The Crystal Structure of a Complex of Acetylcholinesterase with a Bis-(-)- nor-meptazinol Derivative Reveals Disruption of the Catalytic Triad

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J.Med.Chem.2009,52,2543-2549 The Crystal Structure of a Complex of Acetylcholinesterase with a Bis-()-nor-meptazinol Derivative Reveals Disruption of the Catalytic Triad Aviv Paz, .+ Qiong Xie, .Harry M. Greenblatt, t Wei Fu,' Yun Tang, Israel Silman, Zhuibai Qiu, and Joel L Sussman* T d Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel, Department of medicinai Chemistry, School of Pharmacy, Fudan University, Shanghai 200032, P. R. China, School of Pharmacy, East China University of Science and Technology, Shanghai 200237 P.R. China Received December 31. 2008 A bis-()-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and aB peptide aggregation The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 A resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the"anionic subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440N, which is hydrogen-bonded to Ser2000 in the native enzyme. The second nor-meptazinol binds at the peripheral anionic"site at the gorge entrance. A number of GOLD models of the complex, using both native TCAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors The enzyme acetylcholinesterase(AChE") terminates im transmission at central and peripheral cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine(ACh). It has served as a therapeutic target for the symptomatic treatment of Alzheimer's disease(AD) due to the cholinergic ypothesis, which argues that the cognitive decrements observed in AD patients are associated with impairment of cholinergic Figure 1. Chemical structure of 5h transmission. The hypothesis thus predicts that inhibition of AChE should prolong the effect of the neurotransmitter ACh, Table 1. Data Collection and Refinement Statistics resulting in partial restoration of the cognitive abilities of the P121 a=112.17 patients. As a consequence, the first generation of AD drugs cell parameters(A) were all acetylcholinesterase inhibitors(AChEIs), and four are c=137.57 currently used extensively, including the synthetic compound molecules/asymmetric unit 2020 and rivastigmine and the alkaloids galanthamine'and 40-2.7 al reflections 338424 AChE has been demonstrated to colocalize with the amyloid-P unique reflections 993%099.76) peptide(AB) in the brains of AD patients, and has been shown av lo(n) 17.5(3.55) to accelerate the assembly of AB to amyloid fibrils. Several (% ligands that bind at the peripheral anionic site(PAS)of AChE 18.5 have been shown to retard aggregation. Furthermore, a 23.5(5.2% of data) 0.019 monoclonal antibody directed against the PAs also reversed the rmsd, bond angles(deg effect of AChE on AB deposition. In the active-site gorge of PDB code 2W6C AChE the "anionic" subsite of the active site. also known as Values in parenthe the catalytic"anionic"site( CAS), is located at the bottom of A). Rmerge=2ll-( ) V2 I, where I is the observed intensity, and()is the gorge, and the PAS is near its entrance. 4. I This led to the the average intensity obtained from multiple observations of symmetry. development of the bivalent ligand approach in which identical related reflections after rejections.“R=∑F。-uFM∑ where F and Fc are the observed and calculated structure factors, respectively. As Fax: 972-8-934-4159. E-mail: joel. sussman(@ weizmann. acil or distinct pharmacophores are linked, via an appropriate spacer, Department of Structural Biology, Weizmann Institute of Science to produce a bifunctional drug with enhanced affinity. This Department of Neurobiology, Weizmann Institute of Science in turn, proved valuable for developing drugs with dual action s Department of Medicinal Chemistry, School of Pharmacy, Fudan both in inhibition of catalytic activity and in arresting the Ache- School of Pharmacy, East China University of Science and Technology catalyzed assembly of amyloid fibrils(for recent reviews see These authors made equal contribut refs 17 and 18) yloid-B peptide: ACh, acetylcholine: AChE, Xie and co-workers recently reported the synthesis and acetylcholinesterase: AChEL, acetylcholinesterase inhibitor, AD, Alzheimer's characterization of novel bis-(-)-nor-meptazinols that inhibit house: MEP,(-)-meptazinol; PAS, peripheral anionic site; Tc, Torpedo both AChE and butyrylcholinesterase (BChE) as well as californica: a, axial; e, equatorial tarding AB aggregation % The most potent of these compounds l0.102lm801657vCCC:S40 c 2009 American Chemical Society Web03/27/2009

The Crystal Structure of a Complex of Acetylcholinesterase with a Bis-(-)-nor-meptazinol Derivative Reveals Disruption of the Catalytic Triad Aviv Paz,†,‡,⊥ Qiong Xie,§,⊥ Harry M. Greenblatt,† Wei Fu,§ Yun Tang,| Israel Silman,‡ Zhuibai Qiu,§ and Joel L. Sussman*,† Departments of Structural Biology and Neurobiology, Weizmann Institute of Science, RehoVot 76100, Israel, Department of Medicinal Chemistry, School of Pharmacy, Fudan UniVersity, Shanghai 200032, P.R. China, School of Pharmacy, East China UniVersity of Science and Technology, Shanghai 200237 P.R. China ReceiVed December 31, 2008 A bis-(-)-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and A peptide aggregation. The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 Å resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the “anionic” subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440Nε2 , which is hydrogen-bonded to Ser200Oγ in the native enzyme. The second nor-meptazinol binds at the peripheral “anionic” site at the gorge entrance. A number of GOLD models of the complex, using both native TcAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure. These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors. Introduction The enzyme acetylcholinesterase (AChEa ) terminates impulse transmission at central and peripheral cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh).1 It has served as a therapeutic target for the symptomatic treatment of Alzheimer’s disease (AD) due to the cholinergic hypothesis, which argues that the cognitive decrements observed in AD patients are associated with impairment of cholinergic transmission.2,3 The hypothesis thus predicts that inhibition of AChE should prolong the effect of the neurotransmitter ACh, resulting in partial restoration of the cognitive abilities of the patients. As a consequence, the first generation of AD drugs were all acetylcholinesterase inhibitors (AChEIs),4 and four are currently used extensively, including the synthetic compounds E20205 and rivastigmine6 and the alkaloids galanthamine7 and (-)-huperzine A.8 AChE has been demonstrated to colocalize with the amyloid- peptide (A) in the brains of AD patients,9 and has been shown to accelerate the assembly of A to amyloid fibrils.10 Several ligands that bind at the peripheral anionic site (PAS) of AChE have been shown to retard aggregation.11,12 Furthermore, a monoclonal antibody directed against the PAS also reversed the effect of AChE on A deposition.13 In the active-site gorge of AChE, the “anionic” subsite of the active site, also known as the catalytic “anionic” site (CAS), is located at the bottom of the gorge, and the PAS is near its entrance.14,15 This led to the development of the bivalent ligand approach in which identical or distinct pharmacophores are linked, via an appropriate spacer, to produce a bifunctional drug with enhanced affinity.16 This, in turn, proved valuable for developing drugs with dual action both in inhibition of catalytic activity and in arresting the AChEcatalyzed assembly of amyloid fibrils (for recent reviews see refs 17 and 18). Xie and co-workers recently reported the synthesis and characterization of novel bis-(-)-nor-meptazinols that inhibit both AChE and butyrylcholinesterase (BChE) as well as retarding A aggregation.19 The most potent of these compounds * To whom correspondence should be addressed. Phone: 972-8-934-4531. Fax: 972-8-934-4159. E-mail:joel.sussman@weizmann.ac.il. † Department of Structural Biology, Weizmann Institute of Science. ‡ Department of Neurobiology, Weizmann Institute of Science. § Department of Medicinal Chemistry, School of Pharmacy, Fudan University. | School of Pharmacy, East China University of Science and Technology. ⊥ These authors made equal contributions. a Abbreviations: A, amyloid- peptide; ACh, acetylcholine; AChE, acetylcholinesterase; AChEI, acetylcholinesterase inhibitor; AD, Alzheimer’s disease; BuChE, butyrylcholinesterase; CAS, catalytic anionic site; m, mouse; MEP, (-)-meptazinol; PAS, peripheral anionic site; Tc, Torpedo californica; a, axial; e, equatorial. Figure 1. Chemical structure of 5h. Table 1. Data Collection and Refinement Statistics space group P3121 cell parameters (Å) a ) 112.17 b ) 112.17 c ) 137.57 molecules/asymmetric unit 1 resolution (Å) 40-2.7 total reflections 338424 unique reflections 26746 completeness (%)a 99.93% (99.76) av I/σ(I) 17.5 (3.55) Rmerge (%)b 11.7 R (%)c 18.5 Rfree (%)d 23.5 (5.2% of data) rmsd, bond lengths (Å) 0.019 rmsd, bond angles (deg) 2.1 PDB code 2W6C a Values in parentheses relate to the highest resolution shell (2.8-2.7 Å). b Rmerge ) ∑ |I - 〈I〉|/∑ I, where I is the observed intensity, and 〈I〉 is the average intensity obtained from multiple observations of symmetryrelated reflections after rejections. c R ) ∑ ||Fo| - ||Fc||/∑ |Fo|, where Fo and Fc are the observed and calculated structure factors, respectively. d As defined by ref 38. J. Med. Chem. 2009, 52, 2543–2549 2543 10.1021/jm801657v CCC: $40.75  2009 American Chemical Society Published on Web 03/27/2009

2544 Journal of Medicinal Chemistry, 2009, VoL. 52, No 8 Paz et al B Trp279 Tyr70 Ty Tyr334 Trp84 His44 Figure 2. Two representations of the refined structure of the 5h/TcAChE complex.(A) TcAChE is displayed as a beige space-filling surface and sh within the active-site gorge as a magenta stick model. The arrow marks the entrance to the active-site gorge.(B)5h shown as a green stick nodel. and the side chains of selected active- site residues with which it makes contact as red stick models was a homodimer with a nine-carbon spacer (5h)(Figure 1). which displayed ICso values of 3.9+ 1.3 and 10.0+ 3.0 nM CAS GOLD docking simulations of 5h, in which the phenyl groups of both MEP moieties were in equatorial orientations with he azapane rings(ecep), indicated that 5h spans the ctive-site gorge, interacting with both the CAS and PAs of mAChE. The phenyl group of the MEP moiety at the CAs was predicted to make a face-to-face T-stacking interaction wit Trp86(mAChE numbering, Trp84 in TcAChE). The meP The MEP moiety at the Cas and the nonamethylene linker displ moiety at the Pas predicted to make catie almost complete electron density, whereas the MEP moiety at the PAs hydrophobic interactions of its seven-membered azepane ring does not, suggesting that it assumes multiple conformations. The with the indole moiety of Trp286(TcAChE Trp279). In addition, electron density is contoured at 30 h was predicted to form two hydrogen bonds at the CAs,one volving interaction of its hydroxyl group with the main chain Rigid body refinement in CCP4- was based on a previo carbonyl oxygen of His447(TcAChE His440) and the other solved trigonal crystal form of native TcAChE (PDB code lEAS involving interaction of its protonated azepane nitrogen with excluding water molecules and carbohydrates. Initial 2Fo- Fc and Tyr1240-(TcAChE Tyrl21) ata, and the initial Fo Fe map was used, with the aid of the In the following, we present the crystal structure of a complex program Coot,27 to fit 5h into positive density at the CAS of TcAChE structure rather than on the mache structure. as TcAChE, as well as the spacer, and to add 78 water molecules Subsequent restrained refinement rounds with overall B-factor predicted, 5h spans the CAS and the PAS. The crystal structure refinement were performed and 56 carbohydrate atoms were added is compared to those obtained by computer docking of 5h and until convergence to values of Rwork =18.5% and Re to the native crystal structures of TcAChE and mAChE As analyzed by Molprobity, 95.0%o of all residues are in favored regions and 99. 6% are in allowed regions of the ramachandran Materials and Methods plot, the only outliers being Asp380 and Asn457, which are both Crystallization and Data Collection. TcAChe was purifi glycosylated surface residues. Due to the poor electron density at essentially as described by Sussman et al., with the exception of e PAs observed in the 2Fo- Fe map we chose to submit the the affinity column elution which was performed with tetrameth coordinates of 5h without the atoms of the mEP moiety within the lammonium bromide instead of decamethonium bromide. Trigonal PAS. In all figures therein, this mEp moiety is shown for descriptive crystals of the enzyme 4 were soaked for 20 h at 4C in 2 uL of purposes only. Simulated annealing omit maps were constructed I mM dissolved in the crystallization solution(40% PEG 200 PHENIX (v/v)/150 mM MES(Sigma-Aldrich), pH 7.4), employing the Molecular Docking. Molecular simulations were performed on hanging drop procedure. The crystals were then transferred to an R14000 SGI Fuel workstation with the software package SYBYL cryoprotectant oil and flash frozen in liquid nitrogen. 6.9(Tripos Inc, St Louis, MO). Standard parameters were used Data collection was performed at the eSrF in Grenoble, on unless otherwise indicated. The crystal structures of both native beamline ID 14-1. at T=100 K and 2=0.934 A. 180 image TcAChE(PDB code IEA5 )and of the 5h/TCAChE complex were were taken, at an oscillation angle of 1, with an exposure time of used. Heteroatoms and water molecules in the proteins were 6s. DENzo and ScalePacK- were used to integrate and scale removed, and hydrogen atoms were subsequently added. the data. Data were truncated with the CCP4 program TRUN- Three-dimensional structures of sh were generated by connecting CATE, and 5.16% of the reflections were randomly used as test two(-)-nor-MEP units in either equatorial or axial conformations. The equatorial conformation of the(-)-nor-MEP unit was retrieved Structure Determination and Refinement. A model of sh was from the crystal structure of MEP. The axial conformation was constructed using the Gauss View 3.09 pr at using ( Gaussian, Carnegie, generated based on earlier NMR studies. Two sp'Natoms of sh PA)and converted to a PDb file forma Babel. 2b were protonated, and the Gasteiger-Huckel partial charges were

was a homodimer with a nine-carbon spacer (5h) (Figure 1), which displayed IC50 values of 3.9 ( 1.3 and 10.0 ( 3.0 nM toward mouse brain AChE and mouse serum BChE, respectively, and an IC50 of 16.6 µM for inhibition of A aggregation. GOLD20 docking simulations of 5h, in which the phenyl groups of both MEP moieties were in equatorial orientations with respect to the azapane rings (eCeP), indicated that 5h spans the active-site gorge, interacting with both the CAS and PAS of mAChE. The phenyl group of the MEP moiety at the CAS was predicted to make a face-to-face π-stacking interaction with Trp86 (mAChE numbering, Trp84 in TcAChE). The MEP moiety at the PAS was predicted to make cation-π and hydrophobic interactions of its seven-membered azepane ring with the indole moiety of Trp286 (TcAChE Trp279). In addition, 5h was predicted to form two hydrogen bonds at the CAS, one involving interaction of its hydroxyl group with the main chain carbonyl oxygen of His447 (TcAChE His440) and the other involving interaction of its protonated azepane nitrogen with Tyr124O (TcAChE Tyr121).19 In the following, we present the crystal structure of a complex of 5h with TcAChE and new GOLD models based on the TcAChE structure rather than on the mAChE structure. As predicted, 5h spans the CAS and the PAS. The crystal structure is compared to those obtained by computer docking of 5h and to the native crystal structures of TcAChE and mAChE. Materials and Methods Crystallization and Data Collection. TcAChE was purified essentially as described by Sussman et al.,21 with the exception of the affinity column elution, which was performed with tetramethylammonium bromide instead of decamethonium bromide. Trigonal crystals of the enzyme14 were soaked for 20 h at 4 °C in 2 µL of 1 mM 5h19 dissolved in the crystallization solution (40% PEG 200 (v/v)/150 mM MES (Sigma-Aldrich), pH 7.4), employing the hanging drop procedure.22 The crystals were then transferred to cryoprotectant oil and flash frozen in liquid nitrogen. Data collection was performed at the ESRF in Grenoble, on beamline ID 14-1, at T ) 100 K and λ ) 0.934 Å. 180 images were taken, at an oscillation angle of 1°, with an exposure time of 6 s. DENZO and SCALEPACK23 were used to integrate and scale the data. Data were truncated with the CCP424 program TRUNCATE,25 and 5.16% of the reflections were randomly used as test reflections (Table 1). Structure Determination and Refinement. A model of 5h was constructed using the GaussView 3.09 program (Gaussian, Carnegie, PA) and converted to a PDB file format using Babel.26 Rigid body refinement in CCP424 was based on a previously solved trigonal crystal form of native TcAChE (PDB code 1EA5), excluding water molecules and carbohydrates. Initial 2Fo - Fc and Fo - Fc electron density maps were calculated using 40-2.7 Å data, and the initial Fo - Fc map was used, with the aid of the program Coot,27 to fit 5h into positive density at the CAS of TcAChE, as well as the spacer, and to add 78 water molecules. Subsequent restrained refinement rounds with overall B-factor refinement were performed and 56 carbohydrate atoms were added until convergence to values of Rwork ) 18.5% and Rfree ) 23.5%. As analyzed by Molprobity,28 95.0% of all residues are in favored regions and 99.6% are in allowed regions of the Ramachandran plot, the only outliers being Asp380 and Asn457, which are both glycosylated surface residues. Due to the poor electron density at the PAS observed in the 2Fo - Fc map we chose to submit the coordinates of 5h without the atoms of the MEP moiety within the PAS. In all figures therein, this MEP moiety is shown for descriptive purposes only. Simulated annealing omit maps were constructed using PHENIX.29 Molecular Docking. Molecular simulations were performed on an R14000 SGI Fuel workstation with the software package SYBYL 6.9 (Tripos Inc., St. Louis, MO). Standard parameters were used unless otherwise indicated. The crystal structures of both native TcAChE (PDB code 1EA5) and of the 5h/TcAChE complex were used. Heteroatoms and water molecules in the proteins were removed, and hydrogen atoms were subsequently added. Three-dimensional structures of 5h were generated by connecting two (-)-nor-MEP units in either equatorial or axial conformations. The equatorial conformation of the (-)-nor-MEP unit was retrieved from the crystal structure of MEP.30 The axial conformation was generated based on earlier NMR studies.30 Two sp3 N atoms of 5h were protonated, and the Gasteiger-Hu¨ckel partial charges were Figure 2. Two representations of the refined structure of the 5h/TcAChE complex. (A) TcAChE is displayed as a beige space-filling surface and 5h within the active-site gorge as a magenta stick model. The arrow marks the entrance to the active-site gorge. (B) 5h shown as a green stick model, and the side chains of selected active-site residues with which it makes contact as red stick models. Figure 3. Simulated annealing omit map of the 5h/TcAChE complex. The MEP moiety at the CAS and the nonamethylene linker display almost complete electron density, whereas the MEP moiety at the PAS does not, suggesting that it assumes multiple conformations. The electron density is contoured at 3σ. 2544 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 Paz et al

Complex of AChE with a Bis-(-)-hor- meptazinol Journal of Medicinal Chemistry, 2009, Vol. 52, No 8 2545 PAS 118 Tyr279 Tyro Tyr 33 Tyr121 Phe290 Phe331 Figure 4. Overlay of the CAs of the 5h/TCAChE crystal structure on that of native TcAChE. The view is from below the catalytic triad looking up the gorge. The amino acid side chains in the complex are displayed as red sticks and those in the native enzyme as blue sticks The comparison shows that in the complex the catalytic triad is disrupted ue to formation of an H-bond between His440N and the phene xygen of the MEP moiety (cyan dashed line), with concomitant ruption of its native H-bond with Ser2000. As a consequence, the CAS side chain of Ser200 rotates away from His440, widening the distance between His440N22 and Ser2000 from the native distance of 3.0 A Side view of sh and of the residues active-site (blue dashed line)to 4.2 A(red dashed lin addition. Phe330 is 5h/TcAChE complex. sh is display sticks. the tilted away from its native position by the seven-membered ring of the side chains in the complex as and the MEP monet corresponding residues in native TcAChE as blue sticks signed. Following this, the ligand was energy minimized in 1000 G|u73 Molecular docking was carried out using GOLD 3.0(CCDC, Cambridge, UK, 2005) to generate an ensemble of docked conformations for the ligand. The active site was defined as all toms within a radius of 25 A around Tyr12105 of TcAChE.This larged binding pocket was chosen, as a smaller one might neither ccommodate a large bis-ligand nor include both the catalytic and GIn74 peripheral sites of AChE. Because of the high flexibility of the ligand, which contains many rotatable bonds, 600 genetic algorithm (GA)runs were performed rather than the default of 10 For each GA run, the default GA settings were used, except that early termination was prohibited and pyramidal nitrogen inversion was allowed An advanced combination approach of consensus scoring wa used to guide the selection of the most reliable conformation(s) from the set of candidate conformations that GOLD generated. All Gy335 informations were evaluated with five available scoring functions including four scoring functions(G Score, PMF, D Score, and ChemScore)from the CSCORE module in SYBYL and another stand-alone scoring function, X-SCORE 1. 2. 1.The"rank-by-ran Figure 6. View from above of the entrance to the active-site gorge in the h5/TcAChE complex. 5h is displayed as green sticks, and the coring. The final rank of a certain conformation was calculated backbone of TeAChE is in beige. The side chains of residues lining by taking the unweighted average of all five scoring functions. The the entrance to the gorge are layed as sticks and overlaid top"re-ranked"solution was chosen as the representative binding transparent blue surface mode for the ligand. the PAs, with the nonamethylene spacer that links them shakin Results and discussion along the gorge(Figure 2). The simulated annealing omit map generated for the final refined structure shows full electron Examination of the crystal structure of the 5h/TcAChe density for the MEP moiety at the CAs and for most of the complex reveals one MEP moiety in the CAs and the other in linker, except for a lack of electron density for the second carbon

assigned. Following this, the ligand was energy minimized in 1000 steps using the Tripos force field. Molecular docking was carried out using GOLD 3.0 (CCDC, Cambridge, UK, 2005) to generate an ensemble of docked conformations for the ligand. The active site was defined as all atoms within a radius of 25 Å around Tyr121O of TcAChE. This enlarged binding pocket was chosen, as a smaller one might neither accommodate a large bis-ligand nor include both the catalytic and peripheral sites of AChE. Because of the high flexibility of the ligand, which contains many rotatable bonds, 600 genetic algorithm (GA) runs were performed rather than the default of 10. For each GA run, the default GA settings were used, except that early termination was prohibited and pyramidal nitrogen inversion was allowed. An advanced combination approach of consensus scoring was used to guide the selection of the most reliable conformation(s) from the set of candidate conformations that GOLD generated. All conformations were evaluated with five available scoring functions, including four scoring functions (G_Score, PMF, D_Score, and ChemScore) from the CSCORE module31 in SYBYL and another stand-alone scoring function, X-SCORE 1.2.1.32 The “rank-by-rank” strategy reported by Wang et al.33 was adopted for consensus scoring. The final rank of a certain conformation was calculated by taking the unweighted average of all five scoring functions. The top “re-ranked” solution was chosen as the representative binding mode for the ligand. Results and Discussion Examination of the crystal structure of the 5h/TcAChE complex reveals one MEP moiety in the CAS and the other in the PAS, with the nonamethylene spacer that links them snaking along the gorge (Figure 2). The simulated annealing omit map generated for the final refined structure shows full electron density for the MEP moiety at the CAS and for most of the linker, except for a lack of electron density for the second carbon Figure 4. Overlay of the CAS of the 5h/TcAChE crystal structure on that of native TcAChE. The view is from below the catalytic triad looking up the gorge. The amino acid side chains in the complex are displayed as red sticks and those in the native enzyme as blue sticks. The comparison shows that in the complex the catalytic triad is disrupted due to formation of an H-bond between His440Nε2 and the phenol oxygen of the MEP moiety (cyan dashed line), with concomitant disruption of its native H-bond with Ser200Oγ. As a consequence, the side chain of Ser200 rotates away from His440, widening the distance between His440Nε2 and Ser200Oγ from the native distance of 3.0 Å (blue dashed line) to 4.2 Å (red dashed line). In addition, Phe330 is tilted away from its native position by the seven-membered ring of the MEP moiety. Figure 5. Side view of 5h and of the residues lining the active-site gorge in the 5h/TcAChE complex. 5h is displayed as green sticks, the amino acid side chains in the complex as red sticks, and the corresponding residues in native TcAChE as blue sticks. Figure 6. View from above of the entrance to the active-site gorge in the h5/TcAChE complex. 5h is displayed as green sticks, and the backbone of TcAChE is in beige. The side chains of residues lining the entrance to the gorge are displayed as sticks and overlaid with a transparent blue surface. Complex of AChE with a Bis-(-)-nor-meptazinol Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2545

Complex of AChE with a Bis-(-)-hor- meptazinol Journal of Medicinal Chemistry, 2009, Vol. 52, No 8 2547 At the PAs, as already noted, the electron density p that the MEP moiety samples more than one conformation.The principal residues surrounding it are Tyr70, Glu73, GIn74 Trp279, and gly335(Figure 6), but the conformational hetero- geneity precludes assignment of specific interactions. The possibility was considered that the apparent conformational ht reflect chemical inhomogeneity, but mass spectrometry of the compound clearly excluded this possibility (not shown). Ligplot was used to compare the principal ligand-protein interactions of 5h in the TcAChE crystal structure( Figure 7A) and in the previously published modeled complex with mAChE In this model, the MEP moieties are both in the equatorial position because this conformation was that found in the smal molecule X-ray structure of MEP3O(Figure 7B). Superposition of the crystal structures of native mAChE (PDB ode 2HA2)and of the 5h/TcAChE complex(Figure 8)reveals ructures of the Sh/TcAChE complex and of native mAChE looking no significant main chain or side chain differences that could down the gorge. No significant differences in main chain or side chai provide a basis for the differences between the latter and the conformations are observed between the TcAChE structure(blue) and the mAChE structure(red). Only the main chains are traced for clarity: model of the 5h/mAChE complex obtained using GOLD sh is shown in green There is overall similarity in the positioning of the ligand (rmsd 3. A), with hydrophobic interactions with Trp84, Phe330, and Tyr334 (or the corresponding mAChE residues)appearing In e crystal structure an computational model. However, as mentioned above there are significant differences as well. Thus, in the X-ray structure, His440 is hydrogen-bonded to 5h through the m atom of its imidazole side chain, no through its main-chain carbonyl oxygen, as was suggested by the GOLD model. Furthermore, the crystal structure does not reveal T-T stacking interactions between Trp84 and the phenyl group of the MEP group bound at the CAS nor hydrogen bonding of the protonated azepane nitrogen to Tyrl2lc (Tyr1240s in mAChE) On the basis of the 5h/TcAChE crystal structure, we decided to carry out additional GOLD simulations, with the same algorithm and parameters that we previously used, in which both MEP moieties in 5h could adopt either axial or equatorial ntations(aa and ee, respectively) or could ado orientations(ea and ae)both in the native enzyme(PDB code IEA5)and in the template of the 5h/TcAChE complex, devoid Figure 9. Comparison of the conformation of the MEP moiety in the of 5h, waters, and hydrocarbons. This was done to reconcile CAS of the 5h/TcAChE crystal structure with its conformation in two the observation that there was a difference between the axial models obtained by by docking to the protein template of the crystal orientation of the mEP moiety at the CAS determined by the (rmsd= 1. 15 A, rerank=3.2) shows good agreement between the complex X-ray structure and the equatorial orientations of the crystal structure and the model.(B)Superposition of the other model ligand used in the gOld model. while the NMR solution (rmsd= 1. 18 A, rerank= 2.4)on the crystal structure yields a totally structure of mEP detected both the axial and equatorial ncorrect orientation. The crystal structure is displayed as thick sticks conformers. Table 2 lists the conformations and scores and the models as lines obtained(models are available as Supporting Information) In this modeling study, we found rather than using either (Figure 4). Its phenolic oxygen forms a hydrogen bond wit His440N22(2.55 A), thus disrupting the latter's hydrogen bond TCAChE (EA5) or mAChE, that in general the best scores were with Ser2000 within the catalytic triad. The xi angle of the TcAChE complex structure(PDB code 2w6C) When the two crystal structure(PDB code IEA5). As a consequence, the distance between Ser2000" and His440Ne2 increases from 3.0 posed on Sh from the crystal structure of the complex(Figure ), it could be seen that the modeled binding mode of the MEP A in the native structure to 4.2 A. An analogous rupture of moiety at the CAS was quite similar to that in the crystal the catalytic triad and rotation of Ser200 was observed in the structure in the case of 2w6c_ea_220( Figure 9A), even though complex of TcAChE with a bis-tacrine inhibitor with a pen their orientations are ec in the model and ac in the experimen- tamethylene spacer. Phe330 also undergoes a conformational tal structure, respectively. However in the second model change, relative to the native structure, that involves-25and 2w6c_ee_447, the CAS MEP moiety assumes a different 33 rotations, respectively, of its %i and x2 angles. The residues orientation(Figure 9B). Both models have high ranks(Table lining the gorge between the two MEP moieties do not reveal 2), exemplifying the difficulty in choosing a sing gle model, rather any substantial changes relative to their positions and conforma- than an ensemble of models, in the absence of a crystal structure tions in native TcAChE(Figure 5) of a complex. No comparison of the MEP moieties at the PAS

(Figure 4). Its phenolic oxygen forms a hydrogen bond with His440Nε2 (2.55 Å), thus disrupting the latter’s hydrogen bond with Ser200Oγ within the catalytic triad. The 1 angle of the freed Ser200 is rotated ∼120° relative to its position in the native crystal structure (PDB code 1EA5). As a consequence, the distance between Ser200Oγ and His440Nε2 increases from ∼3.0 Å in the native structure to ∼4.2 Å. An analogous rupture of the catalytic triad and rotation of Ser200 was observed in the complex of TcAChE with a bis-tacrine inhibitor with a pentamethylene spacer.34 Phe330 also undergoes a conformational change, relative to the native structure, that involves ∼25° and ∼33° rotations, respectively, of its 1 and 2 angles. The residues lining the gorge between the two MEP moieties do not reveal any substantial changes relative to their positions and conformations in native TcAChE (Figure 5). At the PAS, as already noted, the electron density suggests that the MEP moiety samples more than one conformation. The principal residues surrounding it are Tyr70, Glu73, Gln74, Trp279, and Gly335 (Figure 6), but the conformational heterogeneity precludes assignment of specific interactions. The possibility was considered that the apparent conformational heterogeneity might reflect chemical inhomogeneity, but mass spectrometry of the compound clearly excluded this possibility (not shown). Ligplot35 was used to compare the principal ligand-protein interactions of 5h in the TcAChE crystal structure (Figure 7A) and in the previously published modeled complex with mAChE. In this model, the MEP moieties are both in the equatorial position because this conformation was that found in the small molecule X-ray structure of MEP30 (Figure 7B). Superposition of the crystal structures of native mAChE (PDB code 2HA2) and of the 5h/TcAChE complex (Figure 8) reveals no significant main chain or side chain differences that could provide a basis for the differences between the latter and the model of the 5h/mAChE complex obtained using GOLD.19 There is overall similarity in the positioning of the ligand (rmsd ) 3.01 Å), with hydrophobic interactions with Trp84, Phe330, and Tyr334 (or the corresponding mAChE residues) appearing in both the crystal structure and the computational model. However, as mentioned above, there are significant differences as well. Thus, in the X-ray structure, His440 is hydrogen-bonded to 5h through the Nε2 atom of its imidazole side chain, not through its main-chain carbonyl oxygen, as was suggested by the GOLD model. Furthermore, the crystal structure does not reveal π-π stacking interactions between Trp84 and the phenyl group of the MEP group bound at the CAS nor hydrogenbonding of the protonated azepane nitrogen to Tyr121O (Tyr124O in mAChE). On the basis of the 5h/TcAChE crystal structure, we decided to carry out additional GOLD simulations, with the same algorithm and parameters that we previously used, in which both MEP moieties in 5h could adopt either axial or equatorial orientations (aa and ee, respectively) or could adopt mixed orientations (ea and ae) both in the native enzyme (PDB code 1EA5) and in the template of the 5h/TcAChE complex, devoid of 5h, waters, and hydrocarbons. This was done to reconcile the observation that there was a difference between the axial orientation of the MEP moiety at the CAS determined by the complex X-ray structure and the equatorial orientations of the ligand used in the GOLD model, while the NMR solution structure of MEP detected both the axial and equatorial conformers.36 Table 2 lists the conformations and scores obtained (models are available as Supporting Information). In this modeling study, we found rather than using either TcAChE (1EA5) or mAChE, that in general the best scores were obtained for 5h modeled inside the empty template of the 5h/ TcAChE complex structure (PDB code 2W6C). When the two best models, 2w6c_ea_220 and 2w6c_ee_447, were superimposed on 5h from the crystal structure of the complex (Figure 9), it could be seen that the modeled binding mode of the MEP moiety at the CAS was quite similar to that in the crystal structure in the case of 2w6c_ea_220 (Figure 9A), even though their orientations are eC in the model and aC in the experimental structure, respectively. However in the second model, 2w6c_ee_447, the CAS MEP moiety assumes a different orientation (Figure 9B). Both models have high ranks (Table 2), exemplifying the difficulty in choosing a single model, rather than an ensemble of models, in the absence of a crystal structure of a complex. No comparison of the MEP moieties at the PAS Figure 8. Superposition of the active-site gorge area in the crystal structures of the 5h/TcAChE complex and of native mAChE looking down the gorge. No significant differences in main chain or side chain conformations are observed between the TcAChE structure (blue) and the mAChE structure (red). Only the main chains are traced for clarity; 5h is shown in green. Figure 9. Comparison of the conformation of the MEP moiety in the CAS of the 5h/TcAChE crystal structure with its conformation in two models obtained by docking to the protein template of the crystal structure (PDB code 2W6C). (A) Superposition of one such model (rmsd ) 1.15 Å, rerank ) 3.2) shows good agreement between the crystal structure and the model. (B) Superposition of the other model (rmsd ) 1.18 Å, rerank ) 2.4) on the crystal structure yields a totally incorrect orientation. The crystal structure is displayed as thick sticks and the models as lines. Complex of AChE with a Bis-(-)-nor-meptazinol Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2547

2548 Journal of Medicinal Chemistry, 2009, Vol. 52, No 8 Paz et al was made because, as discussed, the electron density there is (7) Harvey, A L. The phar quite poor, most likely due to local disorder and the rmsd Pharmacol. Ther. 1995. 68 between the 5h from the crystal structure and the models was (8)Zhang R. w: Tang, X C; Han, Y.Y.; Sang, G. W i Zhang, Y D. Ma, Y.X.: Zhang, C L; Yang, R. M. Drug calculated only for the CAS MEP, linker region, and the nitrogen A in the treatment of senile memory disorders. Acta Pharmacol. Si of the CAs MEP Most of the different gold models obtained 1991.12.250-252 for this complex, using different AChE molecules(apo and holo (9)Alvarez, A: Bronfman, F: Perez, C.A. Vicente, M: Garrido, J enzymes, Tc and mouse), share an overall similarity but many, plaque component. brillogenesis of amyloid-beta-peptides. Neurosci. Left in detail, fail to capture the exact nuances of the 5h-TCAChE 1995,201,49-52. Interactions (10)Alvarez, A Opazo, C. Alarcon, R; Garrido, J; Inestrosa, N. C Acetylcholinesterase promotes the aggregation of amyloid-beta-peptio phenol oriented down the gorge toward the catalytic③3mmm吗用M业B Since an earlier modeling study of a MEP/TcAChE compl N.C∴ Alvarez,A; Perez foreno. R. D. 1.: Linker. C: Casanueva. O. I: Soto, C: Garrido, J. Acet the spacers attached via the azepane rings and not through the imer's fibrils: possible role of the peripheral site of the enzym oxygen atoms of the phenol groups. 9 The crystal structure of 1996,16,881-891 the 5h/TcAChE complex described here displays a somewhat (12)Bartolini, M.; Bertucci, C; Cavrini, V: Andrisano, V. beta-Amyloid similar orientation of mep within the cas but also reveals high aggregation induced by human acetylcholinesterase: inhibition studies. Biochem. Pharmacol. 2003. 65. 407- flexibility of the MEP moiety at the PAs. It might, therefore, (13) Reyes, A E. Perez, D. R. Alvarez, A: Garrido, J: Gentry, M.K. be worth considering use of another atom on the azepane ring Doctor, B. P. Inestrosa. N. C. A me for linkage to the spacer or even synthesizing a molecule in acetylcholinesterase inhibits the formation of amyloid fibrils induce by the enzyme. Biochem. Biophys. Res. Commun. 1997, 232, 652- and the other through its azepane ring. These derivatives might (14)Sussman, J. Li; Harel, M. Frolow, F; Oefner, C; Goldman, A. have different affinities and specificities for AChE and possibl L; Silman, I. Atomic structure of acetylcholinesterase from To ind more specifically to the PAS than 5h. This could provide alifomica: a prototypic acetylcholine-binding protein Scien 253,872-879 information as to whether flexible binding of moieties at the (15)Silma PAS might affect the inhibitory effect on AB deposition. Acknowledgment. This work was supported in part by the T grants by the Divadol Foundation, the Israel Science Foundation nd low cost bis-tetrahydroaminacrine inhibitors of acetylcholinesterase. Steps toward novel drugs for treating Alzheimers the Nalvyco Foundation, the Neuman Foundation, the Bruce disease.J.Biol.chem.1996.271,23646-23649 rosen foundation the jean and Jula Goldwurm Memorial (17) Du, D M; Carlier, P R Development Foundation, a research grant from Erwin Pearl, the Benoziyo drugs for Alzhe acetylcholinesterase Pham.Des.2004,lO,3141-3156 Center for Neuroscience, the European Commission Sixth (18)Haviv, H :W D. M.: Silman. I: Sussman. J. L. Bivalent ligands Framework Research and Technological Development Pro- derived from Huperzine A tylcholinesterase inhibitors. Curr. ramme"SPINE2-COMPLEXES project under contract num- Top.Med.Chem.2007,7,375-387 ber LSHG-CT-2006-031220, Teach-SG" project under contract (19)Xie, Q; Wang, H; Xia, Z; Lu, M: Tang. Y. Sheng. W: Li. W: Zhou. W: Zhu, number ISSG-CT-2007-037198 (I.S. and J. LS), and by grants ptazinols as novel nanomolar ch from the national natural science foundation of pr china with high inhibitory potency on amyloid-beta (nos. 30472088 and 30772553)and the Chinese lll Project to ECUST. J L.S. is the Morton and Gladys Pickman (20) Jones, G: Willett, P: Glen, R. C: Leach, A. R: Taylor, R ation of a genetic algorithm for flexible docking Professor of Structural Biology. We are grateful to Esther Roth J.Mol.Biol.1997.267.727-7 and Lilly Toker for the samples of purified TcAChE. (21) an J. L; HareL, M. Frolow. F: Varon, L; Toker. L: Futerman man, I. Purification and crystallization of a dimeric form of Supporting Information Available: Structures acetylcholinesterase from Torpedo californica subsequent to solubi (ZIP): wbc_b_ee_447-pdb, wbc_b_ea_220 pdb, 2wbc Biol.1988,203,821-82 2wbc_leas_ee_531. pdb, wbc_leasea_52l pdb, 2wbc (22)McPherson, A Preparation and Analysis of Protein Crystals; John ThismaterialisavailablefreeofchargeviatheInternetathttp:// Wiley Sons: New York, 1982 pubs. acs. org 23)Otwinowski, Z. Minor, w. Processing of X-ray diffraction data collected in oscillation mode Methods Enzymol. 1997. 276. 307-326 (24)CCP4. The CCP4 suite: ns for protein crystallography. Acta References llgr.1994,50,760-7 (25) Erera io. A icons Kilo. Set at ot of cergstilogin ngis. Mol.Biol.1975.43.103-218 (2)Bartus, R. T: Dean, R. L, Ill; Beer, B: Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 1982, 217, 408 (26)Walters, P. Stahl, M., Babel, version 1.1: Department of Chemistry, niversity of Arizona: Tucson, AZ, 1994 (27)Emsley, P: Cowtan, K. Coot: Model-Building Tools for Moled (3)Dunnett, S. B. Fibiger, H. C. Role of forebrain cholinergic systen Graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126- relevance to the cog (28)Davis, I. W: Leaver-Fay, A: Chen, V.B.; Block, J N Kapral, G.J. (4)Greenblatt, H. M: Dvir, H: Silman, L; Sussman, J. L. Acetylcho- inesterase: a multifaceted target for structu hardson, J.S.: Richardson, D. C. MolProbity: all-atom contacts anticholinesterase agents for the treatment of Alzheimers disease. and structure validation for proteins and nucleic acids. Nucleic Acids Mo. Neurosci.2003.20,369-383. Res.2007.35.W375-W383 (5)Kawakami, Y: Inoue, A: Kawai, T; Wakita, M: Sugimoto, H. (29) Adams, P D. Grosse-Kunstleve, Rw T.R. Hopfinger, A J. The rationale for E2020 as a potent acetylcholinest McCoy, A. J: Moriarty, N. w; Read, R. hettini. J C: Saut erase inhibitor. Bioorg. Med. Chem. 1996. 4. 1429-1446 PHENIX lg new software for (6)Enz, A: Boddeke, H; Gray, J; Spiegel, R linicopharmacologic properties of SDZ ENA 713, Sect. D: Biol. Crystallogr. 2002, 58, 1948-1954 cetylcholinesterase inhibitor. Ann. N.Y. Acad. Sc 0.272 (0)Chen, Y. Studies on the synthesis, resolution and optical isomers of 275 meptazinol. Dissertation. Fudan Univ: Shanghai, 2004: Pp 19-2

was made because, as discussed, the electron density there is quite poor, most likely due to local disorder and the rmsd between the 5h from the crystal structure and the models was calculated only for the CAS MEP, linker region, and the nitrogen of the CAS MEP. Most of the different GOLD models obtained for this complex, using different AChE molecules (apo and holo enzymes, Tc and mouse), share an overall similarity but many, in detail, fail to capture the exact nuances of the 5h-TcAChE interactions. Since an earlier modeling study of a MEP/TcAChE complex placed the azepane ring in the middle of the gorge with the phenol oriented down the gorge toward the catalytic site,37 the bivalent MEP derivatives were designed and synthesized with the spacers attached via the azepane rings and not through the oxygen atoms of the phenol groups.19 The crystal structure of the 5h/TcAChE complex described here displays a somewhat similar orientation of MEP within the CAS but also reveals high flexibility of the MEP moiety at the PAS. It might, therefore, be worth considering use of another atom on the azepane ring for linkage to the spacer or even synthesizing a molecule in which one MEP would be connected through its phenol group and the other through its azepane ring. These derivatives might have different affinities and specificities for AChE and possibly bind more specifically to the PAS than 5h. This could provide information as to whether flexible binding of moieties at the PAS might affect the inhibitory effect on A deposition. Acknowledgment. This work was supported in part by the grants by the Divadol Foundation, the Israel Science Foundation, the Nalvyco Foundation, the Neuman Foundation, the Bruce Rosen Foundation, the Jean and Jula Goldwurm Memorial Foundation, a research grant from Erwin Pearl, the Benoziyo Center for Neuroscience, the European Commission Sixth Framework Research and Technological Development Programme “SPINE2-COMPLEXES” project under contract number LSHG-CT-2006-031220, “Teach-SG” project under contract number ISSG-CT-2007-037198 (I.S. and J.L.S.), and by grants from the National Natural Science Foundation of P.R. China (nos. 30472088 and 30772553) and the Chinese 111 Project grant to ECUST. J.L.S. is the Morton and Gladys Pickman Professor of Structural Biology. We are grateful to Esther Roth and Lilly Toker for the samples of purified TcAChE. Supporting Information Available: Structures of PDB 2W6C (ZIP):2w6c_rr6_ee_447.pdb,2w6c_rr6_ea_220.pdb,2w6c_rr6_aa_147.pdb, 2w6c_1ea5_ee_531.pdb,2w6c_1ea5_ea_521.pdb,2w6c_1ea5_aa_027.pdb. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Rosenberry, T. L. Acetylcholinesterase. AdV. Enzymol. Relat. Areas Mol. Biol. 1975, 43, 103–218. (2) Bartus, R. T.; Dean, R. L., III; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 1982, 217, 408– 414. (3) Dunnett, S. B.; Fibiger, H. C. Role of forebrain cholinergic systems in learning and memory: relevance to the cognitive deficits of aging and Alzheimer’s dementia. Prog. Brain Res. 1993, 98, 413–420. (4) Greenblatt, H. M.; Dvir, H.; Silman, I.; Sussman, J. L. Acetylcholinesterase: a multifaceted target for structure-based drug design of anticholinesterase agents for the treatment of Alzheimer’s disease. J. Mol. Neurosci. 2003, 20, 369–383. (5) Kawakami, Y.; Inoue, A.; Kawai, T.; Wakita, M.; Sugimoto, H.; Hopfinger, A. J. The rationale for E2020 as a potent acetylcholinesterase inhibitor. Bioorg. Med. Chem. 1996, 4, 1429–1446. (6) Enz, A.; Boddeke, H.; Gray, J.; Spiegel, R. Pharmacologic and clinicopharmacologic properties of SDZ ENA 713, a centrally selective acetylcholinesterase inhibitor. Ann. N.Y. Acad. Sci. 1991, 640, 272– 275. (7) Harvey, A. L. The pharmacology of galanthamine and its analogues. Pharmacol. Ther. 1995, 68, 113–128. (8) Zhang, R. W.; Tang, X. C.; Han, Y. Y.; Sang, G. W.; Zhang, Y. D.; Ma, Y. X.; Zhang, C. L.; Yang, R. M. Drug evaluation of huperzine A in the treatment of senile memory disorders. Acta Pharmacol. Sin. 1991, 12, 250–252. (9) Alvarez, A.; Bronfman, F.; Perez, C. A.; Vicente, M.; Garrido, J.; Inestrosa, N. C. Acetylcholinesterase, a senile plaque component, affects the fibrillogenesis of amyloid-beta-peptides. Neurosci. Lett. 1995, 201, 49–52. (10) Alvarez, A.; Opazo, C.; Alarcon, R.; Garrido, J.; Inestrosa, N. C. Acetylcholinesterase promotes the aggregation of amyloid-beta-peptide fragments by forming a complex with the growing fibrils. J. Mol. Biol. 1997, 272, 348–361. (11) Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer’s fibrils: possible role of the peripheral site of the enzyme. Neuron 1996, 16, 881–891. (12) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. beta-Amyloid aggregation induced by human acetylcholinesterase: inhibition studies. Biochem. Pharmacol. 2003, 65, 407–416. (13) Reyes, A. E.; Perez, D. R.; Alvarez, A.; Garrido, J.; Gentry, M. K.; Doctor, B. P.; Inestrosa, N. C. A monoclonal antibody against acetylcholinesterase inhibits the formation of amyloid fibrils induced by the enzyme. Biochem. Biophys. Res. Commun. 1997, 232, 652– 655. (14) Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science 1991, 253, 872–879. (15) Silman, I.; Sussman, J. L. Acetylcholinesterase: “classical” and “nonclassical” functions and pharmacology. Curr. Opin. Pharmacol. 2005, 5, 293–302. (16) Pang, Y. P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. Highly potent, selective, and low cost bis-tetrahydroaminacrine inhibitors of acetylcholinesterase. Steps toward novel drugs for treating Alzheimer’s disease. J. Biol. Chem. 1996, 271, 23646–23649. (17) Du, D. M.; Carlier, P. R. Development of bivalent acetylcholinesterase inhibitors as potential therapeutic drugs for Alzheimer’s disease. Curr. Pharm. Des. 2004, 10, 3141–3156. (18) Haviv, H.; Wong, D. M.; Silman, I.; Sussman, J. L. Bivalent ligands derived from Huperzine A as acetylcholinesterase inhibitors. Curr. Top. Med. 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W.; Hung, L. W.; Ioerger, T. R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter, N. K.; Terwilliger, T. C. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 1948–1954. (30) Chen, Y. Studies on the synthesis, resolution and optical isomers of meptazinol. Dissertation. Fudan Univ: Shanghai, 2004; pp 19-28. 2548 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 Paz et al

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