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 MEPtion for Ligand Docking) program uses a genetic algorithm to explore the full range of ligand conformational flexibil￾ity and the rotational flexibility of selected receptor hydrogen atoms. The mechanism for ligand placement is based on fitting points, which are added to hydrogen￾bonding 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 reproduc￾ing the binding poses of the known seven AChE com￾plexes, 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 flexi￾bility 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