The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase,butyrylcholinesterase, glutathione S-transferase,lactoperoxidase, and carbonic anhydrase isoenzymes I,II, IX,and XII

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates including humans as many diverse isoforms. Acetylcholinesterase (AChE) is responsible for acetyl choline (ACh) hydrolysis and plays a fundamental role in nerve impulse transmission by terminating the action of the ACh neurotransmitter at cholinergic synapses and neuromuscular junctions. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effect of CAPE on human carbonic anhydrase (hCA) isoforms I, II, IX, and XII, AChE, BChE, LPO, and GST was evaluated. CAPE inhibited these enzymes with K_is in the range between micromolar to picomolar. The best inhibitory effect was observed against AChE and BChE.     

Design,synthesis, and evaluation of guanylhydrazones as potential inhibitors or reactivators of acetylcholinesterase

Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic organophosphorus compounds, were designed, synthesized, characterized, and tested as potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman’s test, nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six other guanylhydrazones obtained from different benzaldehydes. The results indicate that all compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and was compared with tacrine, displaying similar activity to this reference material. These results indicate that guanylhydrazones as well as future similar derivatives may function as drugs for the treatment of Alzheimer's disease.     

Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase

A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209–0.291?nM. On the other hand, tacrine, which is used in the treatment of Alzheimer’s disease possessed lower inhibition effect (K_i: 0.398?nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (K_i) in the range of 4.49–5.61?nM for hCA I, and 4.94–7.66?nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated K_i values of 281.33?nM for hCA I and 9.07?nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels.     

Beilschglabrines A and B: Two new bioactive phenanthrene alkaloids from the stem bark of Beilschmiedia glabra

Two new phenanthrene alkaloids, beilschglabrines A (1) and B (2) were isolated from the stem bark of Beilschmiedia glabra, together with lupeol, taraxerol, and 24-methylenelanosta-7,9-diene-3β-15α-diol. The structures of the isolated compounds were elucidated by extensive spectroscopic data analysis and comparison with respective literature data. The compounds were tested for DPPH radical scavenging, acetylcholinesterase and lipoxygenase inhibitory activities. Compound 1 displayed considerable activity in the acetylcholinesterase (IC₅₀ 50.4 μM), the DPPH radical scavenging (IC₅₀ 115.9 μM) and the lipoxygenase (IC₅₀ 32.8 μM) assays.     

Design,synthesis and preliminary structure–activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: a further study based on Flavokawain B Mannich base derivatives

In order to study the structure–activity relationship of Flavokawain B Mannich-based derivatives as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-containing chalcone derivatives (4?a–8d) were designed, synthesized, and evaluated for AChE inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone dramatically influenced the inhibitory activity against AChE. Among them, compound 6c revealed the strongest AChE inhibitory activity (IC₅₀ value: 0.85?μmol/L) and the highest selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The molecular docking assay showed that this compound can both bind with the catalytic site and the peripheral site of AChE.     

Reversible AChE inhibitors in C. elegans vs. rats, mice

We are investigating whether Caenorhabditis elegans could be used as a screen for vertebrates by comparing the responses of components of its cholinergic system to well-characterized toxicants. We assessed whether C. elegans displays similar toxicity as rats and mice to reversible acetylcholinesterase (AChE) inhibitors, and sought to corroborate that the toxicity mechanism is the same. To determine relative potencies, movement-concentration curves were generated, 50th percentiles for movement were located, ranked and compared statistically to rat and mouse oral acute LD50s. The ranking was significantly correlated to rat and mouse rankings (alpha=0.05). We measured a concentration-dependent decrease in AChE activity correlating to a decrease in movement for each carbamate, suggesting that the mechanism of toxicity is the same. Finally, as seen in mammals, inhibition of AChE activity occurred before a movement decrease. The response of C. elegans to carbamate exposure shows significant correlation to rat and mouse data.     

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates as conformationally constrained inhibitors of acetylcholinesterase

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates (1-15) are synthesized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti-conformations of acetylcholine, respectively. All carbamates 1-15 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. For a series of geometric isomers, the inhibitory potencies are as follows: benzene-1,4-di-N-substituted carbamate (para compound) > benzene-1,3-di-N-substituted carbamate (meta compound) > benzene-1,2-di-N-substituted carbamate (ortho compound). Therefore, benzene-1,4-di-N-substituted carbamates (para compounds), with the angle of 180 degrees between two C(benzene)-O bonds, mimic the preferable anti C-O/C-N conformers of acetylcholine for the choline ethylene backbone in the acetylcholinesterase catalysis.     

Pesticide detection with a liposome-based nano-biosensor

Monitoring of the organophosphorus pesticides dichlorvos and paraoxon at very low levels has been achieved with liposome-based nano-biosensors. The enzyme acetylcholinesterase was effectively stabilized within the internal nano-environment of the liposomes. Within the liposomes, the pH sensitive fluorescent indicator pyranine was also immobilized for the optical transduction of the enzymatic activity. Increasing amounts of pesticides lead to the decrease of the enzymatic activity for the hydrolysis of the acetylcholine and thus to a decrease in the fluorescent signal of the pH indicator. The decrease of the liposome biosensors signal is relative to the concentration of dichlorvos and paraoxon down to 10⁻¹⁰ M levels. This biosensor system has been applied successfully to the detection of total toxicity in drinking water samples. Also a colorimetric screening device for pesticide analysis has been evaluated.     

A novel enzyme immunoassay based on potentiometric measurement of molecular adsorption events by an extended-gate field-effect transistor sensor

We developed a novel enzyme immunoassay based on a potentiometric measurement of molecular adsorption events by using an extended-gate field-effect transistor (FET) sensor. The adsorbing rate of a thiol compound on a gold surface was found to depend on the concentration of the compound. To construct an electrochemical enzyme immunoassay system by using the sensor, the enzyme chemistry of acetylcholinesterase (AChE) to generate a thiol compound was used and combined with the enzyme-linked immunosorbent assays (ELISA). After the AChE-catalyzed reaction, the amount of the antigen was obtained by detecting the adsorbing rate of the generated thiol compound on the gold electrode using the FET sensor. The measurement stability was also found to improve when a high frequency voltage of 10 kHz or more was superimposed to the reference electrode. The signal corresponding to a range between 1 and 250 pg/mL of Interleukin 1β was obtained by the FET sensor when a voltage of 1 MHz was superimposed onto the reference electrode. The FET sensor based ELISA used in this measurement technique can successfully detect Interleukin 1β at concentrations as low as 1 pg/mL.     

Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.