Investigation of the binding mode of (−)-meptazinol and bis-meptazinol derivatives on acetylcholinesterase using a molecular docking method

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J Mol model(2006)12:390-397 DOI10.1007/s008940050058y ORIGINAL PAPER Qiong Xie. Yun Tang. Wei Li Xing-Hai Wang Zhui-Bai Qiu Investigation of the binding mode of (-)-meptazinol and bis-meptazinol derivatives on acetylcholinesterase using a molecular docking method Received: 8 December 2004/ Accepted: 9 August 2005/ Published online: 11 January 2006 c) Springer-Verlag 2006 Abstract Molecular docking has been performed to Introduction investigate the binding mode of (-)-meptazinol (MEP) with acetylcholinesterase(AChE) and to screen bis- Alzheimers disease(AD)is a neurodegenerative disorder meptazinol(bis- MEP)derivatives for preferable synthetic that seriously threatens the health of elderly people around candidates virtually. A reliable and practical docking the world. Although with an unclear pathogenesis, AD is method for investigation of AChE ligands was established commonly believed to be associated with the dysfunction by the comparison of two widely used docking programs, of the central cholinergic system [1, 2]. Acetylcholinester- FlexX and GOLD. In our hands, we had more luck using ase (AChE) plays a key role in the regulation of the GOLd than FlexX in reproducing the experimental poses cholinergic system, and hence, inhibition of AChE has of known ligands(RMSD1.0 mM). The Q.Xie.Y.Tang(凶)·W.Li·X.H.Wang:Z.B.Qu() inhibitory potency of(-)-MEp was almost equivalent to that School of Pharmacy, Fudan University of ()-galanthamine and tacrine, about 100 times less potent hanghai, 200032, People's Republic of china than physostigmine [11]. Recently, we determined the 54237419 absolute configurations of ()and (+)-MEP as S and R, respectively, by X-ray crystal structures(Fig. 1)[12] Tel:+86-21-54237595 Therefore, it was of considerable interest to investigate the mechanism of action of (-)-MEP on AChE for the search of Y Tang() hool of Pharmacy new mep derivatives as achE inhibitors East China University of Science and Technology, Molecular docking is an efficient tool for investigating Shanghai, 200237, People's Republic of China receptor-ligand interactions and for virtual screening Tel:+86-21-64251052 which plays a key role in rational drug design [13

oh derivatives of both enantiomers connected by spacers length two to 14 carbons at substituents in NI-position(D) or C4-amino group (II)of the azepine ring. Bis-MEP derivatives were believed to make dual action, allowing interactions with both the ester-cleavage peripheral anionic site(PAS)of AChE Fig. 1 The structures of (=)-MEP, ()-MEP, and(+)-MEP Materials and methods especially when the crystal structure of a receptor or such as Auto Dock [15], DOCK [16], FlexX [17], Glide workstation. Molecular performed on a R14000 SGI Fuel [18] and GOLD [19], treat the ligand with full flexibility carried out with the software package SYBYL version 6.9 but assume the receptor to be rigid or apply very limited [32]. The programs GOLD [33] and CORINA [34] were flexibility to side chains. Various searching algorithms used for gOld docking and 2 d to 3 D structural conver such as fast shape matching(DOCK), simulated annealing sion, respectively. Standard parameters were used unless AutoDock), incremental construction(FlexX), genetic otherwise indicated algorithms(GOLD), Monte Carlo(Ligand-Fit, and Tabu earch, have been employed in docking studies [20] Scoring functions are usually categorized as force-field Preparation of proteins based methods(such as DOCK and GOLD), empirical free-energy scoring functions(such as FlexX), and knowl- Three crystal structures of Torpedo californica AChE edge-based scoring functions [20](such as PMF [21]).(TcAChE)complexed with E2020 [25], HUPA [29]and Among various docking algorithms, FlexX and GOLd are THA [31] were retrieved from PDB [24] with correspond the two most reliable methods in complex validation tests ing entry code IEVE, IVOT and lACJ. The proteins were [22] prepared by removing heteroatoms and water molecules Thusly, in the present study we first explored the and adding all hydrogen atoms. The active site of lEVE feasibility of FlexX and GOLD on AChE with seven was defined as residues with at least one atom within a known AChE inhibitors whose complex structures with radius of 10 A from any atom of E2020. For IVOT and AChE [23]are available from the Protein Data Bank(PDB) lAC], a 12 A radius around the corresponding ligand was 24], namely donepezil(E2020)[25], galanthamine(Gnt) defined. The active sites were saved as PDb files for FlexX [26], BW284c51[27], edrophonium(EDR)[28], huperzine docking and MOL2 files for GOLd docking A(HUPA)[29], huprine X(HUPX)[30], tacrine(THA) [ 31](Fig. 2). The better of the two programs was then used as the docking protocol to investigate the binding mode of Preparation of known ligands for pose reproduction ()-MEP on AChE. In addition, a small focused library with 91 bis-MEP derivatives(Fig. 3)was built and The seven known AChE inhibitors were extracted from the careened virtually using the specified docking method to corresponding complex structures with AChE, whose PDB give preferable candidates for synthesis. The compounds codes are lEVE, IQTl, lE3Q, 2ACK, IVoT, 1E66, and among the screening library included the bis-ligand lACJ, respectively. Atom types and bond types were OH BW284c51 E2020 EDR HUPX THA Fig 2 Structures of seven known AChE inhibitors

especially when the crystal structure of a receptor or enzyme is available. Almost all current docking programs, such as AutoDock [15], DOCK [16], FlexX [17], Glide [18] and GOLD [19], treat the ligand with full flexibility but assume the receptor to be rigid or apply very limited flexibility to side chains. Various searching algorithms such as fast shape matching (DOCK), simulated annealing (AutoDock), incremental construction (FlexX), genetic algorithms (GOLD), Monte Carlo (Ligand-Fit), and Tabu search, have been employed in docking studies [20]. Scoring functions are usually categorized as force-field based methods (such as DOCK and GOLD), empirical free-energy scoring functions (such as FlexX), and knowledge-based scoring functions [20] (such as PMF [21]). Among various docking algorithms, FlexX and GOLD are the two most reliable methods in complex validation tests [22]. Thusly, in the present study we first explored the feasibility of FlexX and GOLD on AChE with seven known AChE inhibitors whose complex structures with AChE [23] are available from the Protein Data Bank (PDB) [24], namely donepezil (E2020) [25], galanthamine (GNT) [26], BW284c51 [27], edrophonium (EDR) [28], huperzine A (HUPA) [29], huprine X (HUPX) [30], tacrine (THA) [31] (Fig. 2). The better of the two programs was then used as the docking protocol to investigate the binding mode of (−)-MEP on AChE. In addition, a small focused library with 91 bis-MEP derivatives (Fig. 3) was built and screened virtually using the specified docking method to give preferable candidates for synthesis. The compounds among the screening library included the bis-ligand derivatives of both enantiomers connected by spacers of length two to 14 carbons at substituents in N1- position (I) or C4- amino group (II) of the azepine ring. Bis-MEP derivatives were believed to make dual action, allowing interactions with both the ester-cleavage site and the peripheral anionic site (PAS) of AChE. Materials and methods The study was mainly performed on a R14000 SGI Fuel workstation. Molecular modeling and FlexX docking were carried out with the software package SYBYL version 6.9 [32]. The programs GOLD [33] and CORINA [34] were used for GOLD docking and 2 D to 3 D structural conversion, respectively. Standard parameters were used unless otherwise indicated. Preparation of proteins Three crystal structures of Torpedo californica AChE (TcAChE) complexed with E2020 [25], HUPA [29] and THA [31] were retrieved from PDB [24] with corresponding entry code 1EVE, 1VOT and 1ACJ. The proteins were prepared by removing heteroatoms and water molecules and adding all hydrogen atoms. The active site of 1EVE was defined as residues with at least one atom within a radius of 10 Å from any atom of E2020. For 1VOT and 1ACJ, a 12 Å radius around the corresponding ligand was defined. The active sites were saved as PDB files for FlexX docking and MOL2 files for GOLD docking. Preparation of known ligands for pose reproduction The seven known AChE inhibitors were extracted from the corresponding complex structures with AChE, whose PDB codes are 1EVE, 1QTI, 1E3Q, 2ACK, 1VOT, 1E66, and 1ACJ, respectively. Atom types and bond types were OH N CH3 * OH N CH3 OH N CH3 (±)-MEP (-)-S-MEP (+)-R-MEP Fig. 1 The structures of (±)-MEP, (−)-MEP, and (+)-MEP O N N O N O O N OH O O N OH NH H O 2N Cl N NH2 N NH2 BW284c51 E2020 GNT EDR HUPA THA HUPX Fig. 2 Structures of seven known AChE inhibitors 391

Fig. 3 Structures of designed bis-meP derivatives ( n=2-14 2-14 n=2-14 corrected manually. Hydrogen atoms and Gasteiger-Marsili GOLd docking [35] atomic charges were then added. The structures were minimized in SYBYL for 100 steps with the Tripos force The default parameter settings for three times speed-up field and saved to a multi-MOL2 file were used, and no flipping was allowed In docking runs for pose reproduction, all ten solutions of each ligand were saved for further analysis. However, for virtual screening, Preparation of focused library for virtual screening only the best solution was kept for each molecule. Bis-MEP derivatives with spacer length of two to 14 arbons. a total of 91 molecules were drawn in ISIS/Base Results and discussion with ISIS/Draw from MDL [36] and exported into a 2 D structure data file(SDF)to form a small focused library. Comparison of prediction accuracy of Flexx The 2 D structures of the library were subsequently con- and GOLD verted into 3 D-structures with CORINA [37, still in SDF format. The 3 D structures were then read into SYBYL The accurate prediction of protein-ligand interaction geo- olecular SpreadSheet table for further treatment, such as metries is essential for the success of virtual-screening energy minimization for 100 steps with Tripos force field approaches in structure-based drug design. It requires and Gasteiger-Marsili [35 charges for each molecule. docking tools that are able to generate suitable conforma These structures were put into databases and then written to tions of a ligand within a protein binding site and reliable MOL2 files as input energetic evaluation indicating the quality of the interaction. Flexx [17] uses an incremental construction algorithm where ligands are docked starting with a base fragment Flexx docking The complete ligand is subsequently constructed by adding e remainin omponents. The best solution is selected All Flexx runs used the version 1. ll.I embedded within according to the docking score, using a pure empirical SYBYL. Formal charges were assigned and the Flexx scoring function by Bohm or a knowledge-based scorin scoring function was chosen to evaluate the docking poses. function Drug Score. The GolD [19]( Genetic Optimiza- Table 1. a Fitness scores, RMSD from crystal structures and ICso of known ligands by gold IEVE IVOT IACJ ICso(nM) MOL NAME Fitness RMSD(A) Fitness RMSD(A) Fitness RMSD (A) E2020 66.15 0.5437 48.64 3.3821 46.45 3.7311 13.6 GNT 5753 0.7819 4.8748 4.2323 19954 Half-open BW284c51 80.28 3.5743 85.36 4.1937 63423.6457 0.0036 EDR 44.09 4.2878 43.93 46.02 4.1636 46.35 3.6063 1.21 51550.528 58.4 HUPX 5994 5.3004 59.79 5.0281 0.9428 0.026(K1) THA 45.13 5.8934 49.265.638 50.47 5.6056 From Reference [47] bFrom Reference [48] From Reference [49] dRank 2 scored 50.19 with an rMsD of 0.5404

corrected manually. Hydrogen atoms and Gasteiger-Marsili [35] atomic charges were then added. The structures were minimized in SYBYL for 100 steps with the Tripos force field and saved to a multi-MOL2 file. Preparation of focused library for virtual screening Bis-MEP derivatives with spacer length of two to 14 carbons, a total of 91 molecules, were drawn in ISIS/Base with ISIS/Draw from MDL [36] and exported into a 2 D structure data file (SDF) to form a small focused library. The 2 D structures of the library were subsequently converted into 3 D-structures with CORINA [37], still in SDF format. The 3 D structures were then read into SYBYL Molecular SpreadSheet table for further treatment, such as energy minimization for 100 steps with Tripos force field and Gasteiger-Marsili [35] charges for each molecule. These structures were put into databases and then written to MOL2 files as input. FlexX docking All FlexX runs used the version 1.11.1 embedded within SYBYL. Formal charges were assigned and the FlexX scoring function was chosen to evaluate the docking poses. GOLD docking The default parameter settings for three times speed-up were used, and no flipping was allowed. In docking runs for pose reproduction, all ten solutions of each ligand were saved for further analysis. However, for virtual screening, only the best solution was kept for each molecule. Results and discussion Comparison of prediction accuracy of FlexX and GOLD The accurate prediction of protein-ligand interaction geometries is essential for the success of virtual-screening approaches in structure-based drug design. It requires docking tools that are able to generate suitable conformations of a ligand within a protein binding site and reliable energetic evaluation indicating the quality of the interaction. FlexX [17] uses an incremental construction algorithm where ligands are docked starting with a base fragment. The complete ligand is subsequently constructed by adding the remaining components. The best solution is selected according to the docking score, using a pure empirical scoring function by Böhm or a knowledge-based scoring function DrugScore. The GOLD [19] (Genetic OptimizaOH N * OH N * H2 C n (3',3'') n (S,S) n=2~14 (R,S) n=2~14 (R,R) n=2~14 OH N * OH N * * * H N H N H2 C n (3'4',4''3'') n (SR,RS) n=2~14 (SS,SS) n=2~14 (RR,RR) n=2~14 (RS,SR) n=2~14 3' 3'' 3' 4' 3'' 4'' · · · · Fig. 3 Structures of designed bis-MEP derivatives Table 1.a Fitness scores, RMSD from crystal structures and IC50 of known ligands by GOLD 1EVE 1VOT 1ACJ IC50 (nM) MOL_NAME Fitness RMSD (Å) Fitness RMSD (Å) Fitness RMSD (Å) Open E2020 66.15 0.5437 48.64 3.3821 46.45 3.7311 13.6a GNT 57.53 0.7819 54.82 4.8748 53.29 4.2323 1995a Half- open BW284c51 80.28 3.5743 85.36 4.1937 63.42 3.6457 0.0036b EDR 44.09 4.2878 43.93 1.2034 46.02 4.1636 240b HUPA 46.35 3.6063 50.78 1.2146 51.55 0.528 58.4a Closed HUPX 59.94 5.3004 59.79 5.0281 70.48 0.9428 0.026 (Ki)c THA 45.13 5.8934 49.26 5.638 50.47 5.6056d 93a a From Reference [47], b From Reference [48], c From Reference [49], d Rank 2 scored 50.19 with an RMSD of 0.5404 392

Table 1. b. Flexx scores and rMSd from crystal structures and ligands by Flexx MOL NAME EVE F Score RMSD(A) F Score RMSD(A) RMSD(A) E2020 17545 -114 9.6344 -9.7 13.3023 GNT 0.5628 113 154656 0.7694 Half-Oper BW284c51 -8.9 4.6601 3.764 EDR -16.5 1.2315 HUPA -12.2 134537 -18.3 3.5703 -117 184646 Closed HUPX -14 11.8 15.3927 11.3687 THA -16.3 174666 1.854 13.5 14.7315 tion for Ligand Docking) program uses a genetic algorithm plexes, namely the complexes of E2020(IEVE)[25], GNT to explore the full range of ligand conformational flexibil-(1QTD)[26], BW284c51(1E3Q),[27] EDR(2ACK),[28] ity and the rotational flexibility of selected receptor HUPA(IVOT), [29] HUPX (1E66), [30] and THA (IACd hydrogen atoms. The mechanism for ligand placement is [31]. Among them, the first two complexes show open-gate based on fitting points, which are added to hydrogen- conformations, whereas the last two form closed-gateway onding or hydrophobic groups on both protein and ligand. binding styles. And half-open gate structures are found for And then the points between acceptors and donors map the middle three complexes. In the present study, we each other. The docking poses are ranked based on a performed both FlexX and goLd docking protocols on nolecular mechanics-like scoring function, named fitness seven known AChE inhibitors. Taking three dominant function. These two protocols are verified as two of the orientations of the Phe330 side chain(open, half-open, and most reliable methods in complex validation tests among closed gate)into account, we have considered all three various docking algorithms [22] possibilities in separate docking runs for each of the seven From a comparison of the published X-ray crystal ligands structures of various TcAChE complexes, it is obvious that Scoring calculations showed that six of the seven the side-chain orientation of Phe330 is the only site with ligands'optimal poses determined by GOLD were very flexibility, which is responsible for substrate trafficking close to the original orientation found in the crystal. The down the gorge. Three major orientations of Phe330 have root mean square deviation(RMSD) for all heavy atoms been observed. The TCcAChE-E2020 complex (IEVE)is between the docked and crystal ligand coordinates was characterized by the open-gate conformation, while the below 1.5 A with the exception of BW284c51, when the complex with THA (lACD) displays a closed one, and the actual gate conformations of AChE were considered half-open conformation is observed in the TcAChE-HUPA(highlighted in Table 1. a). And the fitness scores concluded cOmplex(IVOT)[38]. Since is still difficult to deal with from the actual gate conformations conformed to the docking methods, the open, half-open and closed gate of inhibitors was, the higher their fitness score showed For conformations of AChe were investigated separately in the case of BW284c51, molecular complexity and flexi this study to determine the importance of the side-chain bility might play a part in the failure of binding pose flexibility of Phe330 for trafficking prediction. It is worth noting that Flexx performs better To explore the feasibility of the two methods Flexx and than GOLD in many cases. However, in the present OLD, we compared their predictive power of reproduc- exercise, it seemed that Flexx performed less well than ing the binding poses of the known seven AChE com- GOLD (Table 1. b) Table 2 Fitness score and evaluation of the interactions of(-)MEP and(+)-MEP with TcAChE EVE IVOT lACE (-)MEP (+)-MEP ()-MEP (+)-MEP ()-MEP (+)-MEP Fitness score 47.37450244.95 45.7348.34 H-bond interactions Donor Ph-OH Trp84-NH Ph-OH, Ser122-OH Acceptor Glu199-0 None Ph-o Trp84-CO, Ph-0 None Asp72-CO0 Distance 2.57 2.681,2.470 2.054 Hydrophobic interactions(distance/A)Ar-Trp84.320 Ar - Phe330 4.086 3.958 Az-Phe3304.003 Ar-aromatic cycle of MEP bAz--azepine cycle of MEP

tion for Ligand Docking) program uses a genetic algorithm to explore the full range of ligand conformational flexibility and the rotational flexibility of selected receptor hydrogen atoms. The mechanism for ligand placement is based on fitting points, which are added to hydrogenbonding or hydrophobic groups on both protein and ligand. And then the points between acceptors and donors map each other. The docking poses are ranked based on a molecular mechanics-like scoring function, named fitness function. These two protocols are verified as two of the most reliable methods in complex validation tests among various docking algorithms [22]. From a comparison of the published X-ray crystal structures of various TcAChE complexes, it is obvious that the side-chain orientation of Phe330 is the only site with flexibility, which is responsible for substrate trafficking down the gorge. Three major orientations of Phe330 have been observed. The TcAChE-E2020 complex (1EVE) is characterized by the open-gate conformation, while the complex with THA (1ACJ) displays a closed one, and the half-open conformation is observed in the TcAChE-HUPA complex (1VOT) [38]. Since is still difficult to deal with flexibility of the protein thoroughly in current molecular docking methods, the open, half-open and closed gate conformations of AChE were investigated separately in this study to determine the importance of the side-chain flexibility of Phe330 for trafficking. To explore the feasibility of the two methods FlexX and GOLD, we compared their predictive power of reproducing the binding poses of the known seven AChE complexes, namely the complexes of E2020 (1EVE) [25], GNT (1QTI) [26], BW284c51 (1E3Q), [27] EDR (2ACK), [28] HUPA (1VOT), [29] HUPX (1E66), [30] and THA (1ACJ) [31]. Among them, the first two complexes show open-gate conformations, whereas the last two form closed-gateway binding styles. And half-open gate structures are found for the middle three complexes. In the present study, we performed both FlexX and GOLD docking protocols on seven known AChE inhibitors. Taking three dominant orientations of the Phe330 side chain (open, half-open, and closed gate) into account, we have considered all three possibilities in separate docking runs for each of the seven ligands. Scoring calculations showed that six of the seven ligands’ optimal poses determined by GOLD were very close to the original orientation found in the crystal. The root mean square deviation (RMSD) for all heavy atoms between the docked and crystal ligand coordinates was below 1.5 Å with the exception of BW284c51, when the actual gate conformations of AChE were considered (highlighted in Table 1.a). And the fitness scores concluded from the actual gate conformations conformed to the inhibition activities as well (Table 1.a). The lower the IC50 of inhibitors was, the higher their fitness score showed. For the case of BW284c51, molecular complexity and flexibility might play a part in the failure of binding pose prediction. It is worth noting that FlexX performs better than GOLD in many cases. However, in the present exercise, it seemed that FlexX performed less well than GOLD (Table 1.b). Table 1.b. FlexX scores and RMSD from crystal structures and ligands by FlexX MOL_NAME 1EVE 1VOT 1ACJ F_Score RMSD (Å) F_Score RMSD (Å) F_Score RMSD (Å) Open E2020 −12 17.545 −11.4 9.6344 −9.7 13.3023 GNT −27 0.5628 −11.3 15.4656 −24.7 0.7694 Half-Open BW284c51 −8.9 7.049 −14.3 4.6601 −8.7 3.764 EDR −16.5 3.1461 −18 1.2315 −15.2 0.7693 HUPA −12.2 13.4537 −18.3 3.5703 −11.7 18.4646 Closed HUPX −14 16.688 −11.8 15.3927 −13.9 11.3687 THA −16.3 17.4666 −12.8 11.854 −13.5 14.7315 Table 2 Fitness score and evaluation of the interactions of (−)-MEP and (+)-MEP with TcAChE Index 1EVE 1VOT 1ACJ (−)-MEP (+)-MEP (−)-MEP (+)-MEP (−)-MEP (+)-MEP Fitness score 47.37 45.02 44.95 44.34 45.73 48.34 H-bond interactions Donor Ph-OH – Trp84-NH Ph-OH, Ser122-OH – Ph-OH Acceptor Glu199-O None Ph-O Trp84-CO, Ph-O None Asp72-COO Distance/Å 1.953 – 2.557 2.681, 2.470 – 2.054 Hydrophobic interactions (distance/Å) Ara - Trp84 4.320 – 4.045 – – 4.720 Ar - Phe330 – 3.519 4.118 – – 4.107 Azb -Trp84 – 4.086 – 3.958 3.777 – Az - Phe330 4.003 –– – 4.281 – a Ar—aromatic cycle of MEP b Az—azepine cycle of MEP 393

Fig 4 Best docking solution of MEP into the open pocket (EVE): a(-MEP (leff); b(+)- MEP (right) Phe330 Phe330 The low RMSD and the consistency of calculated site consisting of some aromatic residues such as Trp84 and affinity with experimental activity indicated that the gold Phe330 method and parameter set were reasonable to reproduce the In order to explore the mechanism of action of MEP X-ray structure and could be extended to search and isomers on AChE and explain the reason of their differ- evaluate the binding poses of other AChE ds ences in activity, which is valuable for structure-based accordingly design of MEP derivatives with desired pharmacological On the other hand, comparison of the results of a specific properties, both S and R isomers of MEP were considered ligand from three conformations of AChe demonstrated for GOLD docking runs. ()-MEP and (+)-MEP were that the highest scoring pose was derived from the actual submitted to GOLd separately with the three gate gate conformation and indicated the lowest RMSD. Thus, conformations of AChE to consider the flexibility of the the experimental gate conformation of AChE complexed binding site of AChE with an unknown ligand could be predicted roughly by Fitness scores of the best solutions and detailed inter comparing the fitness score concluded from three gate action indices of both isomers are listed in Table 2.The conformations of achE hydrogen-bond (HB)interaction was evaluated by the dis tance between HB donor and acceptor atoms. The hydro phobic interaction was estimated according to the distance Predicted binding conformation of(-)-MEP between the centroid of aromatic or azepine cycle of MEP with achE and the centroid of the side train of the hydrophobic residue Trp84 or Phe330. Figs. 4, 5, 6 illustrated the binding maps The X-ray structure of AChE illustrated that the active site of (-)-MEP and (+)-MEP with the binding site of three of the enzyme was a deep and narrow gorge with such TcAChE conformations features [39]: a catalytic triad composed of residues The highest score of ()-MEP was found for the open Ser200, Glu327 and His440; a peripheral anionic site at pocket(IEVE), while that of (+)-MEP was found for the the entry centered by residue Trp279; and a hydrophobic closed one (lAC)(Table 2). Although the actual gate 5 Best docking solution of into the half-o T): a()-MEP (lefn); b(+)- Phes 30 Phe330

The low RMSD and the consistency of calculated affinity with experimental activity indicated that the GOLD method and parameter set were reasonable to reproduce the X-ray structure and could be extended to search and evaluate the binding poses of other AChE ligands accordingly. On the other hand, comparison of the results of a specific ligand from three conformations of AChE demonstrated that the highest scoring pose was derived from the actual gate conformation and indicated the lowest RMSD. Thus, the experimental gate conformation of AChE complexed with an unknown ligand could be predicted roughly by comparing the fitness score concluded from three gate conformations of AChE. Predicted binding conformation of (−)-MEP with AChE The X-ray structure of AChE illustrated that the active site of the enzyme was a deep and narrow gorge with such features [39]: a catalytic triad composed of residues Ser200, Glu327 and His440; a peripheral anionic site at the entry centered by residue Trp279; and a hydrophobic site consisting of some aromatic residues such as Trp84 and Phe330. In order to explore the mechanism of action of MEP isomers on AChE and explain the reason of their differences in activity, which is valuable for structure-based design of MEP derivatives with desired pharmacological properties, both S and R isomers of MEP were considered for GOLD docking runs. (−)-MEP and (+)-MEP were submitted to GOLD separately with the three gate conformations of AChE to consider the flexibility of the binding site of AChE. Fitness scores of the best solutions and detailed interaction indices of both isomers are listed in Table 2. The hydrogen-bond (HB) interaction was evaluated by the distance between HB donor and acceptor atoms. The hydrophobic interaction was estimated according to the distance between the centroid of aromatic or azepine cycle of MEP and the centroid of the side train of the hydrophobic residue Trp84 or Phe330. Figs. 4, 5, 6 illustrated the binding maps of (−)-MEP and (+)-MEP with the binding site of three TcAChE conformations. The highest score of (−)-MEP was found for the open pocket (1EVE), while that of (+)-MEP was found for the closed one (1ACJ) (Table 2). Although the actual gate Fig. 4 Best docking solution of MEP into the open pocket (1EVE): a (−)-MEP (left); b (+)- MEP (right) Fig. 5 Best docking solution of MEP into the half-open pocket (1VOT): a (−)-MEP (left); b (+)- MEP (right) 394

95 Fig. 6 Best docking solution of MEP into the closed pocket (IAC: a(-)-MEP (lefi; b(+)- MEP (right) Phe330 羚yt好 b nformation of AChe binding with ()-MEP and (+) Based on the crystallographic structure of TcAChE, two MEP might not be the same, their highest-score values did important active sites are involved: a catalytic triad located not conform to the activity data. In addition, up to now only at the bottom of the cavity, and a PAs at the opening of the rigid plane-shaped molecules like THA and HUPX were gorge. The distance between Trp 84 and Trp 279 is 12 A und bound in the closed gateway, forming a sandwich- [31]. We therefore presumed that the bis-ligands could like hydrophobic interaction. MEP, a flexible structure, did interact simultaneously with the active and peripheral sites not seem to have enough chance of generating a closed- and thereby optimize the inhibiting potency. The potency gate conformation(Fig 6a and b) of bis-THA [41, 42], bis-GNT [43, bis-HUPA [44, bis- The best-ranking poses of MEP isomers in the half-open HUPB [45], and bis-hupyridone, a simplified HUPA-like pocket(IVOT)(Fig 5a and b)adopted weaker and less monomer [46], all significantly increased when linked by pecific HB interactions. The HB-forming distance was proper methylene spacers due to interactions with both the on-specific. Both the fitness score or the HB specificity inesterase(BChE)selectiv larger than those in other pockets. HB interactions with the catalytic site and the PAs. Bis(7)-THA possessed both amino or carbonylic group of the backbone of Trp84 were optimum AChE inhibition potency and AChE/butyrylcho and intensity suggest that I VOT might not be the correct Aiming to find any molecules with high potency and pocket for MEP selectivity for synthesis and further test, we designed 91 As illuminated by Ennis' pharmacological experiments bis-MEP derivatives and screened them virtually on the [ll],()-MEP should score much higher than(+)-MEP, basis of the methods established above. Two series of which was consistent with the scores derived from the open derivatives were classified based on the linkage position in ocket. Therefore we thought that(-)-MEP and (+)-MEP the azepine cycle. Series I was linked at the NI-tertiary th bound to the open-gate conformation of AChE similarly to the E2020-AChE complex (IEVE). In this Table 3 Rank, ID number, and the fitness scores of the top 15bis- binding site(Fig. 4a), the phenyl ring of (-)-MEP formed a MEP derivatives face-to-face 7- stacking interaction with Trp84(4.320 A), Rank ID number and the seven-membered azepine ring of (-)-MEP formed Seies number n Fitness score a hydrophobic interaction with the side chain of Phe330 bis 1(S,R)n=3 I(S, R) 369.19 (4.003 A). A strong HB, the absence of which caused the 2 bis 2(RS. SR)n=3 II(RS.SR) 3 68.17 poor score of (+)-MEP(Fig 4b), was formed between the 3 bis 1_(SS)_/=3 I(S,S) 363.85 hydroxyl of ()-MEP and the carboxyl group of Glul99 4 bis 1(S, S)n=2 262.24 (1.953A) 5 bis 2(RS, SR)n=7 II(RS, SR) 7 61.52 6 bis 1(S,S)n=7 I(S,S) 61.12 7 bis 2(SR, RS)n=3 8 bis 1(R, R)I=4 I(R, R) 457.56 Results from virtual screening of bis- meP by GOLD 9 bis 2(SR, RS)n=2 I(SR, RS) 2 56.51 In an effort to improve the potency and selectivity, the 10 bis_1_(S,S)_=5 11 bis 2(SR, RS)n=4 II(SR, R 45547 bivalent ligand strategy is applied to the development of 12 bis_2_(SR,RS)_m=6 Il(SR,RS)655.35 new the peutic agents. For AChe, drug units that have little or no intrinsic affinity can function as effective 13 bis_2_(SR, RS)_m=8 II(SR,RS)855.27 catalytic and/or peripheral site ligands when incorporated 14 bis 1(S, S )_=4 I(S, S) into a bivalent drug[40] bisL(RR)=21(R25508

conformation of AChE binding with (−)-MEP and (+)- MEP might not be the same, their highest-score values did not conform to the activity data. In addition, up to now only rigid plane-shaped molecules like THA and HUPX were found bound in the closed gateway, forming a sandwichlike hydrophobic interaction. MEP, a flexible structure, did not seem to have enough chance of generating a closedgate conformation (Fig. 6a and b). The best-ranking poses of MEP isomers in the half-open pocket (1VOT) (Fig. 5a and b) adopted weaker and less specific HB interactions. The HB-forming distance was larger than those in other pockets. HB interactions with the amino or carbonylic group of the backbone of Trp84 were non-specific. Both the fitness score or the HB specificity and intensity suggest that 1VOT might not be the correct pocket for MEP. As illuminated by Ennis’ pharmacological experiments [11], (−)-MEP should score much higher than (+)-MEP, which was consistent with the scores derived from the open pocket. Therefore we thought that (−)-MEP and (+)-MEP both bound to the open-gate conformation of AChE similarly to the E2020-AChE complex (1EVE). In this binding site (Fig. 4a), the phenyl ring of (−)-MEP formed a face-to-face π-π stacking interaction with Trp84 (4.320 Å), and the seven-membered azepine ring of (−)-MEP formed a hydrophobic interaction with the side chain of Phe330 (4.003 Å). A strong HB, the absence of which caused the poor score of (+)-MEP (Fig. 4b), was formed between the hydroxyl of (−)-MEP and the carboxyl group of Glu199 (1.953 Å). Results from virtual screening of bis-MEP by GOLD In an effort to improve the potency and selectivity, the bivalent ligand strategy is applied to the development of new therapeutic agents. For AChE, drug units that have little or no intrinsic affinity can function as effective catalytic and/or peripheral site ligands when incorporated into a bivalent drug [40]. Based on the crystallographic structure of TcAChE, two important active sites are involved: a catalytic triad located at the bottom of the cavity, and a PAS at the opening of the gorge. The distance between Trp 84 and Trp 279 is 12 Å [31]. We therefore presumed that the bis-ligands could interact simultaneously with the active and peripheral sites and thereby optimize the inhibiting potency. The potency of bis-THA [41, 42], bis-GNT [43], bis-HUPA [44], bisHUPB [45], and bis-hupyridone, a simplified HUPA-like monomer [46], all significantly increased when linked by proper methylene spacers due to interactions with both the catalytic site and the PAS. Bis(7)-THA possessed both optimum AChE inhibition potency and AChE/butyrylcholinesterase (BChE) selectivity. Aiming to find any molecules with high potency and selectivity for synthesis and further test, we designed 91 bis-MEP derivatives and screened them virtually on the basis of the methods established above. Two series of derivatives were classified based on the linkage position in the azepine cycle. Series I was linked at the N1-tertiary Table 3 Rank, ID number, and the fitness scores of the top 15 bisMEP derivatives Rank ID number Seies number n Fitness score 1 bis_1_(S,R)_n=3 I (S,R) 3 69.19 2 bis_2_(RS,SR)_n=3 II (RS,SR) 3 68.17 3 bis_1_(S,S)_n=3 I (S,S) 3 63.85 4 bis_1_(S,S)_n=2 I (S,S) 2 62.24 5 bis_2_(RS,SR)_n=7 II (RS,SR) 7 61.52 6 bis_1_(S,S)_n=7 I (S,S) 7 61.12 7 bis_2_(SR,RS)_n=3 II (SR,RS) 3 60.76 8 bis_1_(R,R)_n=4 I (R,R) 4 57.56 9 bis_2_(SR,RS)_n=2 II (SR,RS) 2 56.51 10 bis_1_(S,S)_n=5 I (S,S) 5 55.5 11 bis_2_(SR,RS)_n=4 II (SR,RS) 4 55.47 12 bis_2_(SR,RS)_n=6 II (SR,RS) 6 55.35 13 bis_2_(SR,RS)_n=8 II (SR,RS) 8 55.27 14 bis_1_(S,S)_n=4 I (S,S) 4 55.1 15 bis_1_(R,R)_n=2 I (R,R) 2 55.08 Fig. 6 Best docking solution of MEP into the closed pocket (1ACJ): a (−)-MEP (left); b (+)- MEP (right) 395

amine position and Series II at the primary amine sub- 6. Greenblatt HM, Kryger G, Lewis T, Silman I, Sussman JL stituted position. Both enantiomers were combined suc (1999 FEBS Lett463:321-326 cessionally to consider the chiral influence of each 7. Zhang RW, Tang XC, Han YY, Sang GW, Zhang YD, Ma YX, Zhang CL, Yang RM (1991) Acta Pharmacol Sin 12: 250-252 monomer fully Spacer lengths of the carbon bridge ranged 8.Ford JM, Truman CA, Wilcock GK, Roberts CJC(1993)Clin from two to 14-carbons The conformation of ache was harmacol Ther 53: 691-69 assumed to be open to allow the entrance of these bulky 9 Hoskin P), Hanks gw(991) Drugs 41: 326-344 bis-ligands with long chain linkages Pharmacol 79: 191-199 e Table an sti ths shils coar ter tethys bi MEp I. ER -S7 Haroun E, Latimer N (19%6) J Pharm Pharmacol derivatives. Remarkably improved score values, the top 12. Chen Y(2004)Studies on the synthesis, resolution and optical wo of which almost exceed that of E2020. suggested the somers of meptazinol. Dissertation, Shanghai, Fudan Uni- potential optimization of potency. Interaction analysis 13. Kuntz ID(1992) Science 257: 1078-1082 (pictures omitted) showed that bis-MEP derivatives made 14. Drews J(2000) Science 287: 1960-196 good hydrophobic interactions with the residue Trp279 at 15. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart We the PAs. Comparing the chirality of the two series, we Belew RK, Olson AJ(1998)J Comput Chem 19: 1639-16 found that compounds derived from()-S-MEP with the s, 16. Kuntz ID, Blaney JM, Oatley SJ, Langidge R, Ferrin TE (1982) J Mol Biol 161: 269-288 S-conformation of Series I and the SR, RS-conformation of 17. Rarey M, Kramer B, Lengauer T, Klebe GA(1996)J Mol Biol Series Il dominated at the top of the list, which conformed 261:470489 mEP derivatives with a spacer of two to seven showed high Mainz DT, Repasky MP. Knoll EH, Shel ry求k,Shaw 1739-1749 score values, among which 3-and 7-methylene compounds 19. Jones G. Willett P, Glen RC, Leach AR, R(1997)J Mol were found to be the favored candidates. Under the Biol 267: 727-748 guidance of these results, synthesis of the corresponding 20. Hu x, Balaz s, Shelver WH(2004)JMol Graph Model compounds is underway 21. Muegge I, Martin YC(1999)J 2. Kontoyianni M, McClellan LM s Chem Conclusions Greenblatt HM, Dvir H, Silman I, Sussman JL (2003)J Mol Neurosci 20: 369-383 In this paper, we have established a useful docking method 24. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN to predict the binding poses of AChE inhibitors. Using the essig H, shindyalov IN, Bourne Pe (2000) Nucleic Acids Res28:235-242 GOLD docking protocol, the binding conformation and 25 Kryger G, Sliman L, Sussman JL(1999)Structure 7: 297- orientation of()-MEP with TcAChE was illuminated and 26 Bartolucci C, Perola e, Pilger C, Lamba D the potentially preferable bis-MEP derivative candidates screenin To 27. Felder CE, Harel M, Silman 1, Sussman JL (2002)Acta his is the first exploration of the mechanism of action of 28. Ravelli RBG, Raves ML, Ren Z, Bourgeois D, Roth M, Kroon MEP on AChE. Synthesis and biological evaluation of J, Siman I, Sussman JL(1998)Acta Crystallogr Sect these selected analogues are currently underway and the results will be reported in the due course. Preliminary :9. Raves ML, Harel M, Pang YP, Silman I, Kozikowski AP usman JL (1997) Nat Struct Biol 4: 57-63 iological results implied that part of the candidates 30 Dvir H, Wong DM, Harel M, Barril X, Orozco M, Munoz- showed higher AChE inhibition than the core molecule Torrero FJ, Luque D, Camps P, Rosenberry TL, Silman L, )MEP. Although further biological results are needed to Sussman JL (2002) Biochemistry 41: 2970-2981 attest the actual predictive power of this method, the 31 Harel M. Schalk L Ehret-Sabatier L, Bouet F, Goeldner M present study provides an altemate tool for structural Acad Sci90:9031-9035 optimization of (-)-MEP as new AChE inhibitors 32 SYBYL, version 6.9(2002) Tripos Inc, St. Louis, MO, USA 33. GOLD, version 2.1.(2004) Cambridge Crystallographic Data Centre. Cambridge. UK Acknowledgement We gratefully acknowledge financial support 34 CORINA, version 3.0.(2004)Molecular Networks GmbH, from the National Natural science Foundation of china 35. Gasteiger J, Marsili M(1980) Tetrahedron 36: 3219-3228 36 MDL 5.0(2002)MDL Information Systems Inc, San Leandro, A. USA References 37 Gasteiger J, Rudolph C, Sadowski J(1990)Tetrahedron Comp 1. Marchbanks RM(1982)J Neurochem 39: 9-15 38 Pilger C, Bartolucci C, Lamba D, Tropsha A, Fels G(2001)J Mol Graph Model 19: 288-296 2. Coyle JT, Price DL, Delong MR(1983) Science 219: 1184- 39. Sy 4. Kawakami Y, Inoue A, Kawai T, Wakita M, Sugimoto H 40 Carlier PR, Chow ESH, Han YF, Jing Liu J, EI Yazal J, Pang 3. Davis KL, Powchik P(1995) Lancet 345: 625-630 YP(1999) J Med Chen42:42254231 5.EnzA,Boddeke H, Gray J, Spiegel R(1991)Ann NY Acad Sci 41. Pang YP, Quiram P, Jelacic T, Hong E, Brimijoin S(1996)J Hopfinger AJ(1996) Bioorg Med Chem 4: 1429-144 Biol chen271:23646-23649

amine position and Series II at the primary amine substituted position. Both enantiomers were combined successionally to consider the chiral influence of each monomer fully. Spacer lengths of the carbon bridge ranged from two to 14-carbons. The conformation of AChE was assumed to be open to allow the entrance of these bulky bis-ligands with long chain linkages. Table 3 lists the chiral character, methylene group length, and fitness scores of the top 15 bis-MEP derivatives. Remarkably improved score values, the top two of which almost exceed that of E2020, suggested the potential optimization of potency. Interaction analysis (pictures omitted) showed that bis-MEP derivatives made good hydrophobic interactions with the residue Trp279 at the PAS. Comparing the chirality of the two series, we found that compounds derived from (−)-S-MEP with the S, S-conformation of Series I and the SR,RS-conformation of Series II dominated at the top of the list, which conformed with the activity of (−)-S-MEP versus (+)-R-MEP. BisMEP derivatives with a spacer of two to seven showed high score values, among which 3- and 7-methylene compounds were found to be the favored candidates. Under the guidance of these results, synthesis of the corresponding compounds is underway. Conclusions In this paper, we have established a useful docking method to predict the binding poses of AChE inhibitors. Using the GOLD docking protocol, the binding conformation and orientation of (−)-MEP with TcAChE was illuminated and the potentially preferable bis-MEP derivative candidates were picked out by virtual screening. To our knowledge, this is the first exploration of the mechanism of action of MEP on AChE. Synthesis and biological evaluation of these selected analogues are currently underway and the results will be reported in the due course. Preliminary biological results implied that part of the candidates showed higher AChE inhibition than the core molecule (−)MEP. Although further biological results are needed to attest the actual predictive power of this method, the present study provides an alternate tool for structural optimization of (−)-MEP as new AChE inhibitors. Acknowledgement We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant 30472088). References 1. Marchbanks RM (1982) J Neurochem 39:9–15 2. Coyle JT, Price DL, Delong MR (1983) Science 219:1184– 1190 3. Davis KL, Powchik P (1995) Lancet 345:625–630 4. Kawakami Y, Inoue A, Kawai T, Wakita M, Sugimoto H, Hopfinger AJ (1996) Bioorg Med Chem 4:1429–1446 5. Enz A, Boddeke H, Gray J, Spiegel R (1991) Ann NY Acad Sci 640:272–275 6. Greenblatt HM, Kryger G, Lewis T, Silman I, Sussman JL (1999) FEBS Lett 463:321–326 7. Zhang RW, Tang XC, Han YY, Sang GW, Zhang YD, Ma YX, Zhang CL, Yang RM (1991) Acta Pharmacol Sin 12:250–252 8. Ford JM, Truman CA, Wilcock GK, Roberts CJC (1993) Clin Pharmacol Ther 53:691–695 9. Hoskin PJ, Hanks GW (1991) Drugs 41:326–344 10. Bill DJ, Hartley JE, Stephens RJ, Thompson AM (1983) Br J Pharmacol 79:191–199 11. Ennis C, Haroun F, Lattimer N (1986) J Pharm Pharmacol 38:24–27 12. Chen Y (2004) Studies on the synthesis, resolution and optical isomers of meptazinol. Dissertation, Shanghai, Fudan University 13. Kuntz ID (1992) Science 257:1078–1082 14. Drews J (2000) Science 287:1960–1964 15. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ (1998) J Comput Chem 19:1639–1662 16. Kuntz ID, Blaney JM, Oatley SJ, Langidge R, Ferrin TE (1982) J Mol Biol 161:269–288 17. Rarey M, Kramer B, Lengauer T, Klebe GA (1996) J Mol Biol 261:470–489 18. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS (2004) J Med Chem 47:1739–1749 19. Jones G, Willett P, Glen RC, Leach AR, Taylor R (1997) J Mol Biol 267:727–748 20. Hu X, Balaz S, Shelver WH (2004) J Mol Graph Model 22:293–307 21. Muegge I, Martin YC (1999) J Med Chem 42:791–804 22. Kontoyianni M, McClellan LM, Sokol GS (2004) J Med Chem 47:558–565 23. Greenblatt HM, Dvir H, Silman I, Sussman JL (2003) J Mol Neurosci 20:369–383 24. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) Nucleic Acids Res 28:235–242 25. Kryger G, Sliman I, Sussman JL (1999) Structure 7:297–307 26. Bartolucci C, Perola E, Pilger C, Fels G, Lamba D (2001) Proteins 42:182–191 27. Felder CE, Harel M, Silman I, Sussman JL (2002) Acta Crystallogr Sect D 58:1765–1771 28. 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Pilger C, Bartolucci C, Lamba D, Tropsha A, Fels G (2001) J Mol Graph Model 19:288–296 39. Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I (1991) Science 253:872–879 40. Carlier PR, Chow ESH, Han YF, Jing Liu J, El Yazal J, Pang YP (1999) J Med Chem 42:4225–4231 41. Pang YP, Quiram P, Jelacic T, Hong F, Brimijoin S (1996) J Biol Chem 271:23646–23649 396

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