XIONG Zi-jun et al. reliable and reasonable order to be considered for further analysis and docking studies As a result, 371 compounds were identified by the model with 3.5 Database Searching and Candidate Inhibitors an average fit value of 2.367. In order to reduce false positive Identifying hits and nondrug- like compounds, several selection criteria Model shown in Fig. 6(A)was used to search Asinex Gold were used to filter out redundancies. An initial filtering was Collection database, which contains approximate 20000 performed according to Lipinski's Rule of Five by means of compounds, via the best flexible search method implemented in Pipeline Pilot software: molecular weight(Mw)<500, hydro- CATALYST module encoded Discovery Studio 3.0 program phobic constant Alog P<5, number of HBA<10, number HBD<5, and the number of rotatable bonds( RBsk10. As a The hit compounds from database were then fitted to the phar- result, the numbers of hits of the model reduced to 184.Then macophore model with CATALYST module encoded Discovery the scoring function G-Score, fit value, scaffold diversity and Studio 3.0 program. For each compound, a fit value was re- visual observation were considered generally. Finally, 7 hits turned that represented how well the compound fit to the phar- macophore model Compounds had to map the four features were selected and purchased for pharmacological assay. The ore mo fit value of more than 2.0 in relative drug-like properties of the 7 hits are listed in Table I Drug-like properties, scores and fit values of the top 7 compounds Compund II weight Number of HDs Number of HAs ALO Number of RB G-Score Fit value Awo1l19 431.86 3.891 8 47.9510 2.646 456.52 12 50.2230 2.909 305245768 42.7878 466.57 473983 01511644 2202 5686668 3.945 7 46.1974 2.462 03008103 12.49 7 51.2033 89l 01817599 479.53 2.217 3.6 Pharmacological Profiles compounds had potent inhibitory activity against 5-HT2A with inhibitory ratios above 80%. Among them, compound Our pharmacological test(Table 2)shows that tw 05245768 was the most potent with a percentage inhibition of Table 2 Biological test results of virtual screening hits 94.59% and a Ki value of (593.89*34.10) nmol/L. Fig.7 Compound shows the fitness of this compound to the pharmacophore rate(%)(nmol-L-) model and its fit value is 3.076, which verifies the rationality Awol119 X人心F of our pharmacophore model 47.2 HYDRI HYDR2 05245768 4.59593.89±34.10 01176572 28.6 HBD Fig 7 Compound 05245768 mapped with the pharmacophore model Pharmacophore features are colored the same as those in Fig. 6. l511644 12.24 3.7 Receptor-ligand Interaction Our docking studies suggest that there are three main im- portant interactions: salt bridge, hydrophobic and hydrogen ond interactions[Fig 8(A) bridge and hydrophobic 27.5 interactions are similar to those of ketanserin in the binding pocket of 5-HT2A. The salt bridge is found between the proto- nated nitrogen atom of the ligand and the carboxylate group of the conserved D3. 32 and the hydrophobic interaction is formed 0l817599 1981 between the benzopyrimidine ring and a hydrophobic cluster formed by w3. 28, 13.29 and F6.51. The cavity formed by the hydrophobic cluster in 05245768-5-HT2A complex is slightly larger than that in ketanserin-5-HT2A complex. CompoundNo.4 XIONG Zi-jun et al. 659 reliable and reasonable. 3.5 Database Searching and Candidate Inhibitors Identifying Model shown in Fig.6(A) was used to search Asinex Gold Collection database, which contains approximate 200000 compounds, via the best flexible search method implemented in CATALYST module encoded Discovery Studio 3.0 program[23]. The hit compounds from database were then fitted to the phar￾macophore model with CATALYST module encoded Discovery Studio 3.0 program. For each compound, a fit value was re￾turned that represented how well the compound fit to the phar￾macophore model. Compounds had to map the four features in the pharmacophore model with a fit value of more than 2.0 in order to be considered for further analysis and docking studies. As a result, 371 compounds were identified by the model with an average fit value of 2.367. In order to reduce false positive hits and nondrug-like compounds, several selection criteria were used to filter out redundancies. An initial filtering was performed according to Lipinski's Rule of Five by means of Pipeline Pilot software: molecular weight(MW)<500, hydro￾phobic constant AlogP<5, number of HBA<10, number of HBD<5, and the number of rotatable bonds(RBs)<10. As a result, the numbers of hits of the model reduced to 184. Then the scoring function G-Score, fit value, scaffold diversity and visual observation were considered generally. Finally, 7 hits were selected and purchased for pharmacological assay. The relative drug-like properties of the 7 hits are listed in Table 1. Table 1 Drug-like properties, scores and fit values of the top 7 compounds No. Compund ID Molecular weight Number of HDs Number of HAs ALogP Number of RBs G-Score Fit value 1 AW01119 431.86 1 5 3.891 8 47.9510 2.646 2 02987623 456.52 1 6 4.373 12 50.2230 2.909 3 05245768 409.48 2 8 2.925 7 42.7878 3.076 4 01176572 466.57 2 6 3.118 8 47.3983 2.718 5 01511644 449.34 0 6 3.945 7 46.1974 2.462 6 03008103 412.49 2 6 1.556 7 51.2033 2.891 7 01817599 479.53 1 8 2.217 9 53.4037 2.982 3.6 Pharmacological Profiles Our pharmacological test(Table 2) shows that two Table 2 Biological test results of virtual screening hits Compound ID Structure Inhibitory rate(%) Ki/ (nmol·L–1) AW01119 81.35 NT* 02987623 47.2 NT 05245768 94.59 593.89±34.10 01176572 28.6 NT 01511644 12.24 NT 03008103 27.5 NT 01817599 19.81 NT * NT: not tested. compounds had potent inhibitory activity against 5-HT2A with inhibitory ratios above 80%. Among them, compound 05245768 was the most potent with a percentage inhibition of 94.59% and a Ki value of (593.89±34.10) nmol/L. Fig.7 shows the fitness of this compound to the pharmacophore model and its fit value is 3.076, which verifies the rationality of our pharmacophore model. Fig.7 Compound 05245768 mapped with the pharmacophore model Pharmacophore features are colored the same as those in Fig.6. 3.7 Receptor-ligand Interaction Our docking studies suggest that there are three main im￾portant interactions: salt bridge, hydrophobic and hydrogen bond interactions[Fig.8(A)]. The salt bridge and hydrophobic interactions are similar to those of ketanserin in the binding pocket of 5-HT2A. The salt bridge is found between the proto￾nated nitrogen atom of the ligand and the carboxylate group of the conserved D3.32 and the hydrophobic interaction is formed between the benzopyrimidine ring and a hydrophobic cluster formed by W3.28, I3.29 and F6.51. The cavity formed by the hydrophobic cluster in 05245768-5-HT2A complex is slightly larger than that in ketanserin-5-HT2A complex. Compound