Inhibition of acetylcholinesterase by anisomycin

The antibiotic anisomycin, an inhibitor of protein synthesis in eucaryotic cells, which blocks long-term memory in mice, is shown to interact with the cholinergic system by inhibiting reversibly the acetylcholinesterase. The inhibition is a competitive one, the inhibition constant K_i being 5.0 × 10^?3 for human brain acetylcholinesterase and 1.7 × 10^?3 for acetylcholinesterase of bovine erythrocytes. The anisomycin effect on acetylcholinesterase is compared with the puromycin and cycloheximide-inhibition of the enzyme. The significance of the cholinergic effect of anisomycin in addition to its inhibitory effect on protein synthesis for the interpretation of memory experiments is discussed.     

Structural basis of acetylcholinesterase inhibition by triterpenoidal alkaloids

Acetylcholinesterase plays a crucial role in the metabolism of neurotransmitter, acetylcholine. Inhibition of Torpedo californica acetylcholinesterase by triterpenoidal alkaloids buxamine-B (1) and buxamine-C (2) has been studied by enzyme kinetics and molecular docking experiments. Buxamine-C (2) has been found to be 20-fold potent than buxamine-B (1) (Ki = 5.5 and 110 microM, respectively). The ligand docking experiments predicted that the cyclopentanophenanthrene skeleton of both inhibitors properly fits into the aromatic gorge of the enzyme. The C-3 and C-20 amino groups of both alkaloids mimic the well-known bis-quaternary ammonium inhibitors such as decamethonium and interact with Trp84 and Trp279 residues of the enzyme, respectively. The C-3 amino group in buxamine-C (2) appears to be better positioned at the bottom of the aromatic gorge and thus seems to be crucial for the inhibitory activity of such inhibitors.     

Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates

Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholinesterase. The inhibitors showed bimolecular inhibition constants (k_i) ranging from 0.3 × 10⁵ M^?1 min^?1 to 10.4 × 10⁵ M^?1 min^?1. When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chromophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMoAChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups, showed excellent inhibitory potency against both AChE’s (? 1 × 10⁷ M^?1 min^?1) indicating, in general, that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling.     

Inhibition of acetylcholinesterase from electric eel by (-)-and (+)-physostigmine and related compounds

Unnatural (+)-physostigmine (2) inhibited acetylcholinesterase (AChE) from electric eel considerably less than natural (-)-physostigmine (1), but 2 may because of its possible lower toxicity still be an interesting anticholinesterase agent. (-)-Eseroline (3), a major metabolite of (-)-physostigmine (1) and a potent narcotic analgetic, and its unnatural (+)-antipode (4), were both poor inhibitors of the enzyme. (-)-Dihydrosecophysostigmine (5), a ring-open analog of (-)-physostigmine was less, but (-)-N-methylphysostigmine (6) much more potent than the natural alkaloid. The availability of compounds related to (-)- and (+)-physostigmine by improved chemical synthesis suggests that further structural variation may well lead to other biologically interesting AChE inhibitors.     

Inhibition of acetylcholinesterase by TX-100.

At high detergent concentrations, approximately the equivalent of 2 micelles of TX-100 reversibly bind to acetylcholinesterase and fully inhibit the enzyme. This result suggests that the appropriate lipid environment might regulate this neuronal enzyme's function.     

Inhibition of Candida rugosa lipase by berberine and structurally related alkaloids, evaluated by high-performance liquid chromatography.

It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with beta-naphthyl laurate, and beta-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of beta-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.     

Inhibition of seed germination by quinolizidine alkaloids

Germination of Lactuca sativa L. was inhibited by mixtures of quinolizidine alkaloids. The alkaloid esters resulted in the strongest inhibition: 6 mM 13-tigloyloxylupanine inhibited germination by 100%, whereas the other lupin alkaloids, such as lupanine and sparteine, gave a 45 and 20% inhibition, respectively. Seedlings of Lupinus albus L., which are not affected by quinolizidine alkaloids, excrete lupanine and 13-tigloyloxylupanine into the surrounding medium by their roots. It is assumed that lupin alkaloids are potential compounds of plant-plant interaction (i.e. allelopathy) besides their role in plant-herbivore interrelations.     

Inhibition of electric eel acetylcholinesterase by triarylmethane dyes

The effects of three cationic triarylmethane dyes - pararosaniline (PR), malachite green (MG), methyl green (MetG) - on electric eel AChE (eAChE) activity were tested at 25 degrees C, in 100mM MOPS buffer (pH 8) containing 0.125mM 5-5-dithio-bis(2-nitrobenzoic acid), 20-120muM acetylthiocholine and 0-20muM dye. All three dyes caused reversible, linear- or hyperbolic-mixed inhibition of esteratic activity. The respective inhibitory parameters for PR, MG and MetG were K(i)=8.4+/-0.67, 1.9+/-0.51 and 0.27+/-0.017muM; alpha (competitive coefficient)=5.8+/-2.0, 4.8+/-1.8 and 2.7+/-0.32; beta (noncompetitive coefficient)=0, 0 and 0.20+/-0.011. The data were consistent with ligand binding at the peripheral site and a remote effect on substrate binding and turnover.     

Photoinactivation of acetylcholinesterase by erythrosin B and related compounds

Acetylcholinesterase was rapidly inactivated when exposed to light in the presence of xanthene dyes. Photosensitizing efficiency paralleled the dye triplet state quantum yields, increasing in the order fluorescein less than eosin B less than eosin Y less than erythrosin B less than rose bengal. The observed first-order rate constants of photoinactivation increased hyperbolically with dye concentration. Evidence for the formation of a dye-enzyme complex prior to inactivation was obtained from spectrophotometric and protein fluorescence quenching methods. The latter technique allowed estimates of the dye-enzyme dissociation constants for rose bengal (20 microM) and erythrosin B (30 microM). After photoinactivation, a portion of the dye became covalently bound to the enzyme. The photoinactivation reaction occurs in both aerobic (air saturated) and anaerobic (argon saturated) solution, with the rates of photoinactivation being about three to five times greater under the latter conditions. The aerobic reaction exhibits a large deuterium isotope enhancement effect and is largely (but not completely) quenched by 10(-2) M azide. The anaerobic reaction is unaffected by azide and exhibits only a small deuterium isotope effect. These results indicate that the photoinactivation reaction proceeds mainly by a type II (singlet oxygen mediated) pathway under aerobic conditions and by a type I (radical) pathway under anaerobic conditions. The enzyme was protected from inactivation by edrophonium, a competitive inhibitor, but not by d-tubocurarine, a peripheral-site ligand, indicating that destruction of a crucial residue at or near the catalytic site is an important component of the inactivation process. Extensive destruction of tryptophan undoubtedly occurs, at least under aerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of mammalian digestive disaccharidases by polyhydroxy alkaloids

Several polyhydroxy alkaloids, including the eight presently known to occur in plants, have been compared as inhibitors of mouse gut digestive disaccharidases. The indolizidine castanospermine inhibited all activities tested, but others showed a selectivity which could be of value in studies of carbohydrate digestion and errors of metabolism.     

Inhibition of erythrocyte acetylcholinesterase by n-butanol at high concentrations

Erythrocyte acetylcholinesterase (AChE) is bound to the membrane by a complex glycosylphosphatidylinositol anchor, so the effect of alcohol on AChE activity may reflect direct and/or membrane-mediated effects. The indication of a direct interaction between n-butanol and AChE molecules is the activation/inhibition of AChE by occupation of the enzyme's active and/or regulatory sites by alcohol. The activation of AChE can occur only at low concentrations of alcohols, while at high concentrations AChE is inhibited. In this work the mechanism of inhibition of erythrocyte AChE by n-butanol at high concentrations was studied. The values of activity, calculated assuming parabolic competitive inhibition, which implies that one or two molecules of inhibitor bind to the enzyme, fit well to the experimental values. From the values of the inhibition constants it was concluded that at high n-butanol concentrations two alcohol molecules usually interact with AChE.     

Inhibition of nonenzymic lipid peroxidation by benzylisoquinoline alkaloids.

A group of benzylisoquinoline alkaloids, including five simple benzylisoquinolines, three phtalideisoquinolines, six aporphines, three protoberberines, and two benzophenanthridines, have been studied as inhibitors of lipid peroxidation stimulated by Fe2+/cysteine in rat liver microsomal fractions. Protopapaverine, apomorphine, laudanosoline, tetrahydroberberine, isoboldine, bulbocapnine, boldine, anonaine, glaucine, and stepholidine showed antiperoxidative effects, and structure-activity relationships were established. In simple benzylisoquinolines, the presence of phenolic hydroxyls or similar reactive groups is necessary for inhibition of peroxidation, while in aporphines and protoberberines nonhydroxylated compounds can exert antiperoxidative effects. The phtalideisoquinolines and benzophenanthridines tested were inactive.     

Inhibition of insect acetylcholinesterase by the potato glycoalkaloid alpha-chaconine.

Homogenates from several insect species were assayed for inhibition of acetylcholinesterase by the potato glycoalkaloid alpha-chaconine. Colorado potato beetle acetylcholinesterase was up to 150-fold less sensitive than other species tested. Acetylcholinesterase from an insecticide-resistant strain of Colorado potato beetles was more sensitive to inhibition than the susceptible strain. Most insect species tested had inhibitory concentrations causing a 50% reduction in activity in the 5 to 40 microM range. Sensitive insect acetylcholinesterases were similar to mammalian cholinesterases in their response to alpha-chaconine. The results indicate that pesticides and host plant resistance factors may interact at the same target. Changes in the target due to selection pressure from either pesticides or host plant resistance factors could affect the efficacy of both control strategies.     

Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate

In order to further understand the interaction processes between the Lycopodium alkaloids and acetylcholinesterase, the binding properties of N-acetyl huperzine A (1), huperzine B (2) and huperzine F (3) with Torpediniforms Nacline acetylcholinesterase (TnAchE) were investigated by 1H NMR methods. The nonselective, selective and double-selective spin-lattice relaxation rates were acquired in the absence and presence of TnAchE at a ratio of [ligand]/[protein]=1:0.005. The selective relaxation rates show protons of 1-3 have dipole-dipole interaction with protons of TnAchE at the binding interface. The molecular rotational correlation time of bound ligands was calculated by double-selective relaxation rate at 298 K, which showed that 1-3 had high affinity with the protein. The results indicate that investigation of 1H NMR relaxation data is a useful method to locate the new Lycopodium alkaloids as AchE inhibitors.     

Quenching of singlet oxygen by alkaloids and related nitrogen heterocycles

Fifteen plant alkaloids and related heterocyclic compounds were tested for their ability to quench singlet oxygen. Most of the compounds showed high activity; brucine and strychnine were especially efficient quenchers. Brucine, at a concentration of ca 2.6 x 10^?5 M, is capable of inactivating half the singlet oxygen molecules it encounters. This quenching may serve in nature to protect plants from the deleterious effects of singlet oxygen or other reactive oxidants.