Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.7 (acetylcholinesterase)
28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of N-benzylpiperidine benzisoxazoles has been developed as potent and selective inhibitors of the enzyme acetylcholinesterase (AChE). The benzisoxazole heterocycle was found to be an appropriate bioisosteric replacement for the benzoyl functionality present in the N-benzylpiperidine class of inhibitors. The title compounds were synthesized by alkylating 3-methyl-1,2-benzisoxazoles with an iodo piperidine derivatives as the key step. Benzisoxazoles 1b-j,o displayed potent inhibition of AChE in vitro with IC50's = 0.8-14 nM. Particularly interesting were N-acetyl and morpholino derivatives 1g (IC50 = 3 nM) and 1j (IC50 = 0.8 nM), respectively, which displayed outstanding selectivity for acetyl-over butyrylcholinesterase, in excess of 3 orders of magnitude. N-Acetyl 1g also displayed a favorable profile in vivo. This analog showed a dose-dependent elevation of total acetylcholine in mouse forebrain after oral administration with an ED50 = 2.4 mg/kg. In addition, 1g was able to reverse amnesia in a mouse passive avoidance model at doses of 3.2 and 5.6 mg/kg with an average reversal of 89.7%. Molecular dynamics simulations were used to study the possible binding modes of N-benzylpiperidine benzisoxazoles to AChE from Torpedo californica. Key structural insights were obtained regarding the potency of this class of inhibitors. Specifically, Asp-72, Trp-84, Trp-279, Phe-288, and Phe-330 are implicated in the binding of these inhibitors. The N-benzylpiperidine benzisoxazoles may be suitable compounds for the palliative treatment of Alzheimer's Disease.
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PMID:Novel benzisoxazole derivatives as potent and selective inhibitors of acetylcholinesterase. 806

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.
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PMID:Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. 821 85

A series of new peptidyl (alpha-aminoalkyl)phosphonate diphenyl esters containing the 4-amidinophenyl group were synthesized and tested as irreversible inhibitors for thrombin and other trypsin-like enzymes. These phosphonates irreversibly inhibited several coagulation enzymes and trypsin. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 is the best human thrombin inhibitor in the series with a k(obs)/[I] value of 11,000 M-1 s-1, and it inhibits thrombin more than 5-fold more effectively than the other enzymes tested. Z-(4-AmPhGly)P(OPh)2 is the best inhibitor for plasma kallikrein with a k(obs)/[I] value of 18,000 M-1 s-1. Generally, the (4-AmPhGly)P(OPh)2 derivatives are better inhibitors of thrombin and trypsin than the corresponding (4-AmPhe)P(OPh)2 derivatives which contain an extra CH2 separating the amidinophenyl group from the peptide backbone. The amidino phosphonates did not inhibit acetylcholinesterase and were chemically stable in neutral buffers. In addition, the inhibited trypsin derivative did not regain any enzyme activity after removal of excess inhibitor and incubation in a pH 7.5 buffer for 1 day. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 and D-Phe-Pro-(4-AmPhe)P(OPh)2 prolonged the prothrombin time ca. 2-fold and prolonged the activated partial thromboplastin time ca. 3-4-fold in human plasma at concentrations of 63 and 125 microM, respectively. The novel amidine-containing peptidyl phosphonates reported here are thus effective anticoagulants in vitro, and they may have utility for use in vivo.
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PMID:Novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases. 829 9

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.
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PMID:Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket. 834 97

Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.
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PMID:Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase. 841 49

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.
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PMID:The peptidase activity of human serum butyrylcholinesterase: studies using monoclonal antibodies and characterization of the peptidase. 842 27

The objectives of this investigation were to characterize neuropeptide-degrading enzymes on the surface of gastric muscle cells and to determine their physiological function. Neutral endopeptidase (NEP, EC 3.4.24.11) activity was measured using the fluorogenic substrate glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine. The NEP inhibitors phosphoramidon and DL-thiorphan (1 microM) inhibited degradation of the substrate by gastric muscle membranes by 100% and by freshly dispersed gastric muscle cells by 55-60%. The phosphoramidon or DL-thiorphan-inhibitable activity, attributed to NEP, of membranes was 112 +/- 4.0 pmol h-1 (micrograms protein)-1 and of cells was 4.2 +/- 0.8 nmol h-1 (10(6) cells)-1. This activity was associated with membranes prepared from cells and was not detected in the cytoplasm or in the cell incubation solution. Gastric muscle membranes were fractionated by electrophoresis and analysed by Western blotting using two NEP antisera. Both antisera recognized a protein in membranes with an electrophoretic mobility identical to that of recombinant human NEP and an apparent molecular mass of approximately 95 kDa. Neuropeptides were degraded by membranes with specific activities in the order of [Leu5]enkephalin > [Met5]enkephalin > gastrin-releasing peptide-10 (GRP-10) > [D-Ala2][Leu5]enkephalin > somatostatin-14. Phosphoramidon and DL-thiorphan similarly inhibited the degradation of GRP-10 (mean of 35% inhibition), somatostatin-14 (57%) and the aminopeptidase-resistant analogue, [D-Ala2][Leu5]enkephalin (75%). When aminopeptidases were inhibited with amastatin (10 microM) phosphoramidon inhibited degradation of [Leu5]enkephalin (54%) and [Met5]enkephalin (100%). Phosphoramidon increased the potency of the contractile effects of neuropeptides on muscle cells by > 280-fold for somatostatin-14, 17-fold for GRP-10, 18-fold for [Met5]enkephalin and 14-fold for [Leu5]enkephalin. The results show that an NEP-like enzyme on the surface of gastric muscle cells degrades and inactivates enkephalins, GRP-10 and somatostatin-14 and acts in a manner analogous to that of acetylcholinesterase in the neuromuscular junction of skeletal muscle.
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PMID:Neutral endopeptidase (EC 3.4.24.11) modulates the contractile effects of neuropeptides on muscle cells from the guinea-pig stomach. 844 12

The precise localization of an endothelin (ET) receptor subtype, the ETB receptor, in porcine lung was elucidated by in vitro microautoradiography using a novel ETB-selective radioligand, [125I]BQ-3020 ([125I-Tyr]-N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp -Ile-Ile-Trp). Of the labeled native ET isopeptides, [125I]ET-3 is selective for ETB receptors. However, [125I]ET-3 was not suitable for autoradiography due to its high degree of non-specific binding. On the other hand, [125I]BQ-3020 showed extremely low non-specific binding on autoradiography. The distribution of [125I]BQ-3020 binding in porcine lung was clearly different from that of [125I]ET-1, which showed more widespread binding than [125I]BQ-3020 due to a high affinity to both ETA and ETB receptors. [125I]BQ-3020 was found to bind to parenchyma, parasympathetic ganglia, pulmonary and submucosal plexuses, but bound only slightly to circular smooth muscle layers and the epithelium of airway tracts. Although [125I]ET-1 bound to the smooth muscle layer of all blood vessels, the binding of [125I]BQ-3020 differed among blood vessels. [125I]BQ-3020 binding in blood vessels paralleled acetylcholinesterase activity, suggesting that ETB receptors in blood vessels are located on parasympathetic nerves. Thus, the radioligand [125I]BQ-3020 is very useful for studying the precise localization of ETB receptors.
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PMID:A novel ligand, [125I]BQ-3020, reveals the localization of endothelin ETB receptors. 851 85

The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
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PMID:The architecture of human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors. 866 93

Multiconfiguration thermodynamic integration was used to determine the relative binding strength of tacrine and 6-chlorotacrine by Torpedo californica acetylcholinesterase. 6-Chlorotacrine appears to be bound stronger by 0.7+/-0.4 kcal/mol than unsubstituted tacrine when the active site triad residue His-440 is deprotonated. This result is in excellent agreement with experimental inhibition data on electric eel acetylcholinesterase. Electrostatic Poisson-Boltzmann calculations confirm that order of binding strength, resulting in deltaG of binding of -2.9 and -3.3 kcal/mol for tacrine and chlorotacrine, respectively, and suggest inhibitor binding does not occur when His-440 is charged. Our results suggest that electron density redistribution upon tacrine chlorination is mainly responsible for the increased attraction potential between pronated inhibitor molecule and adjacent aromatic groups of Phe-330 and Trp-84.
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PMID:Binding of tacrine and 6-chlorotacrine by acetylcholinesterase. 867 40


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