T Liu et aL/ Pharmacology, Biochemistry and Behavior 104(2013)138-143 were recorded and analyzed o 0 nM (Morris Water Maze Video Ana tem, DigBeh-MM, Shanghai ▲5.6nM Jiliang Software Technology Co. ) After the above trials, mice wer 720|·11,2nM decapitated within 5 min and the hippocampi were rapidly homoge ized at 3000 rpm within 5 min. The ache activity was measured as described above and protein concentration was quantified using BCA protein assay. 2.7. Statistical analysis Two-way ANOVA with repeated measures was used to analyze 三x目 latency values that were calculated as the mean latency periods for each mouse One-way ANOVA followed by post hoc Dunnett,s multi ole group comparison was used to analyze group differences in the data from probe trials and AChE activity assays. Data were analyzed using SAS version 8.1. p<0.05 was accepted as statistically significant. 3. Results /ASCh(1M×103 3. 1. Concentration-effect relationship for AChE inhibition in vitro Fig 3. Bis-Mep exhibited a mixed type competitive inhibition on electric eel AchE. Lineweaver-Burk double-reciprocal plot was expressed by recip f the initial enzyme velocity vs. ASCh concentration at different concentrations of Bis-Mep. The Concentration-effect curves showed that both(-)-meptazinol values were expressed as mean+SEM (n=3 independent exper nd Bis-Mep inhibited AChE activity in a concentration-dependent nanner(Fig. 2). Compared with(-)-meptazinol, a parallel shift to 3.3. "Water bridges"in the dual-binding on AChE the left was observed in concentration-effect curve of Bis-Mep which was consistent with our previous study(Xie et al 2008). In To provide insights into the enzyme-inhibitor interactions, we ddition, the shift indicated that the inhibition of AChE activity by rformed molecular docking study. As shown in Fig 4a, the active Bis-Mep was 10,000 times more than that by the monomer, possibly site of Torpedo californica(Pacific Electric Ray) acetylcholinesterase due to its binding to the Pas which augments the inhibition of AChE (TcAchE)consists of two subsites: CAS and PAS. The core of CAs is a activity(Du and Carlier, 2004). catalytic triad(Ser200, Glu327 and His440), where the hydrogen bond between Ser200 and His440 is dedicated to the stabilization 3. 2. Mixed type inhibition on electric eel AChE of the conformation. Two conserved residues(Trp84 and Phe330) The characteristics of AChE activity inhibition by Bis-Mep were nition. Trp279 is the main conserved residue of PAS. Our results revealed by the enzyme kinetics assay and analysis of linear transfor- showed that Bis-Mep could bind simultaneously at the CAs and mation of the Michaelis-Menten equation( Lineweaver-Burk)(Fig 3). PAS of AChE with the binding free energy of.0 kcal/mol. The The results showed that both the slopes and the intercept with the dual-binding induced the conformational changes of the residues. x(1/ASCh))and y(1/vmax) axes were increased with the increased As a result, the hydrogen bond between Ser200 and His440 was concentrations At concentrations of 5.6 and 11.2 nM. Bis-Mep elevated disrupted, although the backbone of Ache did not have significant the km value by 51% and 118%, respectively, and decreased the vmax by changes( RMSD: 0.256 A). 11% and 28%, respectively, consistent with the typical features of com- "Water bridges"have been proved to play an important role in the petitive inhibitor. binding mode. Two"water bridges"were located at the two wings of Bis-Mep to stabilize the interaction, and the contacts were 2.75A ·(-)- meptazinol (inside)and 2.40 A(outside)( Fig. 4b). As shown in Fig. 4c, one ()-meptazinol moiety was buried deep into the CAs to form T-IT Bis- Me (3.62 A)with the hydrophobic interactions with His400 and Phe330 The nearest hy- drophobic contacts were 3.40 A and 3.55 A. A hydrogen bond was also observed between the phenolic hydroxyl of Bis-Mep and the hydroxyl hydrogen of Tyr130 The distance between the hydrogen acceptor and donor was 2.53 A Stabilized by the outside"water e, another moiety of Bis-Mep extended to the outer PAs and ormed, and the nearest hydrophobic contact was 3.34A Ctions were ly tied to Trp279. Face-to-face hydrophol 3.4.An of Bis-Mep Two-way repeated analysis with mixed model showed latency to reach the platform was significantly different among the experimental groups of mice(F(5,263)=24.48, P<00001)and days log [AchElsI of acquisition training(F(3. 263)=20.85, p<00001)(Fig. 5a).There was no significant interaction between groups and days(F(15,263 Fig 2 Concentration-effect curves of AchE inhibitory activity. Inhibition of AchE ac- 0.6341, p=0.8457), suggesting that the differences among groups were plotted by the inhibition of AchE activity against the nat ntration. The valu expressed as mean±S &SEM((n=4 The mice in control group exhibited a progressive reduction of mean latency from 55.05 to 23 25 s over the course of 4 trainingwere recorded and analyzed with automated tracking software (Morris Water Maze Video Analysis System, DigBeh-MM, Shanghai Jiliang Software Technology Co.). After the above trials, mice were decapitated within 5 min and the hippocampi were rapidly homoge￾nized at 3000 rpm within 5 min. The AChE activity was measured as described above and protein concentration was quantified using BCA protein assay. 2.7. Statistical analysis Two-way ANOVA with repeated measures was used to analyze latency values that were calculated as the mean latency periods for each mouse. One-way ANOVA followed by post hoc Dunnett's multi￾ple group comparison was used to analyze group differences in the data from probe trials and AChE activity assays. Data were analyzed using SAS version 8.1. pb0.05 was accepted as statistically significant. 3. Results 3.1. Concentration–effect relationship for AChE inhibition in vitro Concentration–effect curves showed that both (−)-meptazinol and Bis-Mep inhibited AChE activity in a concentration-dependent manner (Fig. 2). Compared with (−)-meptazinol, a parallel shift to the left was observed in concentration–effect curve of Bis-Mep which was consistent with our previous study (Xie et al., 2008). In addition, the shift indicated that the inhibition of AChE activity by Bis-Mep was 10,000 times more than that by the monomer, possibly due to its binding to the PAS which augments the inhibition of AChE activity (Du and Carlier, 2004). 3.2. Mixed type inhibition on electric eel AChE The characteristics of AChE activity inhibition by Bis-Mep were revealed by the enzyme kinetics assay and analysis of linear transfor￾mation of the Michaelis–Menten equation (Lineweaver–Burk) (Fig. 3). The results showed that both the slopes and the intercept with the x (1/[ASCh]) and y (1/Vmax) axes were increased with the increased concentrations. At concentrations of 5.6 and 11.2 nM, Bis-Mep elevated the Km value by 51% and 118%, respectively, and decreased the Vmax by 11% and 28%, respectively, consistent with the typical features of com￾petitive inhibitor. 3.3. “Water bridges” in the dual-binding on AChE To provide insights into the enzyme–inhibitor interactions, we performed molecular docking study. As shown in Fig. 4a, the active site of Torpedo californica (Pacific Electric Ray) acetylcholinesterase (TcAChE) consists of two subsites: CAS and PAS. The core of CAS is a catalytic triad (Ser200, Glu327 and His440), where the hydrogen bond between Ser200 and His440 is dedicated to the stabilization of the conformation. Two conserved residues (Trp84 and Phe330) are adjacent to the catalytic triad, and participate in ligand recog￾nition. Trp279 is the main conserved residue of PAS. Our results showed that Bis-Mep could bind simultaneously at the CAS and PAS of AChE with the binding free energy of −94.0 kcal/mol. The dual-binding induced the conformational changes of the residues. As a result, the hydrogen bond between Ser200 and His440 was disrupted, although the backbone of AChE did not have significant changes (RMSD: 0.256 Å). “Water bridges” have been proved to play an important role in the binding mode. Two “water bridges” were located at the two wings of Bis-Mep to stabilize the interaction, and the contacts were 2.75 Å (inside) and 2.40 Å (outside) (Fig. 4b). As shown in Fig. 4c, one (–)-meptazinol moiety was buried deep into the CAS to form π–π interaction (3.62 Å) with the indole ring of Trp84 and edge-to-edge hydrophobic interactions with His400 and Phe330. The nearest hy￾drophobic contacts were 3.40 Å and 3.55 Å. A hydrogen bond was also observed between the phenolic hydroxyl of Bis-Mep and the hydroxyl hydrogen of Tyr130. The distance between the hydrogen bond acceptor and donor was 2.53 Å. Stabilized by the outside “water bridge”, another moiety of Bis-Mep extended to the outer PAS and closely tied to Trp279. Face-to-face hydrophobic interactions were formed, and the nearest hydrophobic contact was 3.34 Å. 3.4. Amelioration of Bis-Mep on Scop-induced cognitive deficits in mice Two-way repeated analysis with mixed model showed that the latency to reach the platform was significantly different among the experimental groups of mice (F(5,263)= 24.48, pb0.0001) and days of acquisition training (F(3,263)=20.85, pb0.0001) (Fig. 5a). There was no significant interaction between groups and days (F(15,263)= 0.6341, p= 0.8457), suggesting that the differences among groups were dependent on the treatment. The mice in control group exhibited a progressive reduction of mean latency from 55.05 to 23.25 s over the course of 4 training Fig. 2. Concentration–effect curves of AChE inhibitory activity. Inhibition of AChE ac￾tivity was expressed as the percentage of inhibition, and concentration–effect curves were plotted by the inhibition of AChE activity against the natural logarithm of the molar compound concentration. The values were expressed as mean± S.E.M. (n=4 independent experiments). Fig. 3. Bis-Mep exhibited a mixed type competitive inhibition on electric eel AChE. Lineweaver–Burk double-reciprocal plot was expressed by reciprocals of the initial enzyme velocity vs. ASCh concentration at different concentrations of Bis-Mep. The values were expressed as mean± S.E.M. (n=3 independent experiments). 140 T. Liu et al. / Pharmacology, Biochemistry and Behavior 104 (2013) 138–143