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.
Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse and Torpedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290I, and the double mutant T. californica F288L/F290V (all mutants given T. californica numbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290I had increased rate constants ( approximately 14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of butyrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290I mouse mutant AChEs, representing an increase of approximately 4000-fold over the T. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE.
Title: The influence of peripheral site ligands on the reaction of symmetric and chiral organophosphates with wildtype and mutant acetylcholinesterases Radic Z, Taylor P Ref: Chemico-Biological Interactions, 119-120:111, 1999 : PubMed
The rates of inhibition of mouse acetylcholinesterase (AChE) (EC 3.1.1.7) by paraoxon, haloxon, DDVP, and enantiomers of neutral alkyl methylphosphonyl thioates and cationic alkyl methylphosphonyl thiocholines were measured in the presence and absence of AChE peripheral site inhibitors: gallamine, D-tubocurarine, propidium, atropine and derivatives of coumarin. All ligands, except the coumarins, at submillimolar concentrations enhanced the rates of inhibition by neutral organophosphorus compounds (OPs) while inhibition rates by cationic OPs were slowed down. When peripheral site ligand concentrations extended to millimolar, the extent of the enhancement decreased creating a bell shaped activation profile. Analysis of inhibition by DDVP and haloxon revealed that peripheral site inhibitors increased the second order reaction rates by increasing maximal rates of phosphylation.
Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.
Title: Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors Radic Z, Pickering NA, Vellom DC, Camp S, Taylor P Ref: Biochemistry, 32:12074, 1993 : PubMed
By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.
