Butyrylcholinesterase Active Site Research Articles

Synthesis and biological evaluation of substituted acetamide derivatives as potential butyrylcholinestrase inhibitors

Alzheimer’s disease (AD) is the most common type of age-related dementia. Inhibition of butyrylcholinesterase (BChE) emerge as an effective therapeutic target for AD. A series of new substituted acetamide derivatives were designed, synthesized and evaluated for their ability to inhibit BChE. The bioassay results revealed that several compounds displayed attractive inhibition against BChE). Among them, compound 8c exhibited the highest BChE inhibition with IC50 values of 3.94 μM. Lineweaver Burk plot indicated that 8c acted as a mixed-type BChE inhibitor. In addition, docking studies confirmed the results obtained through in vitro experiments, and showed that 8c bound to the catalytic anionic site (CAS) and peripheral anionic site (PAS) of BChE active site. Meanwhile, its ADME parameters were approximated using in silico method. Molecular dynamics simulation studies on the complex of 8c-BChE were performed, RMSD, RMSF, Rg, SASA, and the number of hydrogen bonds were calculated as well. These results implied that 8c could serve as appropriate lead molecule for the development of BChE inhibitor.

Design, Synthesis, and Biological Evaluation of Some Benzothiazolone Derivatives as Cholinesterase Inhibitors

AbstractIn this study, nine new benzothiazolone derivatives (6 a–i) were designed and synthesized to identify potent cholinesterase inhibitors. The compounds were tested in vitro against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and found to be selective to BChE. Compound 6 f proved the most potent derivative (IC50=12.25±0.23 μM) against BChE and was identified as a mixed‐type inhibitor with a Ki value of 4.45±0.35 μM according to the kinetic studies. Molecular modelling suggested that the derivatives were druglike and non‐PAINS. Compound 6 f showed good fit in BChE active site interacting with the key sites important for enzyme activity according to the molecular docking study.

Selective butyrylcholinesterase inhibition by isatin dimers and 3-indolyl-3-hydroxy-2-oxindole dimers

Polymethylene-linked isatin dimers 1a-g and 3-indolyl-3-hydroxy-2-oxindole dimers 2a-g were synthesized and are reported here as selective butyrylcholinesterase (BChE) inhibitors. The best compound from each series, 1f and 2d, showed IC50 values of 3.20 µM and 4.49 µM, respectively. Both compounds showed > 11-fold selectivity towards BChE, and mixed-type inhibition by Lineweaver-Burk analysis. These two inhibitors shared π-π stacking interactions with Trp82 and H-bonding to Gly116 and Ser198, the catalytic Ser of the enzyme triad, within the BChE active site as predicted by molecular docking. Compound 1d was also shown to be good inhibitor with an IC50 of 7.56 µM, and it shares the same pentamethylene linker as 2d. Inhibitors 1d, 1f, and 2d were predicted to have moderate, low, and moderate blood–brain barrier (BBB) permeability, respectively, making them, overall, promising lead compounds for further exploration of selective BChE inhibitors.

Phytochemical Analysis and Evaluation of Biological Activity of Lawsonia inermis Seeds Related to Alzheimer's Disease.

Using Lawsonia inermis L. (henna) seeds has been frequently recommended for the improvement of memory in Iranian Traditional Medicine (ITM). In this respect, different fractions of the plant were prepared and evaluated for their in vitro biological assays related to Alzheimer's disease (AD), including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity as well as metal chelating ability and DPPH antioxidant activity. The dichloromethane and ethyl acetate fractions were able to inhibit the BChE selectively with IC50 values of 113.47 and 124.90 μg/mL, respectively, compared with donepezil as the reference drug (IC50 = 1.52 μg/mL). However, all fractions were inactive toward AChE. Phytochemical analysis of the dichloromethane fraction indicated the presence of β-sitosterol (1), 3-O-β-acetyloleanolic acid (2), 3-O-(Z)-coumaroyl oleanolic acid (3), betulinic acid (4), and oleanolic acid (5). The inhibitory activity of isolated compounds was also evaluated toward AChE and BChE. Among them, compounds 2 and 5 showed potent inhibitory activity toward BChE with IC50 values of 77.13 and 72.20 μM, respectively. However, all compounds were inactive toward AChE. Moreover, molecular docking study confirmed desired interactions between those compounds and the BChE active site. The ability of fractions and compounds to chelate biometals (Cu2+, Fe2+, and Zn2+) was also investigated. Finally, DPPH antioxidant assay revealed that the ethyl acetate (IC50 = 3.08 μg/mL) and methanol (IC50 = 3.64 μg/mL) fractions possessed excellent antioxidant activity in comparison to BHA as the positive control (IC50 = 3.79 μg/mL).

Incorporating Substrate Features in Fmoc Amino Acids to Develop Potent and Specific Butyrylcholinesterase Inhibitors for the Potential Treatment of Alzheimer's Disease

Alzheimer's Disease (AD) is a chronic neurodegenerative disease leading to irreversible memory loss and is the most common form of dementia. While a cure for AD is not available, inhibitors of the two major classes of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are under investigation to mitigate the effects of this disease. In individuals with AD, studies showed that BChE activity increased while AChE activity was similar. The increased BChE activity is suggested to deplete acetylcholine, a neurotransmitter, and contribute to dementia. We identified Fmoc-amino acids as BChE inhibitors, and a lysine-based compound that shares structural similarities with the substrate was one of the most potent inhibitors. Although the original inhibitor had the same positive charge as acetylcholine, the inhibitor lacked methyl groups found in the actual substrate. We hypothesized that methylation of the lysine side chain better mimics leading to better inhibitors. Due to challenges in synthesizing a trimethylated product, we first synthesized a dimethyl amino analog of lysine using 34% formaldehyde, acetic acid, NaBH4, and 1,4-dioxane. The product was purified by chromatography and characterized by NMR. Enzyme kinetics assays monitored by UV-Vis spectroscopy were then used to determine inhibition constants (KI values). The KI values for the dimethylated product Fmoc-Lys(CH3)2-O- was four-fold lower than Fmoc-Lys-O- (40 μM versus 150 μM, respectively), indicating the dimethylammonium compound is a better inhibitor. Studies comparing BChE and AChE inhibition indicated that the compound selectively targets BChE relative to AChE. Computational docking was then used to visualize possible binding sites of the inhibitor. The results suggested that the cationic amino group was binding in the BChE active site similar to the substrate and supports the hypothesis that adding substrate features lead to a more potent BChE inhibitor. Currently, we are testing different synthesis approaches to obtain the trimethyl ammonium compound to then further test the effect of introducing substrate features. Together, the results are aimed at systematically evaluating the contributions of introducing substrate features in enzyme inhibitors to develop more potent inhibitors.

Azole antifungal compounds could have dual cholinesterase inhibitory potential according to virtual screening, enzyme kinetics, and toxicity studies of an inhouse library

Recent advances in cholinesterase inhibitors opened new venues for the treatment of cognitive disorders like Alzheimer's disease. Certain azole antifungals like miconazole were reported to have cholinesterase inhibitory effects and hence ameliorate cognitive deficits. In this study, we tested a set of azole antifungal derivatives selected through virtual screening of an inhouse library for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory effects. Compound 61 showed potent and selective AChE inhibition (IC50 = 8.77 µM). The study also yielded dual AChE/BChE inhibitors in addition to a number of potent AChE inhibitors. Enzyme kinetics assays revealed that AChE inhibitors were competitive inhibitors. All the active compounds were imidazole derivatives and the modeling study showed that imidazole at protonated state contributed greatly to the binding interactions with some key residues of AChE and BChE active site. The active derivatives had negligible cytotoxic effects on murine fibroblast viability. According to our results, compounds featuring the classical scaffold of azole antifungal drugs could hold high potential for anticholinesterase drug design.

Microwave-assisted synthesis, biological assessment, and molecular modeling of aza-heterocycles: Potential inhibitory capacity of cholinergic enzymes to Alzheimer's disease

A highly regioselective solvent-free microwave-assisted synthesis of pyrazoles and tetrahydroindazoles based on the condensation of 1,3-diketones with arylhydrazines is described. Compounds were evaluated as cholinesterase inhibitors in order to identify an alternative treatment for Alzheimer's disease. All compounds displayed moderated acetylcholinesterase inhibitory activity and most of the compounds displayed remarkable butyrylcholinesterase inhibitory activity and selectivity. The compounds 3y and 3i with IC50 of 1.65 and 3.59 µM, respectively, were the most active and selective compounds as butyrylcholinesterase inhibitors. Likewise, the compounds were tested as antioxidants agents, results showed that they have the ability to trap free-radicals. Molecular Docking studies showed a key π-π stacking interaction of most of the compounds with residue Trp82 within of butyrylcholinesterase active site. Molecular quantum similarity field, global and local reactivity descriptors, and the Fukui functions were calculated in the Density Functional Theory framework to analyze the reactivity patterns along with the molecular set.

Aporphinoid Alkaloids Derivatives as Selective Cholinesterases Inhibitors: Biological Evaluation and Docking Study.

Alzheimer's dementia is a neurodegenerative disease that affects the elderly population and causes memory impairment and cognitive deficit. Manifestation of this disease is associated to acetylcholine decrease; thus, Cholinesterase inhibition is the main therapeutic strategy for the treatment of Alzheimer's disease. In the present study, a series of aporphinoid alkaloids were tested as potential acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors in vitro. Alkaloids liriodenine (3) and cassythicine (10) were the best inhibitors of both cholinesterases with IC50 values lower than 10 μM. In addition, these alkaloids demonstrated better inhibition of BChE than reference drug galantamine. In addition, some alkaloids showed selective inhibition. Laurotetatine clorhydrate (13) selectively inhibit AChE over BChE. On the contrary, pachyconfine (7) interacted more efficiently with BChE active site. Molecular modelling studies were performed in order to illustrate key interactions between most active compounds and the enzymes and to explain their selectivity. These studies reveal that the benzodioxole moiety exhibits strong interactions due to hydrogen bonds that form with the Glu201 (AChE) and Tyr440 (BChE) residues, which is reflected in the IC50 values.

Synthesis and In Vitro Screening of Novel Heterocyclic β-d-Gluco- and β-d-Galactoconjugates as Butyrylcholinesterase Inhibitors.

The development of selective butyrylcholinesterase (BChE) inhibitors may improve the treatment of Alzheimer’s disease by increasing lower synaptic levels of the neurotransmitter acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and improved cognitive functions. A series of 14 novel heterocyclic β-d-gluco- and β-d-galactoconjugates were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and 1-benzylbenzimidazolium substituted β-d-gluco- and β-d-galacto-derivatives in a 10–50 micromolar range. The analysis performed by molecular modelling indicated key residues of the BChE active site, which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor should enable better blood–brain barrier permeability, and thus increase bioavailability in the central nervous system of these compounds.

Design, synthesis, evaluation and molecular modeling study of 4-N-phenylaminoquinolines for Alzheimer disease treatment

Dual binding site acetylcholinesterase (AChE) inhibitors and butyrylcholinesterase (BChE) inhibitors have recently emerged as two classes of new anti-Alzheimer agents to positively modify the disease’s course. In this work, a new series of 4-N-phenylaminoquinolines was synthesized and evaluated for their abilities to inhibit AChE and BChE. Compound 11b showed significant inhibitory activities on AChE and BChE with IC50 values of 0.86 and 2.65 μM, respectively, a lot better than that of reference drug galanthamine. Furthermore, docking study showed that compound 11b interacted simultaneously not only with active and peripheral sites of AChE, but also with all five regions of BChE active site. These findings suggest that these derivatives could be regarded as promising starting points for further drug discovery developments.

X-ray structures of human bile-salt activated lipase conjugated to nerve agents surrogates

The efficiency of human butyrylcholinesterase (BChE) as a stoichiometric bioscavenger of nerve agents is well established. However, wide use is currently limited by production and purification costs. Aiming at identifying an alternative human protein bioscavenger, we looked for an original scaffold candidate by virtual screening of the Protein Data Bank for functional similarity using the "Surfing the Molecules" software (sumo-pbil.ibcp.fr) and a search model based on the BChE active site topology. Besides the expected acetylcholinesterase and butyrylcholinesterase, we identified a set of bile salt activated lipases structures, among which the human pancreatic lipase (hBAL) that shares 34% identity with BChE. We produced the recombinant enzyme in mammalian cells, purified it, and measured the inhibition constants for paraoxon and surrogates of VX, sarin and tabun. We solved the X-ray structure of apo hBAL and conjugates with paraoxon and the surrogates at resolutions in the 2-Å range. These structures allow the assessment of hBAL for scavenging nerve agents. They revealed that hBAL has inverted stereoselectivity for the surrogates of nerve agent compared to human cholinesterases. We observed a remarkable flip of the catalytic histidine driven by the chelation of Zn2+. Dealkylation of the conjugate, aka aging, was solely observed for paraoxon.

Influence of the γ-carboline and carbazole pharmacophore moieties on anticholinesterase and antiradical activity of multifunctional agents for the treatment of neurodegenerative diseases

A comparative analysis of the esterase profile and antiradical activity of two groups of hybrid compounds, viz., tetrahydro-γ-carboline conjugates with carbazoles and tetrahydrocarbazoles (I) and carbazole conjugates with carbazoles and tetrahydrocarbazoles (II), was performed. The replacement of the tetrahydro-γ-carboline moiety (conjugates I) by the carbazole group (conjugates II) was shown to significantly reduce the ability of the compounds to inhibit butyrylcholinesterase (BChE) and scavenge free radicals. The tetrahydro-γ-carboline–tetrahydrocarbazole combination is optimal in terms of both high anti-BChE activity and free radical scavenging ability. According to molecular modeling calculations, the stronger binding of tetrahydro-γ-carboline conjugates (I) in the BChE active site compared to carbazole conjugates (II) is attributed to the ability of I to form ionic and π-cation interactions with amino acid residues lining the BChE gorge. Therefore, conjugates of tetrahydro-γ-carboline and tetrahydrocarbazole derivatives are the most promising compounds for the design of new multitarget drugs combining cognitive-stimulating and antioxidant properties.

Design, synthesis and biological evaluation of benzofuran appended benzothiazepine derivatives as inhibitors of butyrylcholinesterase and antimicrobial agents

A series of bezofuran appended 1,5-benzothiazepine compounds 7a–v was designed, synthesized and evaluated as cholinesterase inhibitors. The biological assay experiments showed that most of the compounds displayed a clearly selective inhibition for butyrylcholinesterase (BChE), while a weak or no effect towards acetylcholinesterase (AChE) was detected. All analogs exhibited varied BChE inhibitory activity with IC50 value ranging between 1.0 ± 0.01 and 72 ± 2.8 μM when compared with the standard donepezil (IC50, 2.63 ± 0.28 μM). Among the synthesized derivatives, compounds 7l, 7m and 7k exhibited the highest BChE inhibition with IC50 values of 1.0, 1.0 and 1.8 μM, respectively. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for compound 7l with BChE. In addition, docking studies confirmed the results obtained through in vitro experiments and showed that most potent compounds bind to both the catalytic anionic site (CAS) and peripheral anionic site (PAS) of BChE active site. The synthesized compounds were also evaluated for their in vitro antibacterial and antifungal activities. The results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms and showed high activity against both gram positive and gram negative bacteria and fungi.

Computational Study of Butyrylcholinesterase Inhibition by Aryl Alkyl Cholinyl Phosphorus Derivatives

Acetylcholine (ACh) is a neurotransmitter that is released from nerve cells to send signals to other cells, and acetylcholinesterase (AChE) is a highly specific enzyme that hydrolyzes ACh to regulate intercellular communication. However, butyrylcholinesterase (BChE), a non-specific AChE imposter, also breaks down choline based esters such as ACh. The activity of BChE has been found to gradually increase over time in patients with Alzheimer's disease (AD), which has been linked to a significant decrease in synaptic ACh levels, thus disrupting intercellular communication. In the treatment of AD, discovering inhibitors that prevent the activity of BChE and that do not interfere with the activity of AChE is vital. Aryl alkyl cholinyl phosphorous (AACP) derivatives are organophosphates that are expected to reversibly bind within the BChE binding pocket in the same manner as natural physiological substrates. This study utilizes massive flexible inhibitor docking calculations to predict the relative binding affinities between the enzyme and a number of AACP derivatives and to predict the optimal binding orientation(s) of each AACP derivative within the BChE active site. When compared to their previously studied dialkyl phenyl phosphate (DAPP) analogs, AACP derivatives are predicted to interact more strongly with the binding site of BChE, thus serving as more potent inhibitors. Our results reproduce experimental trends in binding affinity, indicating that derivatives with longer alkyl chains show increased inhibitory strength (as observed for DAPP derivatives), and the neutralization of the charge of the choline group and protonation of the neutral choline group had little effect on inhibitor strength, offering insight into the structural preferences of BChE for specific AACP derivatives. This study thus provides a framework for both future inhibitor design and more rigorous molecular dynamics studies of cholinesterase-inhibitor interaction.

New Cinchona Oximes Evaluated as Reactivators of Acetylcholinesterase and Butyrylcholinesterase Inhibited by Organophosphorus Compounds.

For the last six decades, researchers have been focused on finding efficient reactivators of organophosphorus compound (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In this study, we have focused our research on a new oxime scaffold based on the Cinchona structure since it was proven to fit the cholinesterases active site and reversibly inhibit their activity. Three Cinchona oximes (C1, C2, and C3), derivatives of the 9-oxocinchonidine, were synthesized and investigated in reactivation of various OP-inhibited AChE and BChE. As the results showed, the tested oximes were more efficient in the reactivation of BChE and they reactivated enzyme activity to up to 70% with reactivation rates similar to known pyridinium oximes used as antidotes in medical practice today. Furthermore, the oximes showed selectivity towards binding to the BChE active site and the determined enzyme-oxime dissociation constants supported work on the future development of inhibitors in other targeted studies (e.g., in treatment of neurodegenerative disease). Also, we monitored the cytotoxic effect of Cinchona oximes on two cell lines Hep G2 and SH-SY5Y to determine the possible limits for in vivo application. The cytotoxicity results support future studies of these compounds as long as their biological activity is targeted in the lower micromolar range.

Plasma butyrylcholinesterase regulates ghrelin to control aggression

Ongoing mouse studies of a proposed therapy for cocaine abuse based on viral gene transfer of butyrylcholinesterase (BChE) mutated for accelerated cocaine hydrolysis have yielded surprising effects on aggression. Further investigation has linked these effects to a reduction in circulating ghrelin, driven by BChE at levels ∼ 100-fold above normal. Tests with human BChE showed ready ghrelin hydrolysis at physiologic concentrations, and multiple low-mass molecular dynamics simulations revealed that ghrelin's first five residues fit sterically and electrostatically into BChE's active site. Consistent with in vitro results, male BALB/c mice with high plasma BChE after gene transfer exhibited sharply reduced plasma ghrelin. Unexpectedly, such animals fought less, both spontaneously and in a resident/intruder provocation model. One mutant BChE was found to be deficient in ghrelin hydrolysis. BALB/c mice transduced with this variant retained normal plasma ghrelin levels and did not differ from untreated controls in the aggression model. In contrast, C57BL/6 mice with BChE gene deletion exhibited increased ghrelin and fought more readily than wild-type animals. Collectively, these findings indicate that BChE-catalyzed ghrelin hydrolysis influences mouse aggression and social stress, with potential implications for humans.

Pharmacophore-based virtual screening and density functional theory approach to identifying novel butyrylcholinesterase inhibitors

To identify the critical chemical features, with reliable geometric constraints, that contributes to the inhibition of butyrylcholinesterase (BChE) function. Ligand-based pharmacophore modeling was used to identify the critical chemical features of BChE inhibitors. The generated pharmacophore model was validated using various techniques, such as Fischer's randomization method, test set, and decoy set. The best pharmacophore model was used as a query in virtual screening to identify novel scaffolds that inhibit BChE. Compounds selected by the best hypothesis in the virtual screening were tested for drug-like properties, and molecular docking study was applied to determine the optimal orientation of the hit compounds in the BChE active site. To find the reactivity of the hit compounds, frontier orbital analysis was carried out using density functional theory. Based on its correlation coefficient (0.96), root mean square (RMS) deviation (1.01), and total cost (105.72), the quantitative hypothesis Hypo1 consisting of 2 HBA, 1 Hy-Ali, and 1 Hy-Ar was selected as the best hypothesis. Thus, Hypo1 was used as a 3D query in virtual screening of the Maybridge and Chembridge databases. The hit compounds were filtered using ADMET, Lipinski's Rule of Five, and molecular docking to reduce the number of false positive results. Finally, 33 compounds were selected based on their critical interactions with the significant amino acids in BChE's active site. To confirm the inhibitors' potencies, the orbital energies, such as HOMO and LUMO, of the hit compounds and 7 training set compounds were calculated. Among the 33 hit compounds, 10 compounds with the highest HOMO values were selected, and this set was further culled to 5 compounds based on their energy gaps important for stability and energy transfer. From the overall results, 5 hit compounds were confirmed to be potential BChE inhibitors that satisfied all the pharmacophoric features in Hypo1. This study pinpoints important chemical features with geometric constraints that contribute to the inhibition of BChE activity. Five compounds are selected as the best hit BchE-inhibitory compounds.

Carbamates with Differential Mechanism of Inhibition Toward Acetylcholinesterase and Butyrylcholinesterase

Most carbamates are pseudoirreversible inhibitors of cholinesterases. Phenothiazine carbamates exhibit this inhibition of acetylcholinesterase but produce reversible inhibition of butyrylcholinesterase, suggesting that they do not form a covalent bond with the catalytic serine. This atypical inhibition is attributable to pi-pi interaction of the phenothiazine moiety with F329 and Y332 in butyrylcholinesterase. These residues are in a helical segment, referred to here as the E-helix because it contains E325 of the catalytic triad. The involvement of the E-helix in phenothiazine carbamate reversible inhibition of butyrylcholinesterase is confirmed using mutants of this enzyme at A328, F329, or Y332 that show typical pseudoirreversible inhibition. Thus, in addition to various domains of the butyrylcholinesterase active site gorge, such as the peripheral anionic site and the pi-cationic site of the Omega-loop, the E-helix represents a domain that could be exploited for development of specific inhibitors to treat dementias.

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.

Development of a MALDI-TOF-MS Method to Identify and Quantify Butyrylcholinesterase Inhibition Resulting from Exposure to Organophosphate and Carbamate Pesticides

A novel, proteomics based method was developed for the detection, quantification, and categorization of serum butyrylcholinesterase (BChE) inhibitors, including organophosphates (OPs) and carbamates (CBs). This method was based on the MALDI-TOF-MS analysis of the trypsin generated BChE active site peptide (191-SVTLFGESAGAASVSLHLLSPR-212) previously modified by reaction with an OP or CB. The ionization efficiency of OP modified active site peptides by MALDI was greatly improved by adding diammonium citrate to the MALDI matrix, which made the quantification of OP exposure feasible. Excellent linearity (r2 > 0.98) between the normalized abundance ratios (NARs) and OP concentrations or logarithm of carbaryl concentration was obtained. The accuracy of the developed assay was evaluated by comparison of IC50 and IC100 values from the assay with those determined by the Ellman method. Results from this method were comparable with those from the Ellman method. The advantage of the assay was that both the origin and the extent of pesticide exposure can be determined in one analysis. Our MALDI method can provide critical evidence for the pesticide exposure at low BChE inhibition levels even down to 3%, not available with the Ellman method.