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(R25508conformation 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 sandwich￾like hydrophobic interaction. MEP, a flexible structure, did not seem to have enough chance of generating a closed￾gate 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], bis￾HUPB [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/butyrylcho￾linesterase (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 bis￾MEP 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