Microcalorimetric assay of acetylcholinesterase

Comparative assays were made in a spectrophotometer and a microcalorimeter for the reaction between acetylcholinesterase (EC 3.1.1.7) and acetylthiocholine. The rate of light absorbance change and the rate of heat flow were measured from similar and simultaneous reactions in spectrophotometer and microcalorimeter, respectively. At the enzyme activity levels studied, i.e., 0.05–0.15 I.U. in calorimetry and 1–4 I.U. in spectrophotometry, the reaction rates were linear and showed first-order kinetics. A highly significant positive correlation was seen between the two methods (r = 0.997). More importantly, spectrophotometric assay with acetylthiocholine (which utilized a secondary reaction with chromagen, dithiobisnitrobenzoic acid) stood in highly significant positive correlation with calorimetric assays (which did not require a chromagen) either with the same substrate (r = 0.976) or with acetylcholine (r = 0.900). It appears that microcalorimetry can be used in preference to spectrophotometry for enzyme kinetic studies to overcome the complexity of reaction mixture and interference problems and with the advantage of using natural substrates.     

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.     

Isolation,identification, and quantification of Pentylcurcumene from Geophila repens: A new class of cholinesterase inhibitor for Alzheimer’s disease

The aerial part of Geophila repens (L.) I.M. Johnst (Rubiaceae) has been used in India to improve intelligence and memory for a long time. As part of our ongoing efforts in discovering potential bioactive compounds from G. repens, we have studied the isolation, identification, and quantification of a new class of cholinesterase inhibitor from G. repens for Alzheimer’s disease (AD). Terpene was isolated from hydroalcohol extract of G. repens (GRHA) and its structure was identified “Pentylcurcumene” by spectroscopic data. HPTLC fingerprint analysis was performed and good separation was achieved in mobile phase (benzene:methanol; 7.5:2.5, v/v, 254 and 366 nm; R_f 0.51). The method was validated using ICH guidelines in terms of linearity, specificity, sensitivity, accuracy, precision, robustness and stability. In cellular antioxidant studies e.g. DPPH, oxygen-radical-absorbance-capacity (ORAC) and cell-based-antioxidant-protection-in-erythrocytes (CAP-e) assays showed that, Pentylcurcumene showed remarkably different degrees of antioxidant activities in dose-dependent manner. Pentylcurcumene demonstrated anticholinesterase activities e.g. IC₅₀ of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition were 73.12 ± 0.56 and 97.65 ± 0.46 μg/ml, respectively. To better understand enzyme kinetics, Lineweaver-Burk plot of Pentylcurcumene displayed the highest affinity with competitive inhibition (reversible) towards both AChE (V_max 0.8) and BChE (V_max 0.6). An improved and advanced HPTLC tool of bioautography detection of Pentylcurcumene has been successfully demonstrated its anticholinesterase activities. Molecular docking simulations of Pentylcurcumene (ligand) and enzymes (proteins) exhibited the binding of ligand at active sites of AChE (human/rat) and BChE (human/homology) efficiently and also predicted the hydrophobic interaction of drug towards different amino acid residue within proteins. As per the results of antioxidant study and with the support of molecular docking analysis, it is concluded that Pentylcurcumene could be a potential first-line cholinesterase-inhibitor for AD.     

Colorimetric and fluorometric assays for acetylcholinesterase and its inhibitors screening based on a fluorescein derivate

A fluorescein-based sensor was developed for the AChE activity assay and the inhibitor screening. The sensor provided the dual assay methods for the screening of AChE activity in the presence or absence of inhibitor. The colorimetric and fluorometric assays were based on the following processes: (1) owing to the hydrolysis of acetylthiocholine in the presence of AChE, the fluorescein-based probe can rapidly induce 1,4-addition of the hydrolysis product thiocholine to α,β-unsaturated ketone in the compound 1, resulting in strong fluorescence and absorption changes; (2) in the presence of the corresponding inhibitor, the fluorescence enhancement or the absorption change would be inhibited in that the formation of thiocholine was hindered.     

Cation-π and π-π stacking interactions allow selective inhibition of butyrylcholinesterase by modified quinine and cinchonidine alkaloids

Scaffold varied quaternized quinine and cinchonidine alkaloid derivatives were evaluated for their selective butyrylcholinesterase (BChE) inhibitory potential. K_i values were between 0.4–260.5 μM (non-competitive inhibition) while corresponding K_ivalues to acetylcholinesterase (AChE) ranged from 7.0–400 μM exhibiting a 250-fold selectivity for BChE.Docking arrangements (GOLD, PLANT) revealed that the extended aromatic moieties and the quaternized nitrogen of the inhibitors were responsible for specific π–π stacking and π–cation interactions with the choline binding site and the peripheral anionic site of BChE’s active site.