XIONG Zi-jun et al. GF/B glass fiber filter paper and washing was performed 3 model of 5-HT2A receptor was assembled taking the X-ray times with 3 mL of eluent(50 mmol/L Tris-HCL, pH=7.7), dry- crystal structure of B2-adrenergic receptor as a template. Step- ing was conducted in the microwave oven for 8-9 min, then wise energy minimizations of the homology 5-HT2A model was the filter paper was transferred into a 0.5 mL centrifuge tube, to carried out for 500 steps with Kollman All-Atom force field hich 500 uL of soluble scintillation fluid was then added. and the SYBYL program. The final model was verified by After 30 min of rest in darkness, the radioactivity was mea- professional structure validation program Procheck 3.5.4. The sured. Then pharmacological parameters: percentage inhibition, Procheck statistics shows that 96.1% of the residues of the ICso and Ki were calculated. Concentrations were measured 5-HT2A model were either in the most favored or in the addi every two tubes, two independent tests of each compound were tionally allowed regions of the Ramachandran map(Fig. 2) conducted suggesting that the overall main chain and side chain struc- The inhibitor was docked into the 5-ht2a receptor via the tures were all reasonable flexible docking program GOLD 4.12. Before docking, the (A) final conformation of the ligand was obtained after 1000 steps of energy minimization via the Tripos force field and optimiza tion at the dFT/B3LYP/6-311G level by means of Gaussian 2009/201 Instead of the default 10600 genetic algorithm(GA) runs were performed to fully take into account the flexibility of the ligand. For each GA run, the default Ga settings were used with no early termination allowed and internal ligand energy offset turned on. Finally, GOLD-Score was used as the fitness function 3 Results and discussion Fig2 3D model of 5-HT2A receptor(A)and ramchandran 3.1 3D Structure of 5-HT2A Receptor The different colored areas indicate disallowed( white), generously allowed (light yellow), additional allowed(yellow)and most favored(red)regions Sequence alignment( Fig. 1)indicates that the sequence entity and similarity are 39.0% and 62. 4%, for the TMs be TM-I TM-2 B2-Adrenerg IVLATFCNLVITATAKEI HIOEKUSAL TAWIILTI Ar EL-I TM-3 EL-2 Fig3 Binding pocket of 5-HT2A model obtained B2-Adrer 5-HT2A TKAFL from CAsTp program 3.2 Active Site of 5-HT, Model TM-5 IL-3 TM-6 The active site recognized by Castp program is shown in B2-Adrenergic Fig 3, which is in good agreement with the available mutation experiments of 5-HT2A and other GPCRs 21 22).The conserved TM-7 active site is surrounded by TM-3, TM-5, TM-6 and TM-7 and OISGNPLIECR covered by EL-2. A hydrophobic cluster, composed of residues 5-HT2A KESCNEDVIGALLNPVVICYLSSASLVYTL w6.48. F6.51 and F6.52 is located at the active site and Fig1 Sequence alignments of p2-adrenergic(2RHI) 3.3 Antagonistic Conformation of 5-HT2A with 5-HT2A generated by Clustalw The asterisk(*) indicates strict identity;a In order to obtain the antagonistic conformation of similarity, the do.)indicates a semi-conserved similarity(sequence identity 5-HT2A, the well-known antagonist ketanserin, which has been is 39.0%, similarity is 62. 4% in the TM region between 5-HT2A and p2-adr). shown to have high affinity for 5-HT2a in an amount of availa- The most conserved residues X 50 for each TM are marked by a black star ble mutation experiments, was docked into the active site of under the sequences. The seven transmembrane helices( TM), extracellular loops(EL) and intracellular loops(IL) are marked above the sequences. 5-hT2a by the advanced docking program GOLD 4. 12, which tween B2-adrenergic receptor and 5-HT2A receptor. Based on considers the flexibility of both receptor and ligand. The key the high homology revealed by sequence alignments, the 3D residues of 5-HT2A involved in the interaction between 5-HT2ANo.4 XIONG Zi-jun et al. 657 GF/B glass fiber filter paper and washing was performed 3 times with 3 mL of eluent(50 mmol/L Tris-HCl, pH=7.7), dry￾ing was conducted in the microwave oven for 8―9 min, then the filter paper was transferred into a 0.5 mL centrifuge tube, to which 500 μL of soluble scintillation fluid was then added. After 30 min of rest in darkness, the radioactivity was mea￾sured. Then pharmacological parameters: percentage inhibition, IC50 and Ki were calculated. Concentrations were measured every two tubes, two independent tests of each compound were conducted. The inhibitor was docked into the 5-HT2A receptor via the flexible docking program GOLD 4.12. Before docking, the final conformation of the ligand was obtained after 1000 steps of energy minimization via the Tripos force field and optimiza￾tion at the DFT/B3LYP/6-311G level by means of Gaussian 2009[20]. Instead of the default 10600 genetic algorithm(GA) runs were performed to fully take into account the flexibility of the ligand. For each GA run, the default GA settings were used with no early termination allowed and internal ligand energy offset turned on. Finally, GOLD-Score was used as the fitness function. 3 Results and Discussion 3.1 3D Structure of 5-HT2A Receptor Sequence alignment(Fig.1) indicates that the sequence identity and similarity are 39.0% and 62.4%, for the TMs be￾Fig.1 Sequence alignments of β2-adrenergic(2RH1) with 5-HT2A generated by ClustalW The asterisk(*) indicates strict identity; a colon(:) indicates a conserved similarity; the dot(.) indicates a semi-conserved similarity(sequence identity is 39.0%, similarity is 62.4% in the TM region between 5-HT2A and β2-adr). The most conserved residues X.50 for each TM are marked by a black star under the sequences. The seven transmembrane helices(TM), extracellular loops(EL) and intracellular loops(IL) are marked above the sequences. tween β2-adrenergic receptor and 5-HT2A receptor. Based on the high homology revealed by sequence alignments, the 3D model of 5-HT2A receptor was assembled taking the X-ray crystal structure of β2-adrenergic receptor as a template. Step￾wise energy minimizations of the homology 5-HT2A model was carried out for 500 steps with Kollman All-Atom force field and the SYBYL program. The final model was verified by a professional structure validation program Procheck 3.5.4. The Procheck statistics shows that 96.1% of the residues of the 5-HT2A model were either in the most favored or in the addi￾tionally allowed regions of the Ramachandran map(Fig.2), suggesting that the overall main chain and side chain struc￾tures were all reasonable. Fig.2 3D model of 5-HT2A receptor(A) and Ramchandran plots of 5-HT2A model(B) The different colored areas indicate disallowed(white), generously allowed (light yellow), additional allowed(yellow) and most favored(red) regions. Fig.3 Binding pocket of 5-HT2A model obtained from CASTp program 3.2 Active Site of 5-HT2A Model The active site recognized by CASTp program is shown in Fig.3, which is in good agreement with the available mutation experiments of 5-HT2A and other GPCRs[21,22]. The conserved active site is surrounded by TM-3, TM-5, TM-6 and TM-7 and covered by EL-2. A hydrophobic cluster, composed of residues W6.48, F6.51 and F6.52, is located at the active site and sup￾posed to participate in a coordinated conformational change. 3.3 Antagonistic Conformation of 5-HT2A In order to obtain the antagonistic conformation of 5-HT2A, the well-known antagonist ketanserin, which has been shown to have high affinity for 5-HT2A in an amount of availa￾ble mutation experiments, was docked into the active site of 5-HT2A by the advanced docking program GOLD 4.12, which considers the flexibility of both receptor and ligand. The key residues of 5-HT2A involved in the interaction between 5-HT2A