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 carbonassigned. 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