore based virtual screening method by integrating a set of the electrostatic interaction, the hydroxyl group should also be computational approaches and acological test. The reserved for the hydrogen bond interaction, with regard to results described in this paper that this method is phenyl ring, it can be substituted by lipophilic groups such as very efficient for identifying novel bioactive agents. This methyl, isopropyl, phenyl and halogen for stronger hydropho- workflow can also be applied in the study of hit identification bic interaction with the hydrophobic pocket formed by w3.28, and lead optimization. 13.29 and Leu228[ Fig 8(B) (A) Reference [2] Horacek J, Pharmacopsychiatry, 2000, 33(Suppl. 1), 34 [3] Aghajanian G. K, Marek G. J, Brain. Res. Rev., 2000, 31(2/3), [4] Reynolds G. P, J Psychopharmacol., 2004, 18(3), 340 F [5] Meltzer H. Y, Matsubara S, Lee J C.,J. Pharmacol. Exp. Thera- peat,1989,25/(1),238 [6] Huang E. S, Protein. Sci., 2003, 12(7), 1360 [7] Dunn M. F, Handbook of Proteins, 2007, 2, 678 18] Cherezov v, Rosenbaum D M, Hanson M. A, Rasmussen S. G F, Thian F. S, Kobilka T. S, Choi H. J, Kuhn P, Weis I. W ian K. K, et al, Science, 2007, 318(5854), 1258 [9] Accelrys Software, Discovery Studio 3.0 Edition, San Diego, CA, 0] Ballesteros J. A, Weinstein H, Meth. Neurosci [11] Sali A, Blundell T. L, J. Mol. Biol, 1993, 234(3), [12] Fiser A, Sali A,, Meth. Eneymol, 2003, 374, 46 Fig 8 Binding modes of compound 05245768 with 13 Pearson W.R., Meth. Ensymol, 1990, 183, 63 . hT2A(A)and hydrophobic pocket around [14 Thompson J. D, Higgins D G, Gibson T J, Nucl. Acids. Res. the benzyl ring of compound 05245768 formed 1994,2(22),4673 by w3.28, 13.29 and Leu228(B) [15] SYBYL Manual, Tripos Inc, 2001 The key residues involved in the interactions are shown. The salt bridge [16] Laskowski R. A, Mac Arthur M. W, Moss D. S, Thornton J. M, J. and hydrogen bond are shown by dashed lines, compound 05245768 is Appl. Crystallogr, 1993, 26(2),283 presented by sticks and the key residues are shown in stick if they are [17 Jones G, Willett P, Glen R. C, Leach A. R, Taylor R, J. Mol. Bi- the active site of 5-HT2A. The unit of the distances is nm. ol,1995,245,43 4 Conclusions [18 Binkowski T. A, Naghibzadeh S, Liang J, Nucl. Acids. Res, 2003, 3l(13),3352 We reported herein the 3D structure of 5-HT2A receptor [19] Leach C, Br J. Clin. Psychol., 1988, 27( Part 2), 173 with the newly resolved high resolution X-ray structure of [20] Frisch M. J, Trucks G. w,Schlegel H B, Scuseria G. E, Robb M B2-adrenergic receptor as template. The structure was verified A, Cheeseman J. R, Scalmani G, Barone V, Mennucci B, Pe- G al. Gaussian 09. Revision AI. Gaussian Inc model was developed based on the grid calculation in which allingford CT. 2009 five types of chemical probes ctive site 21] Shapiro D. A, Kristiansen K, Kroeze W.K., Roth B. L, Mol and the cluster analyses were performed to select the phar- Pharmacol.,2000,58(5),877 122 Wurch T, Palmier C, Pauwels P J, Biochem. Pharmacol., 2000 for the gold collection of asinex database which contains ca 5909),1l17 200000 molecules. The combination of Lipinski's Rule of 23] Bao H J, Tang Y L, XuX. J, Xiang J F, Zheng Z H, Lu X.H., Five with fit value and G-Score was used to filter redundan- Chem. J. Chinese Universities, 2010, 31(50,938 cies and false positives. Finally, 7 molecules were obtained [24 Bruno A, Guadix A. E, Costantino G, J. Chem. Inf Model, 2009 and purchased for the biological binding assay. A potent 496),1602 5-hT2A inhibitor 05245768 with a Ki value of (593.89*34.10) 5] Dezi C, Brea J, Alvarado M, Ravina E, Masaguer C. F, Loza mol/L was found I, Sanz F, Pastor M J. Med. Chem., 2007, 50(14), 3242 reasonable receptor-ligand revealed by660 CHEM. RES. CHINESE UNIVERSITIES Vol.27 05245768 differs with ketanserin in that its hydroxyl tail forms a hydrogen bond with S5.46 instead of the aforemen tioned halogen bond between the fluorine atom and S5.43. This result is consistent well with previous studies[24,25]. From the docking result, we can predict that in order to keep or increase the acti￾vity, the protonated nitrogen atom of the ligand is necessary for the electrostatic interaction, the hydroxyl group should also be reserved for the hydrogen bond interaction, with regard to the phenyl ring, it can be substituted by lipophilic groups such as methyl, isopropyl, phenyl and halogen for stronger hydropho￾bic interaction with the hydrophobic pocket formed by W3.28, I3.29 and Leu228[Fig.8(B)]. Fig.8 Binding modes of compound 05245768 with 5-HT2A(A) and hydrophobic pocket around the benzyl ring of compound 05245768 formed by W3.28, I3.29 and Leu228(B) The key residues involved in the interactions are shown. The salt bridge and hydrogen bond are shown by dashed lines, compound 05245768 is represented by sticks and the key residues are shown in stick if they are the active site of 5-HT2A. The unit of the distances is nm. 4 Conclusions We reported herein the 3D structure of 5-HT2A receptor with the newly resolved high resolution X-ray structure of β2-adrenergic receptor as template. The structure was verified by a professional program Procheck 3.5.4. A pharmacophore model was developed based on the grid calculation in which five types of chemical probes were used to map the active site and the cluster analyses were performed to select the phar￾macophore feature. The obtained model was used to search for the Gold collection of Asinex Database which contains ca. 200000 molecules. The combination of Lipinski’s Rule of Five with fit value and G-Score was used to filter redundan￾cies and false positives. Finally, 7 molecules were obtained and purchased for the biological binding assay. A potent 5-HT2A inhibitor 05245768 with a Ki value of (593.89±34.10) nmol/L was found. A reasonable receptor-ligand interaction was revealed by molecular docking, it guided the optimization of the ligand by enhancing the lipophilicity of its aromatic ring. More com￾pounds as designed will be purchased or synthesized for fur￾ther biological test to discover more effective inhibitors of 5-HT2A receptor. In this study, we developed a pharmaco￾phore based virtual screening method by integrating a set of computational approaches and pharmacological test. The results described in this paper indicate that this method is very efficient for identifying novel bioactive agents. This workflow can also be applied in the study of hit identification and lead optimization. Reference [1] Abi-Dargham A., Laruelle M., Eur. Psychiatr., 2005, 20(1), 15 [2] Horacek J., Pharmacopsychiatry, 2000, 33(Suppl. 1), 34 [3] Aghajanian G. K., Marek G. J., Brain. Res. Rev., 2000, 31(2/3), 302 [4] Reynolds G. P., J. Psychopharmacol., 2004, 18(3), 340 [5] Meltzer H. Y., Matsubara S., Lee J. C., J. Pharmacol. Exp. Thera￾peut., 1989, 251(1), 238 [6] Huang E. S., Protein. Sci., 2003, 12(7), 1360 [7] Dunn M. F., Handbook of Proteins, 2007, 2, 678 [8] Cherezov V., Rosenbaum D. M., Hanson M. A., Rasmussen S. G. F., Thian F. S., Kobilka T. S., Choi H. J., Kuhn P., Weis I. W., Brian K. K., et al., Science, 2007, 318(5854), 1258 [9] Accelrys Software, Discovery Studio 3.0 Edition, San Diego, CA, 2010 [10] Ballesteros J. A., Weinstein H., Meth. Neurosci., 1995, 25, 366 [11] Sali A., Blundell T. L., J. Mol. Biol., 1993, 234(3), 779 [12] Fiser A., Sali A., Meth. Enzymol., 2003, 374, 461 [13] Pearson W. R., Meth. Enzymol., 1990, 183, 63 [14] Thompson J. D., Higgins D. G., Gibson T. J., Nucl. Acids. Res., 1994, 22(22), 4673 [15] SYBYL Manual, Tripos Inc., 2001 [16] Laskowski R. A., MacArthur M. W., Moss D. S., Thornton J. M., J. Appl. Crystallogr., 1993, 26(2), 283 [17] Jones G., Willett P., Glen R. C., Leach A. R., Taylor R., J. Mol. Bi￾ol., 1995, 245, 43 [18] Binkowski T. A., Naghibzadeh S., Liang J., Nucl. Acids. Res., 2003, 31(13), 3352 [19] Leach C., Br. J. Clin. Psychol., 1988, 27( Part 2), 173 [20] Frisch M. J., Trucks G. W., Schlegel H. B., Scuseria G. E., Robb M. A., Cheeseman J. R., Scalmani G., Barone V., Mennucci B., Pe￾tersson G. A., et al., Gaussian 09, Revision A.1., Gaussian Inc., Wallingford CT, 2009 [21] Shapiro D. A., Kristiansen K., Kroeze W. K., Roth B. L., Mol. Pharmacol., 2000, 58(5), 877 [22] Wurch T., Palmier C., Pauwels P. J., Biochem. Pharmacol., 2000, 59(9), 1117 [23] Bao H. J., Tang Y. L., Xu X. J., Xiang J. F., Zheng Z. H., Lu X. H., Chem. J. Chinese Universities, 2010, 31(5), 938 [24] Bruno A., Guadix A. E., Costantino G., J. Chem. Inf. Model., 2009, 49(6), 1602 [25] Dezi C., Brea J., Alvarado M., Raviña E., Masaguer C. F., Loza M. I., Sanz F., Pastor M., J. Med. Chem., 2007, 50(14), 3242