T Liu et al/ Pharmacology, Biochemistry and Behavior 104(2013)138-143 of AChE simultaneously have been shown to exhibit higher potency and 2. 4. Kinetic parameter selectivity for AChE inhibition than monomers, such as bis-tacrine (Rydberg et al, 2006)and bis-galanthamine(Greenblatt et al. 2004). To estimate the kinetic parameters Km(Michaelis constant) and In our previous studies, a series of Bis(-)-(-)-nor-meptazinols were Vmax(maximum velocity of reaction), the enzyme activity of electric designed and synthesized, in an effort to identify novel drug candidates eel AChE was determined at substrate(ASch) concentrations ranging nor-meptazinols(Bis-Mep, Fig. 1)connected by 9 alkane chain have lated by regression analysis of Lineweaver-Burk plots(1/velocite a a for AD by connecting two (-)-nor-meptazinol monomers with from 0.03 to 0. 17 mM in the presence of different concentration alkylene linkers of different lengths(Xie et al, 2008). Bis(9)-(-)- of inhibitors. The Km and Vmax values for AChE inhibition were calce been shown to have dual-binding on AChE and exhibited the highest 1/substrate) potency for AChE inhibition(ICso value of 3.9 nM). Bis-Mep also inhibited AChE induced AB aggregation, indicating its potential for AD therapy(Xie et al, 2008) 2.5. Molecular docking In the present study, we report the manner of Bis-Mep on AChE The X-ray crystallographic structure of AChE complex with a inhibition using the kinetic and molecular docking methods, and Bis(-)-(-)-nor-meptazinol derivative(PDB ID: 2W6C)was re- evaluated its effectiveness on cognitive amelioration on scopolamine- trieved from PDB. The binding pocket was predicted by MOE( Molec duced memory deficits and brain AChE inhibition in mice. lar Operating ment, Canada) and MOE's automatic docking algorithm was employed to dock Bis-Mep to binding pocket. Thirt 2. Materials and methods preferred poses were identified from conformation searches. These poses were then introduced into the pocket for energy minimization 2.1. Animals and binding evaluation, and the final binding mode was identified from the best ligand pose. Potential energy and ligand interaction Kunming mice at body weight of 18-22g(six weeks old, either gender) was calculated by MOE. vere purchased from Shanghai JiaoTong University School of Medidi YXK 2008-0050) Mice were maintained under standard conditior with a 12 h: 12 h light-dark cycle, a temperature and humidity controlled 2.6. Morris water maze task environment and ad-libitum access to food and water. The animal exper- imental protocol was approved by Institutional Animal Care and Use dures described previously(Zhang et al. 2008). Briefly, mice were Committee of ethics committees of Shanghai JiaoTong University School acclimatized to the experimental environments 2 weeks prior to the of Medicine(Approval number: 2010126), and carried out in accordance start of the study. The swimming pool(140 cm diameter)was placed with Guide for the Care and Use of Laboratory Animals(NRC 2011) in the middle of an experimental room(475 420 cm) containing a few of cues, including wall posters, blue window curtain, and a com- 22. Materials uter rack and the observer remained in the same location for each trial. a circular pool with black wall was filled with water to a depth Bis-Mep and(-)-meptazinol were synthesized in our laboratory. of 30 cm( consistently maintained at 22+1.0"C). The tank was di- Acetylthiocholine iodide(ASCh), eserine, 5. -dithio-bis-2-nitrobenzo vided into four equal quadrants, and a black platform(9 cm diameter) acid(DTNB). scopolamine(Scop)and electric eel AChE were purchased was submerged 1 cm below the water surface at the center of one from Sigma(St Louis, MO, USA). Huperzine A(Hup A)was obtained quadrant. The platform was invisible to the mice and remained in from Fudan Forward Science Technology Co(Shanghai). BCA protein one location for the entire study. A video camera was placed above ssay was obtained from Pierce(USA). Other reagents were analytical the centre of the pool to capture images of the swimming trace of grade and obtained from commercial source mice. Scop(1 mg/kg, ip. was administered 20 min before the test. Bis-Mep(10. 100 ng/kg or 1 Hg/kg) was administrated by subcutaneous injection(.c)120 min and Hup a(1 mg/kg) was intraperitoneally 2.3. Determination of AChE inhibitory activity (ip )administrated 20 min before the injection of Scop uring the acquisition testing phase, each mouse was given four AChE activity was detected according to a modified Ellman's method trials per day for four consecutive days, with 30 s interval between (Ellman et al, 1961 ).Briefly, mouse forebrain homogenates(1: 9 w/v in trials. The starting position was randomized over days but remained 0.05 M phosphate buffer solution)were pre-incubated with Bis-Mep the same order for all the mice. Each trial lasted either until the (10-1-10-4M)or()-meptazinol(10-8-10-2M)for 20 min at mouse had found the platform or for a maximum of 90 s.The 37Cin 0.05 M PBS(pH 7.2), containing 0.25 mM DTNB. The substrate, mouse that was unsuccessful within 90 s was guided to the platform 0.5 mM ASCh, was then quickly added. The reaction was terminated by At the end of each trial, the mice were allowed to rest on the platform 100 memory. Each mouse was allowed to search for 60 s. All the traces Fig. 1. Chemical structure of Bis(9)-(-)-nor-meptazinol(Bis-Mep.of AChE simultaneously have been shown to exhibit higher potency and selectivity for AChE inhibition than monomers, such as bis-tacrine (Rydberg et al., 2006) and bis-galanthamine (Greenblatt et al., 2004). In our previous studies, a series of Bis(−)-(−)-nor-meptazinols were designed and synthesized, in an effort to identify novel drug candidates for AD by connecting two (−)-nor-meptazinol monomers with alkylene linkers of different lengths (Xie et al., 2008). Bis(9)-(−)- nor-meptazinols (Bis-Mep, Fig. 1) connected by 9 alkane chain have been shown to have dual-binding on AChE and exhibited the highest potency for AChE inhibition (IC50 value of 3.9 nM). Bis-Mep also inhibited AChE induced Aβ aggregation, indicating its potential for AD therapy (Xie et al., 2008). In the present study, we report the manner of Bis-Mep on AChE inhibition using the kinetic and molecular docking methods, and evaluated its effectiveness on cognitive amelioration on scopolamine￾induced memory deficits and brain AChE inhibition in mice. 2. Materials and methods 2.1. Animals Kunming mice at body weight of 18–22 g (six weeks old, either gender) were purchased from Shanghai JiaoTong University School of Medicine (SYXK 2008-0050). Mice were maintained under standard conditions with a 12 h:12 h light–dark cycle, a temperature and humidity controlled environment and ad-libitum access to food and water. The animal exper￾imental protocol was approved by Institutional Animal Care and Use Committee of ethics committees of Shanghai JiaoTong University School of Medicine (Approval number: 2010126), and carried out in accordance with Guide for the Care and Use of Laboratory Animals (NRC 2011). 2.2. Materials Bis-Mep and (-)-meptazinol were synthesized in our laboratory. Acetylthiocholine iodide (ASCh), eserine, 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB), scopolamine (Scop) and electric eel AChE were purchased from Sigma (St Louis, MO, USA). Huperzine A (Hup A) was obtained from Fudan Forward Science & Technology Co (Shanghai). BCA protein assay was obtained from Pierce (USA). Other reagents were analytical grade and obtained from commercial sources. 2.3. Determination of AChE inhibitory activity AChE activity was detected according to a modified Ellman's method (Ellman et al., 1961). Briefly, mouse forebrain homogenates (1:9 w/v in 0.05 M phosphate buffer solution) were pre-incubated with Bis-Mep (10−11–10−4 M) or (−)-meptazinol (10−8 –10−2 M) for 20 min at 37 °C in 0.05 M PBS (pH 7.2), containing 0.25 mM DTNB. The substrate, 0.5 mM ASCh, was then quickly added. The reaction was terminated by the addition of 100 μL 1 mM eserine, and the color production was measured spectrophotometrically at 412 nm. The percent inhibition of AChE was determined by comparison with the control. 2.4. Kinetic parameters To estimate the kinetic parameters Km (Michaelis constant) and Vmax (maximum velocity of reaction), the enzyme activity of electric eel AChE was determined at substrate (ASCh) concentrations ranging from 0.03 to 0.17 mM in the presence of different concentrations of inhibitors. The Km and Vmax values for AChE inhibition were calcu￾lated by regression analysis of Lineweaver–Burk plots (1/velocity vs. 1/[substrate]). 2.5. Molecular docking The X-ray crystallographic structure of AChE complex with a Bis(−)-(−)-nor-meptazinol derivative (PDB ID: 2W6C) was re￾trieved from PDB. The binding pocket was predicted by MOE (Molec￾ular Operating Environment, Canada) and MOE's automatic docking algorithm was employed to dock Bis-Mep to binding pocket. Thirty preferred poses were identified from conformation searches. These poses were then introduced into the pocket for energy minimization and binding evaluation, and the final binding mode was identified from the best ligand pose. Potential energy and ligand interaction was calculated by MOE. 2.6. Morris water maze task The memory of mice was evaluated following our modified proce￾dures described previously (Zhang et al., 2008). Briefly, mice were acclimatized to the experimental environments 2 weeks prior to the start of the study. The swimming pool (140 cm diameter) was placed in the middle of an experimental room (475× 420 cm) containing a few of cues, including wall posters, blue window curtain, and a com￾puter rack. And the observer remained in the same location for each trial. A circular pool with black wall was filled with water to a depth of 30 cm (consistently maintained at 22±1.0 °C). The tank was di￾vided into four equal quadrants, and a black platform (9 cm diameter) was submerged 1 cm below the water surface at the center of one quadrant. The platform was invisible to the mice and remained in one location for the entire study. A video camera was placed above the centre of the pool to capture images of the swimming trace of mice. Scop (1 mg/kg, i.p.) was administered 20 min before the test. Bis-Mep (10, 100 ng/kg or 1 μg/kg) was administrated by subcutaneous injection (s.c.) 120 min and Hup A (1 mg/kg) was intraperitoneally (i.p.) administrated 20 min before the injection of Scop. During the acquisition testing phase, each mouse was given four trials per day for four consecutive days, with 30 s interval between trials. The starting position was randomized over days but remained the same order for all the mice. Each trial lasted either until the mouse had found the platform or for a maximum of 90 s. The mouse that was unsuccessful within 90 s was guided to the platform. At the end of each trial, the mice were allowed to rest on the platform for 30 s. On the 5th day, the platform was removed from the pool and a probe trial was used to examine the retention of spatial reference memory. Each mouse was allowed to search for 60 s. All the traces Fig. 1. Chemical structure of Bis(9)-(−)-nor-meptazinol (Bis-Mep). T. Liu et al. / Pharmacology, Biochemistry and Behavior 104 (2013) 138–143 139