Inhibition of Cholinesterase by Dialkylcarbamates
Abstract The pI50 index and separation coefficients of chosen 3-N,N-diethylaminophenyl-N′,N′-dialkylcarbamates were determined. Index pI50 (pI50 = negative logarithm of molar concentration of inhibitor inhibiting the enzyme activity by 50%) describes the effectiveness of the inhibitor. The rate of ability of the inhibitor to pass the blood-brain barrier is usually described by the separation coefficient in a system n-octanol/water (Kow). Obtained results were compared with pI50 and Kow of Exelon®, the commercially used drug against the Alzheimer’s disease.
From the New Method for the Determination of the Half Inhibition Concentration ( IC 50 ) of Cholinesterase Inhibitors
The IC50 is the molar concentration of a given inhibitor (I), which reduces the respective uninhibited reaction rate to one half, and is indicative of the inhibitory potency of a compound. We investigated new inhibitors of the in vitro hydrolysis of acetylcholine (ACh) by cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Ellman ́s method (Ellman et al., 1961) is routinely used for the determination of the cholinesterase activity and effi ciency of cholinesterase inhibitors. It is based on the measurement of the absorbance at 412 nm of the yellow complex produced by the reaction between thiocholine (TCh) and DTNB (5,5 ́-dithiobis-2-nitrobenzoic acid, Ellman ́s reagent), as described in detail in Zdražilová et al. (2004). Ellman ́s method has two disadvantages: 1) The composition of the yellow complex and its absorption coeffi cient at 412 nm depend strongly on the composition of the reaction mixture, above all on the pH value. 2) Acetylthiocholine (ATCh) instead of ACh must be used as the substrate. However, the reaction rates of the ACh and ATCh hydrolysis by AChE (or BChE) are not identical, nor are the IC50 values (Zdražilová et al., 2006a). When using ACh, the determination of the rate of the enzymatic reaction, and hence also of IC50, requires the quantitative determination of the concentration of one of the reaction components, i.e. ACh, choline (Ch), acetic acid (HAc), or protons from HAc, in the reaction mixture. The suitable methods, e.g. HPLC or pH-stat (Zdražilová et al., 2006b), are experimentally much more sophisticated and time-consuming. This paper describes a new simple, and fast method of IC50 determination with the possibility of using either ACh or ATCh. The method is based on the measurement of changes in the pH value as a function of time during the enzymatic hydrolysis of ACh or ATCh, it is called the pH(t) method.
Two New Methods Monitoring Kinetics of Hydrolysis of Acetylcholine and Acetylthiocholine
Hydroxylamine and HPLC methods, measuring in vitro kinetics of enzymatic hydrolysis of acetylcholine or acetylthiocholine by cholinesterases, are described. The hydroxylamine method determines the dependence of substrate concentration vs. time, the HPLC method is able to measure simultaneously the time dependences of substrate and both primary products, choline or thiocholine, and acetic acid. Practical determinations are shown, comparison with known (above all Ellman’s and pH-stat) methods, advantages and disadvantages are discussed.