NMR determination of Electrophorus electricus acetylcholinesterase inhibition and reactivation by neutral oximes

Abstract

Neurotoxic organophosphorus compounds (OPs), which are used as pesticides and chemical warfare agents lead to more than 700,000 intoxications worldwide every year. The main target of OPs is the inhibition of acetylcholinesterase (AChE), an enzyme necessary for the control of the neurotransmitter acetylcholine (ACh). The control of ACh function is performed by its hydrolysis with AChE, a process that can be completely interrupted by inhibition of the enzyme by phosphylation with OPs. Compounds used for reactivation of the phosphylated AChE are cationic oximes, which usually possess low membrane and hematoencephalic barrier permeation. Neutral oximes possess a better capacity for hematoencephalic barrier permeation. NMR spectroscopy is a very confident method for monitoring the inhibition and reactivation of enzymes, different from the Ellman test, which is the common method for evaluation of inhibition and reactivation of AChE. In this work (1)H NMR was used to test the effect of neutral oximes on inhibition of AChE and reactivation of AChE inhibited with ethyl-paraoxon. The results confirmed that NMR is a very efficient method for monitoring the action of AChE, showing that neutral oximes, which display a significant AChE inhibition activity, are potential drugs for Alzheimer disease. The NMR method showed that a neutral oxime, previously indicated by the Ellman test as better in vitro reactivator of AChE inhibited with paraoxon than pralidoxime (2-PAM), was much less efficient than 2-PAM, confirming that NMR is a better method than the Ellman test.

Synthesis of New Quinoline-Piperonal Hybrids as Potential Drugs against Alzheimer’s Disease

“…Recent published works have indicated results consistent with Ellman’s method [25,32,33,34,35,36]. In our study we used the NMR method [29] to determine the concentrations of the substrate (ACh) and the product acetate (Ac), which are obtained from direct integration of the corresponding absorption peaks for methyl groups (2.24 ppm for ACh and 2.16 ppm for Ac) [32,35]. The comparison between the integration of the Ac peak in pure AChE (no inhibitor) and in the presence of the inhibitor allowed the determination of the percentages of inhibition shown in the plot of Figure 3.…”

“…As compound (8) presented the lowest IC 50 value in Ellman’s test, it was selected for an additional kinetic study by NMR [32] in order to verify the percentage of inhibition when compared to the reference drug (tacrine). The enzyme selected for this study was Ee AChE because it displays an identical active site and a very similar activity to the human enzyme [33,34].…”

“…As before [32], the structures of the new compounds were built and optimized, using the density functional theory (DFT) with M06 and 6-311++G** of Spartan08 ® [21]. In addition, the values of Weight (amu), Polar Surface Area (PSA, Å 2 ), Area (Å 2 ) and their Volume (Å 3 ) were obtained.…”

Computational enzymology for degradation of chemical warfare agents: promising technologies for remediation processes

“…The inhibition does not reach the anionic site of AChE, further contributing to reactivation, given the binding to the cationic part of the reactivator (for example: 2-PAM. The enzyme reactivation is achieved from the oxime action and, consequently, the breakup of this OP binding with the enzyme [71].…”

Development of multi‐response optimization and quadratic calibration curve for determination of ten pesticides in complex sample matrices using QuEChERS dispersive liquid–liquid microextraction followed by gas chromatography

“…Additionally, various analytical methods have been developed for the analysis of pesticides in different sample matrices mostly based on CE , LC‐MS , and GC with flame photometric detector , nitrogen–phosphorus detector , electron capture detector , and MS . Additionally, spectroscopic techniques such as FT‐IR , UV‐Vis spectrophotometry , NMR , and inductively coupled plasma have been used with chromatography for the analysis of pesticides.…”