2030 Journal of Medicinal Chemistry, 2008, VoL. 51, No. 7 Xie et al inhibitory activities of 5a and 5b(43 and 42 uM, respectively) buried within the core of the enzyme binds with the catalytic are similar to that of(-)-MEP. Therefore, the effective alkylene site via face-to-face T-stacking interaction with Trp86(distance bridged bis-(-)-nor-MEP analogues required chains of suitable between the two centroids: 4.27 A). The other nor-MEP moiety length, which was 9 for the best, to bind at both the catalytic reaches the peripheral site on the surface of the enzyme by site and the peripheral site of the AChE binding pocket(gorge). cation-T and hydrophobic interactions between the seven- Compared with the AChE activity, the BChE inhibitory membered azepane ring and Trp286(distance between the two potency was less impacted by chain length(Figure 3). The centroids: 4.09 A). The spatial distance between the centroids ajority of bis-(-)-MEP analogues showed inhibition on BChE of the centric phenyl group and the peripheral azepane ring is of about 100 nM, although the highest potency (Cso=10 nM) 13.5 A, consistent with the reported distance between two was achieved in 5h. This isomer was 1500 times and 150 times tryptophane. 45h forms two hydrogen bonds, both in the more potent than(-)-MEP (ICso= 15 uM)and rivastigmine catalytic active site. The hydroxyl is hydrogen-bonded to the (Cso= 1.6 uM), respectively, but only 10 times lower than main-chain carbonyl oxygen of His447(O-O distance: 2.87 that of the majority compounds (Table 1). The reason that the A). Meanwhile, the protonated azepane amino group is hydrogen AChE inhibition seems to be the enzymic conformational A). In addition, other aliphatic and aromatic residues are difference. There is lack of a functional peripheral site involved in hydrophobic interactions BChE,2.29 and the BChE active site is wider throughout In the binding mode of 5h with the active sit Therefore, there is no restriction of linker length for bivalent of hBChE is shown in Figure 4c, d. 5h is folded in the large BChE inhibitors cavity along the aliphatic residue-dominated wall, and the Most bis-ligand analogues showed greater selectivity for separation of two terminal(-)-nor-MEP units is relatively short. BChE because of their low affinity for AChE. Only four Three hydrogen bonds are found: (i)between the phenolic compounds, 5h, 51, 5j, and 5k, demonstrated slightly more hydroxyl within the core of the enzyme and the carboxylic acid selectivity for AChE. Recent evidence suggests that both AChE xygen of Asp70(O-O distance, 2.49 A);(ii) between the R四hh图 nd progression of Carboxylic acid ox gen of GI.a21 the activity of AChE decreases progressively in certain regions and (ii) between the oxygen on the phenolic hydroxyl at the to reach 10-15% of normal values, whereas the activity of BChE entrance and the side chain amide nh of Ginl19(O.N stays unchanged or is even increased by 20%.30 Thus, it may distance,3.22 A). Ala 277 and Ala328 are not so importantly not be an advantage for a ChE inhibitor to be considerably more involved in the hydrophobic interactions with 5h, unlike the elective for AChE; on the contrary, a good balance between way their counterparts Try286 and Tyr337 in the mAChE AChE and BChE may result in higher efficacy. As a dual catalytic site behave. On the contrary, some residues unique to inhibitor of both AChE and BChE, rivastigmine appears to be hBChE, such as GInI 19, Leu286, and Val288, form hydrophobic beneficial for people with mild to moderate AD, and BChE contacts with 5h. nhibition correlates significantly with cognitive improvement The difference in the binding mode as well as in pharmace in these patients. In our study, although 5h is slightly more logical activities between 5h with AChE and BChE is funda selective for AChE than BChE (2.6), it had the greatest mentally caused by conformational differences between the two inhibition at a nanomolar lever on both enzymes. Therefore, it enzymes. One of the most important differences is the lack of as suggested that it was a promising drug candidate worthy peripheral site in BChE. Residues responsible for J-T or of further investigations. cation-T interactions at AChE peripheral site are replaced by Molecular Docking Studies. Molecular docking study was aliphatic residues in BChE. For example, Trp286, Tyr72, and performed to ascertain the possibility for the most potent Tyrl24 in mAChE are the counterparts of Ala277, Asn68, and ompound Sh to bind at both the catalytic and peripheral sites GInl1g in hBChE, respectively. In addition, some bulky of AChE and to explore the difference in the interactions of Sh aromatic residues in AChE active site have been replaced by with AChE and BChE. Mammalian enzymes were used in small aliphatic ones in BChE. As a result, the active site of docking, compatible with the pharmacological test. Because of BChE is wider and able to accommodate bis-ligands with linkers the unavailability of crystallographic data of mouse BChE. of wider-ranging lengths. These might be the main reasons that hBChE was used instead because there is a high sequence the bche inhibition is less sensitive to the linker length identity (82%)especially betwe ouse and human bche and Inhibition of A ChE-lnduced AB Aggregation. Three com- the residues in active sites are highly conservative. Here, we pounds, 5g, 5h, and 5i, were selected to assess their abilities to chose recently resolved X-ray crystal structures of the mAChE inhibit AB aggregation induced by AChE using a thioflavin complex with succinylcholine'with a high resolution of 2.05 T-based fluorometric assay, compared with the reference A and of native hBChE28 with a 2.0 A resolution. GOLD compound propidium iodine(Sigma-Aldrich), a known specific docking protocol was employed because it has been proved by peripheral site-binding inhibitor, and the monomer(-)-MEP our previous study to be accurate and reliable for reproducing (Table 2). Results showed that Sh and 5i markedly prevented the binding modes of seven AChE inhibitors in their X-ray the AChE-induced AB aggregation with ICso values of 16.6 and rystal structures of Torpedo californica AChE (TcAChE) 5.8 uM, similar to that of propidium(IC50= 12. 8 uM). With a complexes. An advanced consensus scoring technology was used small ICso value and a higher efficiency of inhibition, 5i and to guide the selection of the most reliable conformation from a 5h were the wonderful compounds that inhibited the aggregation set of candidate conformations that GOLD generated of AB induced by AChE. In contrast, (-)-MEP and 5g showed Our results show that Sh is able to simultaneously make fairly low inhibitory activity (Table 2), which indicated their ontact with both the catalytic and peripheral sites of mAChE, limited ability to interact with the peripheral site of the enzyme s illustrated in Figure 4a. The key interactions of these dimeric Different behaviors of these four compounds were attributed inhibitors with the catalytic and peripheral sites are T-stacking to different lengths of linker, demonstrating that a linker no and cation- interactions. The phenyl group of the nor-MEP horter than 9 was necessary to inhibit AChE-induced Ainhibitory activities of 5a and 5b (43 and 42 µM, respectively) are similar to that of (-)-MEP. Therefore, the effective alkylene￾bridged bis-(-)-nor-MEP analogues required chains of suitable length, which was 9 for the best, to bind at both the catalytic site and the peripheral site of the AChE binding pocket (gorge). Compared with the AChE activity, the BChE inhibitory potency was less impacted by chain length (Figure 3). The majority of bis-(-)-MEP analogues showed inhibition on BChE of about 100 nM, although the highest potency (IC50 ) 10 nM) was achieved in 5h. This isomer was 1500 times and 150 times more potent than (-)-MEP (IC50 ) 15 µM) and rivastigmine (IC50 ) 1.6 µM), respectively, but only 10 times lower than that of the majority compounds (Table 1). The reason that the BChE inhibition is less sensitive to the linker length than the AChE inhibition seems to be the enzymic conformational difference. There is lack of a functional peripheral site in BChE,28,29 and the BChE active site is wider throughout. Therefore, there is no restriction of linker length for bivalent BChE inhibitors. Most bis-ligand analogues showed greater selectivity for BChE because of their low affinity for AChE. Only four compounds, 5h, 5i, 5j, and 5k, demonstrated slightly more selectivity for AChE. Recent evidence suggests that both AChE and BChE may play roles in the etiology and progression of AD beyond regulation of synaptic ACh levels. In the AD brain, the activity of AChE decreases progressively in certain regions to reach 10–15% of normal values, whereas the activity of BChE stays unchanged or is even increased by 20%.30 Thus, it may not be an advantage for a ChE inhibitor to be considerably more selective for AChE; on the contrary, a good balance between AChE and BChE may result in higher efficacy. As a dual inhibitor of both AChE and BChE, rivastigmine appears to be beneficial for people with mild to moderate AD, and BChE inhibition correlates significantly with cognitive improvement in these patients.31 In our study, although 5h is slightly more selective for AChE than BChE (2.6), it had the greatest inhibition at a nanomolar lever on both enzymes. Therefore, it was suggested that it was a promising drug candidate worthy of further investigations. Molecular Docking Studies. Molecular docking study was performed to ascertain the possibility for the most potent compound 5h to bind at both the catalytic and peripheral sites of AChE and to explore the difference in the interactions of 5h with AChE and BChE. Mammalian enzymes were used in docking, compatible with the pharmacological test. Because of the unavailability of crystallographic data of mouse BChE, hBChE was used instead because there is a high sequence identity (82%) especially between mouse and human BChE and the residues in active sites are highly conservative. Here, we chose recently resolved X-ray crystal structures of the mAChE complex with succinylcholine32 with a high resolution of 2.05 Å and of native hBChE28 with a 2.0 Å resolution. GOLD33 docking protocol was employed because it has been proved by our previous study18 to be accurate and reliable for reproducing the binding modes of seven AChE inhibitors in their X-ray crystal structures of Torpedo californica AChE (TcAChE) complexes. An advanced consensus scoring technology was used to guide the selection of the most reliable conformation from a set of candidate conformations that GOLD generated. Our results show that 5h is able to simultaneously make contact with both the catalytic and peripheral sites of mAChE, as illustrated in Figure 4a. The key interactions of these dimeric inhibitors with the catalytic and peripheral sites are π-stacking and cation-π interactions. The phenyl group of the nor-MEP buried within the core of the enzyme binds with the catalytic site via face-to-face π-stacking interaction with Trp86 (distance between the two centroids: 4.27 Å). The other nor-MEP moiety reaches the peripheral site on the surface of the enzyme by cation-π and hydrophobic interactions between the seven￾membered azepane ring and Trp286 (distance between the two centroids: 4.09 Å). The spatial distance between the centroids of the centric phenyl group and the peripheral azepane ring is 13.5 Å, consistent with the reported distance between two tryptophanes.14 5h forms two hydrogen bonds, both in the catalytic active site. The hydroxyl is hydrogen-bonded to the main-chain carbonyl oxygen of His447 (O ··· O distance: 2.87 Å). Meanwhile, the protonated azepane amino group is hydrogen￾bonded to the hydroxyl oxygen of Tyr124 (N···O distance: 3.22 Å). In addition, other aliphatic and aromatic residues are involved in hydrophobic interactions, as shown in Figure 4b. In comparison, the binding mode of 5h with the active site of hBChE is shown in Figure 4c,d. 5h is folded in the large cavity along the aliphatic residue-dominated wall, and the separation of two terminal (-)-nor-MEP units is relatively short. Three hydrogen bonds are found: (i) between the phenolic hydroxyl within the core of the enzyme and the carboxylic acid oxygen of Asp70 (O ··· O distance, 2.49 Å); (ii) between the phenolic hydroxyl at the entrance of the enzyme and the carboxylic acid oxygen of Glu276 (O ··· O distance, 2.61 Å); and (iii) between the oxygen on the phenolic hydroxyl at the entrance and the side chain amide NH of Gln119 (O ··· N distance, 3.22 Å). Ala 277 and Ala328 are not so importantly involved in the hydrophobic interactions with 5h, unlike the way their counterparts Try286 and Tyr337 in the mAChE catalytic site behave. On the contrary, some residues unique to hBChE, such as Gln119, Leu286, and Val288, form hydrophobic contacts with 5h. The difference in the binding mode as well as in pharmaco￾logical activities between 5h with AChE and BChE is funda￾mentally caused by conformational differences between the two enzymes. One of the most important differences is the lack of peripheral site in BChE. Residues responsible for π-π or cation-π interactions at AChE peripheral site are replaced by aliphatic residues in BChE. For example, Trp286, Tyr72, and Tyr124 in mAChE are the counterparts of Ala277, Asn68, and Gln119 in hBChE, respectively. In addition, some bulky aromatic residues in AChE active site have been replaced by small aliphatic ones in BChE. As a result, the active site of BChE is wider and able to accommodate bis-ligands with linkers of wider-ranging lengths. These might be the main reasons that the BChE inhibition is less sensitive to the linker length. Inhibition of AChE-Induced A
Aggregation. Three com￾pounds, 5g, 5h, and 5i, were selected to assess their abilities to inhibit A
aggregation induced by AChE using a thioflavin T-based fluorometric assay,20 compared with the reference compound propidium iodine (Sigma-Aldrich), a known specific peripheral site-binding inhibitor, and the monomer (-)-MEP (Table 2). Results showed that 5h and 5i markedly prevented the AChE-induced A
aggregation with IC50 values of 16.6 and 5.8 µM, similar to that of propidium (IC50 ) 12.8 µM). With a small IC50 value and a higher efficiency of inhibition, 5i and 5h were the wonderful compounds that inhibited the aggregation of A
induced by AChE. In contrast, (-)-MEP and 5g showed fairly low inhibitory activity (Table 2), which indicated their limited ability to interact with the peripheral site of the enzyme. Different behaviors of these four compounds were attributed to different lengths of linker, demonstrating that a linker no shorter than 9 was necessary to inhibit AChE-induced A
2030 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 Xie et al