Aromatic amino‐acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept®) to cholinesterases

Abstract

E2020 (R,S)‐1‐benzyl‐4‐[(5,6‐dimethoxy‐1‐indanon)‐2‐yl]methyl)piperidine hydrochloride is a piperidine‐based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure‐activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N‐benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site‐specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200‐ to 400‐fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site‐directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE–E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active‐site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild‐type BChE, further confirms our hypothesis.

Structure of a complex of the potent and specific inhibitor BW284C51 withTorpedo californicaacetylcholinesterase

“…5 are involved in favourable contacts. Although there are no data for TcAChE mutants, the measured K i values for the mouse wild-type enzyme, 2.8±4.8 nM, 3.5±31 mM (Radic et al, 1993) and 2.2±23 nM (Saxena et al, 2000), for BW, DECA and E2020, respectively, are similar to the corresponding values for the Torpedo enzyme. However, those for some of the mutants differ by orders of magnitude.…”

“…As shown in Table 3, the experimental inhibition constants K i for TcAChE range from 2.0 to 64 nM for BW and E2020, but from 0.34 to 7.0 mM for DECA (Eichler et al, 1994;Cousin et al, 1996;Saxena et al, 2000), corresponding to $100-fold weaker binding. Several factors determine the relative binding strength of different inhibitors to a given protein binding site, including conformational¯exibility of the inhibitor and amino-acid residues in the ligand-binding site, tightness of steric ®t, number of favourable (e.g.…”

“…et al, 1996). § K i , K H i , Torpedo wild-type or mouse AChE (Saxena et al, 2000). Radic et al, 1993).…”

“…Since DECA lacks aromatic groups and E2020 has only a single tertiary amino group, BW, which has both an aromatic ring and a quaternary amino group at each extremity plus a carbonyl in the middle that could potentially form a hydrogen bond with a suitable donor, might be expected to bind to AChE more tightly than either DECA or E2020. Indeed, the reported inhibition constant K i for DECA, in the micromolar range, is about 100 times weaker than that of BW (Cousin et al, 1996;Radic et al, 1993), but that of E2020 is of the same order of magnitude as that of BW (Saxena et al, 2000). By comparison of their respective crystal structures, we hoped to gain a better understanding of the reasons for their different af®nities.…”

Synthesis of monooxime-monocarbamoyl bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against tabun- and paraoxon-inhibited acetylcholinesterase

“…The (E)-but-2-ene linker connecting the pyridinium rings is also in compound K075, which is slightly shorter than the saturated one, supplying the butane connecting chain in the molecule of K048. Six compounds (6)(7)(8)(9)(10)(11) were prepared using conventional synthetic procedures; five of them (7-11) have not been previously described in the literature (Figure 4). Firstly, the monoquaternary salts (12 -14) were synthesized using an excess of five equivalent of (E)-1,4-dibromobut-2-ene in acetone, where bi-products occur only in minor yields (Scheme 1).…”