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 inhibitorsespecially 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 knowl￾edge-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 conver￾sion, 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 correspond￾ing 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