Bis(9)-(−)-nor-meptazinol as a novel dual-binding AChEI potently ameliorates scopolamine-induced cognitive deficits in mice

Pharmacology, Biochemistry and Behavior 104(2013)138-143 Contents lists available at SciVerse Science Direct Pharmacology Biochemistry and behavior ELSEVIER journalhomepagewww.elsevier.com/locate/pharmbiochembeh Bis(9)-(-)-nor-meptazinol as a novel dual-binding AChEl potently ameliorates scopolamine-induced cognitive deficits in mice Ting Liu a, d, Zheng Xia a, Wei-Wei Zhang 1, Jian-rong Xu, Xin-Xing Ge a, Juan Li a, Yongyao Cui a, Zhui- Bai Qiu, Jun Xu, Qiong Xie*, Hao Wang…*, Hong-Zhuan Chen己常米 ent of pharmacology, Institute of Medical Sciences, Shanghai jiao Tong University School of medicine, PR China ent of Medicinal Chemistry, School of pharmacy, Fudan University, PR China Center for Drug Discovery, School of pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, PR China ent of pharmacy, Ren Hospital, Shanghai Jiao Tong University School of Medicine, PR China ARTICLE INFO A BSTRACT lzheimer's disease(AD)is a multifaceted neurodegenerative disorder which is characterized by the progres- brary 2012 ive deterioration of cognition and the emergence of behavioral and psychological symptoms in aging in revised form 18 November 2012 Accepted 22 November 2012 patients. Given that the clinical effectiveness of acetylcholinesterase inhibitors(AChEls)has still been Available online 19 December 2012 questioned due to dubious disease-modifying effects, the multi-target directed ligand(MTDL) design has become an emerging strategy for developing new drugs for AD treatment. Bis(9)-(-)-nor-meptazinol (Bis-Mep)was firstly reported by us as a novel MTDL for both potent cholinesterase and amyloid-B aggrega- tion inhibition. In this study, we further explored its AChE inhibition kinetic features and cognitive ameliora tion. Bis-Mep was found to be a mixed-type inhibitor on electric eel AChE by enzyme kinetic study. Molecular ual-binding achE inhibitor ocking revealed that two"water bridges"located at the two wings of Bis-Mep stabilized its interaction with Cognitive meliora both catalytic and peripheral anionic sites of AChE. Furthermore, subcutaneous administration of Bis-Mep(10. 100 or 1000 ng/kg)significantly reversed the scopolamine-induced memory deficits in a typical bell-shaped onse manner. The maximal cognitive amelioration of Bis-Mep was achieved at 100 ng/kg, comparable with the effect of a reference drug Huperzine A at 1 mg/kg and also the relevant AChE inhibition in brain. These findings suggested that Bis-Mep might be a promising dual-binding AChE inhibitor for potential AD therapeutics. o 2012 Elsevier Inc. All rights reserved. 1 Introduction contributes to cognition decline. Therefore, acetylcholinesterase inhibi- tors(AChEls). which inhibit catalytic anionic site(CAS)and increase Alzheimer's disease(AD)is a multifaceted neurodegenerative dis- the availability of acetylcholine(Ach) in synaptic cleft, have been wide- order which is characterized by the progressive deterioration of coms Gauthier, 2010). AChEIs including donepezil galamantine, rivastigmine nition and the emergence of behavioral and psychological sympt in aging patients. Although senile plaques and neurofibrillary tangles and tacrine have been approved by FDa for AD patients to ameliorate are generally accepted as the hallmarks of AD, cholinergic deficiency their cognitive deficits. However, the clinical effectiveness of AchEls has still been questioned due to dubious disease-modifying effects (Small, 2005: Terry and Buccafusco, 2003). Given the complex multifactorial etiology of AD, it is generally AD,Alzheimer's disease:BBB, the blood brain barrier: Bis-Mep. Bis(9)-(-)-nor- accepted that a multi-target therapeutic approach is necessary eptazinol; CAS, catalytic anionic site of AChE; PAS, peripheral anionic site of AChE:( Cavalli et aL, 2008: Lee et al, 2010). Drug discovery for AD, following multi-target directed ligand(MTDL) design, gradually focused on Corresponding authors at: Departmentof Pharmacology, Institute of Medical Sciences, modulating multiple targets implicated in AD pathogenesis. Notably, oTon University School of Medicine, 280 South Chongqing Road, Shanghai, the peripheral anionic site of AChE which is located at the active Medicinal Chemistry, School of Pharmacy, Fudan University, $26 Zhangheng road, Shang center gorge entry, has been shown to be associated with amyloid-B 201203, PR China. tel:+862151980122;fax+862151980122 (AB)aggregation(Munoz-Torrero, 2008). Thus, dual-binding AChEls Correspondence to: Prof Hongzhuan Chen, Department of Pharmacology, Institute which can simultaneously bind to the CAs and PAS of Ache, have going Road, Shanghai.200025, PR China.Tel:+862164398859;fax+862164392916. been proposed to be an advanced strategy to ameliorate symptoms -mailaddresses:xiejoanxq@gmail.com(Q,Xie),angela_wanghaoehotm and disease progression of AD(Bourne et al, 2003). H.Wang),hongzhuan_chen@hotmail.com(h.-zChen). Recently, some dimeric AChEls in which two moieties linked Contributed equally to this work by a suitable length chain can interact with the CAs and the pas 0091-3057/S- see front matter a 2012 Elsevier Inc. All rights reserved htp:/ dxdolorg/10.1016/pb2012.11009

Bis(9)-(−)-nor-meptazinol as a novel dual-binding AChEI potently ameliorates scopolamine-induced cognitive deficits in mice Ting Liu a,d,1 , Zheng Xia a,1 , Wei-Wei Zhang a,1 , Jian-rong Xu a , Xin-Xing Ge a , Juan Li a , Yongyao Cui a , Zhui-Bai Qiu b , Jun Xu c , Qiong Xie b, ⁎, Hao Wang a, ⁎, Hong-Zhuan Chen a, ⁎, ⁎⁎ a Department of Pharmacology, Institute of Medical Sciences, Shanghai JiaoTong University School of Medicine, PR China b Department of Medicinal Chemistry, School of Pharmacy, Fudan University, PR China c Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, PR China d Department of Pharmacy, Renji Hospital, Shanghai JiaoTong University School of Medicine, PR China article info abstract Article history: Received 10 February 2012 Received in revised form 18 November 2012 Accepted 22 November 2012 Available online 19 December 2012 Keywords: Bis-Mep MTDL Dual-binding AChE inhibitor Cognitive amelioration Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder which is characterized by the progres￾sive deterioration of cognition and the emergence of behavioral and psychological symptoms in aging patients. Given that the clinical effectiveness of acetylcholinesterase inhibitors (AChEIs) has still been questioned due to dubious disease-modifying effects, the multi-target directed ligand (MTDL) design has become an emerging strategy for developing new drugs for AD treatment. Bis(9)-(−)-nor-meptazinol (Bis-Mep) was firstly reported by us as a novel MTDL for both potent cholinesterase and amyloid-β aggrega￾tion inhibition. In this study, we further explored its AChE inhibition kinetic features and cognitive ameliora￾tion. Bis-Mep was found to be a mixed-type inhibitor on electric eel AChE by enzyme kinetic study. Molecular docking revealed that two “water bridges” located at the two wings of Bis-Mep stabilized its interaction with both catalytic and peripheral anionic sites of AChE. Furthermore, subcutaneous administration of Bis-Mep (10, 100 or 1000 ng/kg) significantly reversed the scopolamine-induced memory deficits in a typical bell-shaped dose–response manner. The maximal cognitive amelioration of Bis-Mep was achieved at 100 ng/kg, comparable with the effect of a reference drug Huperzine A at 1 mg/kg and also the relevant AChE inhibition in brain. These findings suggested that Bis-Mep might be a promising dual-binding AChE inhibitor for potential AD therapeutics. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Alzheimer's disease (AD) is a multifaceted neurodegenerative dis￾order which is characterized by the progressive deterioration of cog￾nition and the emergence of behavioral and psychological symptoms in aging patients. Although senile plaques and neurofibrillary tangles are generally accepted as the hallmarks of AD, cholinergic deficiency contributes to cognition decline. Therefore, acetylcholinesterase inhibi￾tors (AChEIs), which inhibit catalytic anionic site (CAS) and increase the availability of acetylcholine (ACh) in synaptic cleft, have been wide￾ly used for the treatment of mild to moderate AD (Massoud and Gauthier, 2010). AChEIs including donepezil, galamantine, rivastigmine and tacrine have been approved by FDA for AD patients to ameliorate their cognitive deficits. However, the clinical effectiveness of AChEIs has still been questioned due to dubious disease-modifying effects (Small, 2005; Terry and Buccafusco, 2003). Given the complex multifactorial etiology of AD, it is generally accepted that a multi-target therapeutic approach is necessary (Cavalli et al., 2008; Lee et al., 2010). Drug discovery for AD, following multi-target directed ligand (MTDL) design, gradually focused on modulating multiple targets implicated in AD pathogenesis. Notably, the peripheral anionic site of AChE which is located at the active center gorge entry, has been shown to be associated with amyloid-β (Aβ) aggregation (Muñoz-Torrero, 2008). Thus, dual-binding AChEIs, which can simultaneously bind to the CAS and PAS of AChE, have been proposed to be an advanced strategy to ameliorate symptoms and disease progression of AD (Bourne et al., 2003). Recently, some dimeric AChEIs in which two moieties linked by a suitable length chain can interact with the CAS and the PAS Pharmacology, Biochemistry and Behavior 104 (2013) 138–143 Abbreviations: AChE, acetylcholinesterase; AChEIs, acetylcholinesterase inhibitors; AD, Alzheimer's disease; BBB, the blood brain barrier; Bis-Mep, Bis(9)-(−)-nor￾meptazinol; CAS, catalytic anionic site of AChE; PAS, peripheral anionic site of AChE; Scop, scopolamine; Hup A, Huperzine A. ⁎ Corresponding authors at: Department of Pharmacology, Institute of Medical Sciences, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, PR China. Tel.: +86 21 63846590; fax: +86 21 64392916; Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China. Tel.: +86 21 51980122; fax: +86 21 51980122. ⁎⁎ Correspondence to: Prof. Hongzhuan Chen, Department of Pharmacology, Institute of Medical Sciences, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, PR China. Tel.: +86 21 64398859; fax: +86 21 64392916. E-mail addresses: xiejoanxq@gmail.com (Q. Xie), angela_wanghao@hotmail.com (H. Wang), hongzhuan_chen@hotmail.com (H.-Z. Chen). 1 Contributed equally to this work. 0091-3057/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2012.11.009 Contents lists available at SciVerse ScienceDirect Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh

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

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 training

were 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

T Liu et al/ Pharmacology, Biochemistry and Behavior 104(2013)138-143 14 days. Scop-treated mice displayed significantly longer mean latency from 83. 27 s to 63.76 s over training days(p<0.01), indicating that cognitive impairment had been induced by Scop. Subcutaneous RP279 administration of Bis-Mep to Scop-treated mice resulted in signifi cant reduction of mean latencies at doses ranging from 10 ng/kg to 1 ug/kg with no obvious cholinergic adverse responses. The maximal reduction in mean latency was reached at the 100 ng/kg, and the per formance of the mice approached nearly that of the control mice (p>0.05), indicating the complete reversal of Scop-induced cognitive deficits In the probe trial, the percentage of the total distance and spent in the target quadrant was used to evaluate the spatial reten- tion One-way ANOVA analysis showed that the percentage of dis- tance and time was significantly different among the experimental groups(F(5)=7.755.p<0001,Fg5 b and f(1.5)=7465,p<0001 Fig 5c). Compared with the control group, Scop caused 52.5% and HS440 48.7% decrease in the percentage of the total distance and time, re- spectively. Bis-Mep at the dose of 100 ng/kg significantly ameliorated the cognitive deficits as shown by increased percentage of distance (55.6%)and time( 48. 2%)compared to Scop-treated mice As expected, Hup A, a positive control, significantly reversed cognitive impairment by shortening latencies(p<0.05)and increasing the percentage of the total distance(60.6%)and time(48.6%)(p<0.05) compared to Scop-treated mice Furthermore, the cognitive amelioration was obvi- ously observed in the swimming traces on the 5th day after the admin- tration of Bis-Mep( Fig 5d). Taken together, these results demonstrate that Bis-Mep ameliorates Scop-induced cognitive deficits in mice. 3.5. Bis-Mep inhibits Ache activity in mice hippocampus Compared with the control mice, Scop exposure had no obvious effect on AChE activity in hippocampus (p>0.05, Table 1). However, AChE activity in hippocampus was significantly inhibited by 22.0% (p<0.05) and 230%(p<0.05)after the administration of Bis-Mep (100 ng/kg) and Hup A(1 mg/kg), respectively. These data suggested that Bis-Mep would be capable of crossing the blood brain barrier BB)and inhibiting AChE activity in brain. 4. Discussion The neuropathology in AD patients is characterized by a progres ive loss of memory and cognitive abilities(Walsh and Selkoe, 2004). AChEls which can simultaneously bind to the CAs and pas have been shown to be an effective therapeutic approach for mild to moderate Ad by enhancing cholinergic activity as well as preventing .TRP 279 ChE induced aB aggregation( Castro and Martinez, 2006). Therefore, dual-binding AChEls becomes one of the most widely adopted ap- proaches to hit multiple AD biological targets for AD therapy Another challenge for AD therapy, similar to diabetes and psychi- atric illnesses, is treatment compliance. It is known that the non- compliant behavior has been often observed in older AD patients due to the misunderstanding of complicated multiple medication or the confusion with the dosing regimen or forgetfulness. New means of drug administration should be developed and aimed to solve this difficulty. For example, the rivastigmine patch has been approved for Fig. 4. Water bridges"in the dual-binding to AChE. Bis-Mep was docked into AChE at 10E. Bis-Mep was shown dual-site binding to AChE at CAS, the catalytic triad of which of Bis-Mep to stabilize the binding to CAS and PAS, where Hydrogen bond (green dotted arrow )and n-n interaction(green dotted line) were also observed(b). The lual-binding of Bis-Mep to AchE induced the conformational changes of key residu in CAS and PAS (Green residues: before docking: Red residues: after docking), where

days. Scop-treated mice displayed significantly longer mean latency from 83.27 s to 63.76 s over training days (pb0.01), indicating that cognitive impairment had been induced by Scop. Subcutaneous administration of Bis-Mep to Scop-treated mice resulted in signifi- cant reduction of mean latencies at doses ranging from 10 ng/kg to 1 μg/kg with no obvious cholinergic adverse responses. The maximal reduction in mean latency was reached at the 100 ng/kg, and the per￾formance of the mice approached nearly that of the control mice (p> 0.05), indicating the complete reversal of Scop-induced cognitive deficits. In the probe trial, the percentage of the total distance and time spent in the target quadrant was used to evaluate the spatial reten￾tion. One-way ANOVA analysis showed that the percentage of dis￾tance and time was significantly different among the experimental groups (F(71,5)= 7.755, pb0.001, Fig. 5b and F(71,5)= 7.465, pb0.001, Fig. 5c). Compared with the control group, Scop caused 52.5% and 48.7% decrease in the percentage of the total distance and time, re￾spectively. Bis-Mep at the dose of 100 ng/kg significantly ameliorated the cognitive deficits as shown by increased percentage of distance (55.6%) and time (48.2%) compared to Scop-treated mice. As expected, Hup A, a positive control, significantly reversed cognitive impairment by shortening latencies (pb0.05) and increasing the percentage of the total distance (60.6%) and time (48.6%) (pb0.05) compared to Scop-treated mice. Furthermore, the cognitive amelioration was obvi￾ously observed in the swimming traces on the 5th day after the admin￾istration of Bis-Mep (Fig. 5d). Taken together, these results demonstrate that Bis-Mep ameliorates Scop-induced cognitive deficits in mice. 3.5. Bis-Mep inhibits AChE activity in mice hippocampus Compared with the control mice, Scop exposure had no obvious effect on AChE activity in hippocampus (p>0.05, Table 1). However, AChE activity in hippocampus was significantly inhibited by 22.0% (pb0.05) and 23.0% (pb0.05) after the administration of Bis-Mep (100 ng/kg) and Hup A (1 mg/kg), respectively. These data suggested that Bis-Mep would be capable of crossing the blood brain barrier (BBB) and inhibiting AChE activity in brain. 4. Discussion The neuropathology in AD patients is characterized by a progres￾sive loss of memory and cognitive abilities (Walsh and Selkoe, 2004). AChEIs which can simultaneously bind to the CAS and PAS have been shown to be an effective therapeutic approach for mild to moderate AD by enhancing cholinergic activity as well as preventing AChE induced Aβ aggregation (Castro and Martinez, 2006). Therefore, dual-binding AChEIs becomes one of the most widely adopted ap￾proaches to hit multiple AD biological targets for AD therapy. Another challenge for AD therapy, similar to diabetes and psychi￾atric illnesses, is treatment compliance. It is known that the non￾compliant behavior has been often observed in older AD patients due to the misunderstanding of complicated multiple medication or the confusion with the dosing regimen or forgetfulness. New means of drug administration should be developed and aimed to solve this difficulty. For example, the rivastigmine patch has been approved for Fig. 4. “Water bridges” in the dual-binding to AChE. Bis-Mep was docked into AChE at MOE. Bis-Mep was shown dual-site binding to AChE at CAS, the catalytic triad of which is formed by Ser200, Glu327 and His440; and PAS, the conserved residue of which is Trp279 (a). “Water bridges” (brown dotted line) were revealed in two wings of Bis-Mep to stabilize the binding to CAS and PAS, where Hydrogen bond (green dotted arrow) and π–π interaction (green dotted line) were also observed (b). The dual-binding of Bis-Mep to AChE induced the conformational changes of key residues in CAS and PAS (Green residues: before docking; Red residues: after docking), where the hydrogen bond between Ser200 and His440 (dotted line) was disrupted (c). T. Liu et al. / Pharmacology, Biochemistry and Behavior 104 (2013) 138–143 141

T Liu et aL/ Pharmacology, Biochemistry and Behavior 104(2013)138-143 Table Ex vivo inhibition of Bis-Mep on AchE activity. Bis-Mep(sc )inhibited AChE activity in hippocampus. AChE inhibitory activity in hippocampus was assayed by a modified Ellman's method Data are expressed as mean+ SD for 10 mice each group AChE activity (U/mg protein) Control Scop(1 mg/kg) Hup A (l mg/kg)+ Sco 0831±0205 p<0.05, one way ANOVA followed by Dunnett's multiple comparison test vs the (b) up to 85.6%. Bis-Mep reached peak plasma concentration rapidly after subcutaneous administration (Tmax=6.6 min), and the t1/2 of Bis-Mep was 18.6 h due to its extensive distribution and slow clearance (Ge et al. 2012). The results provided us advantageous in formation to develop Bis-Mep to be a subcutaneous implant for long time therapy. Therefore, in the current study, Bis-Mep was designed to be administrated by subcutaneous injection in mice, then its effects on the cognitive amelioration and ache activity in hippocampus were evaluated In the present study, the concentration-effect relationship for AChE inhibition and the kinetic features of Bis-Mep were firstly determined.(-)-Meptazinol which is split from race-meptazinol (c) hasshownsomeAcheinhibitoryactivity(shietal.,2005).com- pared with (-)-meptazinol, Bis-Mep was shifted leftward in the concentration-effect curve of ache inhibition the results indicated hat Bis-Mep enhanced its effectiveness greatly, possibly due to its binding to the pas which could promote the inhibition of AChE tivity(Du and Carlier, 2004). bino tt always shows mixed-type inhibition when the inhibitors can to either the free enzyme or the enzyme-substrate complex. As for AChE, those mixed-type inhibitors are in favor of the interac tion with CAS and PAS Donepezil is a typical mixed-type inhibitor with high affinity to AChE in the nanomolar range. Structural biolog ical studies has revealed that donepezil could bind simultaneously to the CAs and PAs (Kryger et al, 1999), indicating its potential benefits for severe Ad patients(Farlow et al, 2010: Tsuno, 2009). In this study, Lineweaver-Burk analysis showed that Bis-Mep inhibited AChE in a linear mixed fashion. These results indicate that Bis-Mep another mixed-type inhibitor that inhibits AChE either by binding on free AChE or on AChE-ASCh complex, which may explain its potent inhibition of AChE. According to others and our reports, the potency of Bis-Mep is higher than denepezil in inhibiting AChE-induced AB aggregation. Inhibition of denepezil-induced AB aggregation at 100 HM was 22%, and those of Bis-Mep was 90.8%(Bartolini et al, 2003: Xie et al., 2008). Compared with those of donepezil, Bis-Mep may exert preferential binding to PAS which is mainly responsible for AB aggregation. To explore the mode of dual-binding of Bis-Mep to AChe,we F performed molecular docking which revealed that the methylene groups of Bis-Mep played a key role in the mixed type inhibition Fig. 5. Bis-Mep ameliorated Scop-induced cognitive deficits in mice. (a)The effects of and its dual-binding to CAS and PAS on AChE. The distance between Bis-Mep(sc)on the latency to the platform during the 4 days of acquisition testing. CAS and PAS is about 14 A. Therefore, the length of the methylene (b)The effects of Bis-Mep(sc)on the distance in target quadrant. (c) The effects of groups should be long enough to cover the distance. The flexible Bis-Mep(sc)on the time spent in target quadrant(d) Swimming traces for ditterent methylene groups are beneficial for the dual site binding of Bis-Mep (10 ng/kg)+Scop: C: Bis-Mep(100 ng/kg)+Scop: D: Bis-Mep(1000 ng/g)+Scop: to AChE. Unfortunately, this flexibility also facilitates the transforma- E:Hup A(1 mg/kg)+Scop: and F: Scop(1 mg/kg)alone. Data were expressed as tion of linear conformation of the molecule to spherical conformation, mean+ SD for 12 mice in each group. p<0.05. p<0.01 vs. Scop-treated group. which could be stabilized by the intramolecular hydrogen bonds between two phenolic hydroxyl groups of the two(-)-meptazinol the long-term treatment of AD patients with severe dementia(Frolich, moieties To compare it with the linear conformation, large volume 2008). Similar to the patch, implants are advantageous in long-time of the spherical conformation makes it more difficult to dock into treatment since they not only avoid liver and gastrointestinal the binding pocket of AChE and then to interact with CAS and PAs. first-pass effects but also relieve the frequent taking of pills. The plas- especially the former one. Therefore, the inhibition against AChE ma pharmacokinetic study of Bis-Mep in rat after subcutaneous ad- would decrease, when the proportion of spherical conformation ministration showed that the absolute bioavailability of Bis-Mep was increases. For example, the inhibition of Bis(6)-(-)-meptazinol wa

the long-term treatment of AD patients with severe dementia (Frölich, 2008). Similar to the patch, implants are advantageous in long-time treatment since they not only avoid liver and gastrointestinal first-pass effects but also relieve the frequent taking of pills. The plas￾ma pharmacokinetic study of Bis-Mep in rat after subcutaneous ad￾ministration showed that the absolute bioavailability of Bis-Mep was up to 85.6%. Bis-Mep reached peak plasma concentration rapidly after subcutaneous administration (Tmax= 6.6 min), and the T1/2 of Bis-Mep was 18.6 h due to its extensive distribution and slow clearance (Ge et al., 2012). The results provided us advantageous in￾formation to develop Bis-Mep to be a subcutaneous implant for long time therapy. Therefore, in the current study, Bis-Mep was designed to be administrated by subcutaneous injection in mice, then its effects on the cognitive amelioration and AChE activity in hippocampus were evaluated. In the present study, the concentration–effect relationship for AChE inhibition and the kinetic features of Bis-Mep were firstly determined. (−)-Meptazinol which is split from race-meptazinol has shown some AChE inhibitory activity (Shi et al., 2005). Com￾pared with (−)-meptazinol, Bis-Mep was shifted leftward in the concentration–effect curve of AChE inhibition. The results indicated that Bis-Mep enhanced its effectiveness greatly, possibly due to its binding to the PAS which could promote the inhibition of AChE ac￾tivity (Du and Carlier, 2004). It always shows mixed-type inhibition when the inhibitors can bind to either the free enzyme or the enzyme–substrate complex. As for AChE, those mixed-type inhibitors are in favor of the interac￾tion with CAS and PAS. Donepezil is a typical mixed-type inhibitor with high affinity to AChE in the nanomolar range. Structural biolog￾ical studies has revealed that donepezil could bind simultaneously to the CAS and PAS (Kryger et al., 1999), indicating its potential benefits for severe AD patients (Farlow et al., 2010; Tsuno, 2009). In this study, Lineweaver–Burk analysis showed that Bis-Mep inhibited AChE in a linear mixed fashion. These results indicate that Bis-Mep is another mixed-type inhibitor that inhibits AChE either by binding on free AChE or on AChE-ASCh complex, which may explain its potent inhibition of AChE. According to other's and our reports, the potency of Bis-Mep is higher than denepezil in inhibiting AChE-induced Aβ aggregation. Inhibition of denepezil-induced Aβ aggregation at 100 μM was 22%, and those of Bis-Mep was 90.8% (Bartolini et al., 2003; Xie et al., 2008). Compared with those of donepezil, Bis-Mep may exert preferential binding to PAS which is mainly responsible for Aβ aggregation. To explore the mode of dual-binding of Bis-Mep to AChE, we performed molecular docking which revealed that the methylene groups of Bis-Mep played a key role in the mixed type inhibition and its dual-binding to CAS and PAS on AChE. The distance between CAS and PAS is about 14 Å. Therefore, the length of the methylene groups should be long enough to cover the distance. The flexible methylene groups are beneficial for the dual site binding of Bis-Mep to AChE. Unfortunately, this flexibility also facilitates the transforma￾tion of linear conformation of the molecule to spherical conformation, which could be stabilized by the intramolecular hydrogen bonds between two phenolic hydroxyl groups of the two (–)-meptazinol moieties. To compare it with the linear conformation, large volume of the spherical conformation makes it more difficult to dock into the binding pocket of AChE and then to interact with CAS and PAS, especially the former one. Therefore, the inhibition against AChE would decrease, when the proportion of spherical conformation increases. For example, the inhibition of Bis(6)-(−)-meptazinol was Fig. 5. Bis-Mep ameliorated Scop-induced cognitive deficits in mice. (a) The effects of Bis-Mep (s.c.) on the latency to the platform during the 4 days of acquisition testing. (b) The effects of Bis-Mep (s.c.) on the distance in target quadrant. (c) The effects of Bis-Mep (s.c.) on the time spent in target quadrant. (d) Swimming traces for different groups were presented on the 5th day of Morris Water Maze test. A: Control; B: Bis-Mep (10 ng/kg)+Scop; C: Bis-Mep (100 ng/kg)+Scop; D: Bis-Mep (1000 ng/kg)+ Scop; E: Hup A (1 mg/kg)+ Scop; and F: Scop (1 mg/kg) alone. Data were expressed as mean± SD for 12 mice in each group. ⁎pb0.05, ⁎⁎pb0.01 vs. Scop-treated group. Table 1 Ex vivo inhibition of Bis-Mep on AChE activity. Bis-Mep (s.c.) inhibited AChE activity in hippocampus. AChE inhibitory activity in hippocampus was assayed by a modified Ellman's method. Data are expressed as mean± SD for 10 mice each group. Group AChE activity (U/mg protein) % Scop Control 0.996±0.151 92% Scop (1 mg/kg) 1.079±0.234 100% Bis-Mep (100 ng/kg)+Scop 0.842±0.203⁎ 78% Hup A (1 mg/kg)+ Scop 0.831±0.205⁎ 77% ⁎ pb0.05, one way ANOVA followed by Dunnett's multiple comparison test vs the Scop. 142 T. Liu et al. / Pharmacology, Biochemistry and Behavior 104 (2013) 138–143