Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition

The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent results: Michaelis constant K(m)=3.3mM, catalytic constant k(cat)=1.7 x 10(3)s(-1), bimolecular rate constant k(s)=5.1 x 10(5)M(-1)s(-1). The reaction product, choline, was found to be a competitive inhibitor with a dissociation constant K(i)=9.1mM. Betaine had a slightly higher affinity for the enzyme, but the inhibition was only partial. This study confirms the usefulness of microcalorimetry for the kinetic study of enzymes and their inhibitors.     

Microcalorimetric study of the inhibition of butyrylcholinesterase by carbamates

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by three carbamates (eserine, neostigmine, and rivastigmine) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5). The kinetics of carbamylation was studied in the absence or presence of the substrate, butyrylcholine, using an extension of the model described by Stojan and coworkers (FEBS Lett. 440 (1998) 85-88). The model was fitted to the data by a nonlinear regression procedure using simulated annealing followed by Marquardt's method. The affinity of the carbamates for the free enzyme increased in the order neostigmine相似文献   

Microcalorimetric study of the inhibition of butyrylcholinesterase by paraoxon

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by the organophosphorus compound paraoxon (diethyl 4-nitrophenyl phosphate) was studied by flow microcalorimetry at 37 °C in Tris buffer (pH 7.5) using a modification of the kinetic model described by Stojan and coworkers [J. Stojan, V. Marcel, S. Estrada-Mondaca, A. Klaebe, P. Masson, D. Fournier, A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase, FEBS Lett. 440 (1998) 85-88]. The reversible steps of the inhibition were studied in the mixing cell of the calorimeter, whereas the irreversible step was studied in the flow-through cell. A new pseudo-first-order approximation was developed to allow the kinetic analysis of inhibition progress curves in the presence of substrate when a significant amount of substrate is transformed. This approximation also allowed one to compute an analytical expression of the calorimetric curves using a gamma distribution to describe the impulse response of the calorimeter. Fitting models to data by nonlinear regression, with simulated annealing as a stochastic optimization method, allowed the determination of all kinetic parameters. It was found that paraoxon binds to both the enzyme and acyl-enzyme, but with weak affinities (K_i = 0.123 mM and K′_i = 5.5 mM). A slight activation was observed at the lowest paraoxon concentrations and was attributed to the binding of the substrate to the enzyme-inhibitor complex. The bimolecular inhibition rate constant k_i = 2.8 × 10⁴ M⁻¹ s⁻¹ was in agreement with previous studies. It is hoped that the methods developed in this work will contribute to extending the application range of microcalorimetry in the field of irreversible inhibitors.     

Design and synthesis of N-benzylpiperidine-purine derivatives as new dual inhibitors of acetyl- and butyrylcholinesterase

The synthesis and biological evaluation of N-benzyl-(piperidin or pyrrolidin)-purines are described. Compounds derived from N-benzylpiperidine and N-substituted purines showed moderate acetylcholinesterase inhibition. Preliminary structure–activity relationships and a superimposition of the best compound with the active conformation of donepezil have revealed structural features that have been used in the design of more potent N-benzylpiperidine inhibitors bearing an 8-substituted caffeine fragment and a methoxymethyl linker. These new compounds are interesting dual inhibitors of acetylcholinesterase and butyrylcholinesterase and have been chosen for further optimisation.     

Application of PEG-400 as a green biodegradable polymeric medium for the catalyst-free synthesis of spiro-dihydropyridines and their use as acetyl and butyrylcholinesterase inhibitors

A simple, efficient and green approach for the synthesis of spiro-dihydropyridines derivatives by one-pot multi-component reaction of isatin or acenaphthoquinone derivatives (1 equiv) with malononitrile (1 equiv) and N,N′-substituted-2-nitroethene-1,1-diamines (1 equiv) in PEG-400 under catalyst-free conditions is described. This method provides several advantages such as environmental friendliness, short reaction time, and simple workup procedure for the synthesis of biologically important compounds. The ability of synthesized compounds in inhibition of acetyl and butyrylcholinesterase were investigated both in vitro and in silico. All compounds showed moderate to high level activity against both acetyl and butyrylcholinesterase. There was a good correlation between in vitro and in silico studies.     

Whole body and tissue imaging of the butyrylcholinesterase knockout mouse injected with near infrared dye labeled butyrylcholinesterase

Butyrylcholinesterase (BChE) has proven to be an effective bioscavenger against nerve agents and organophosphates. Phase I safety trials of human BChE are currently being conducted and large-scale production of recombinant BChE is underway. Information on the real-time distribution of BChE from the injection site has not been well characterized. This study utilized the BChE nullizygote (BChE-/-) mouse and tetrameric equine BChE labeled with LI-COR((R)) fluorescent IRDye 800CW to track, quantify and determine the retention time of BChE in vivo following intramuscular injection. In vivo images were acquired with Xenogen's IVIS((R)) 200 imager and the LI-COR Odyssey((R)) Imaging System fitted with the MousePODtrade mark. Plasma and tissues were tested for BChE activity. The 2mg of BChE spread from the injection site to heart, liver, intestine, kidneys, lungs, salivary glands, and muscle, but did not enter the brain or the skin. Fluorescence intensity in organs and BChE activity in plasma peaked on day 1. BChE activity in plasma was undetectable by day 16, at a time when there was still significant fluorescent signal and BChE activity in the liver (0.32units/g), injected quadriceps (0.13units/g) and in most of the organs analyzed. It is concluded that the tetrameric BChE glycoprotein of 340kDa diffuses from the muscle injection site to blood and peripheral organs and has a longer residence time in the organs than in blood.     

NO-donating tacrine derivatives as potential butyrylcholinesterase inhibitors with vasorelaxation activity

To search for potent anti-Alzheimer’s disease (AD) agents with multifunctional effects, 12 NO-donating tacrine–flurbiprofen hybrid compounds (2a–l) were synthesized and biologically evaluated. It was found that all the new target compounds showed selective butyrylcholinesterase (BuChE) inhibitory activity in vitro comparable or higher than tacrine and the tacrine–flurbiprofen hybrid compounds 1a–c, and released moderate amount of NO in vitro. The kinetic study suggests that one of the most active and highest BuChE selective compounds 2d may not only compete with the substrate for the same catalytic active site (CAS) but also interact with a second binding site. Furthermore, 2d and 2l exhibited significant vascular relaxation effect, which is beneficial for the treatment of AD. All the results suggest that 2d and 2l might be promising lead compounds for further research.     

Microwave-assisted synthesis of new benzimidazole derivatives with lipase inhibition activity

Abstract

A practical protocol has been used for the synthesis of benzimidazoles. The reaction of iminoester hydrochlorides of phenylacetic with 4,5-dichloro-1,2-phenylenediamine under microwave irradiation leads to the benzimidazole derivatives with good yields and in short reaction times. After the synthesis of benzimidazoles, we synthesized ester and hydrazide derivatives under microwave irradiation with good yields. All compounds were evaluated with regard to pancreatic lipase activity and 3b, 3c, 5a and 6a showed lipase inhibition at various concentrations.     

Calcium-activated butyrylcholinesterase in human skin protects acetylcholinesterase against suicide inhibition by neurotoxic organophosphates

The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5x10(-3)M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. (45)Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H(2)O(2)-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8m(2) surface area with its calcium gradient in the 10(-3)M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue.     

Synthesis,biological activity and molecular modeling studies on 1H-benzimidazole derivatives as acetylcholinesterase inhibitors

A series of N-{2-[4-(1H-benzimidazole-2-yl)phenoxy]ethyl}substituted amine derivatives were designed to assess cholinesterase inhibitor activities. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitor activities were evaluated in vitro by using Ellman’s method. It was discovered that most of the compounds displayed AChE and/or BuChE inhibitor activity and few compounds were selective against AChE/BuChE. Compound 3c and 3e were the most active compounds in the series against eeAChE and hAChE, respectively. Molecular docking studies and molecular dynamics simulations were also carried out.     

Lipsome-formulated enzymes for organophosphate scavenging: butyrylcholinesterase and Demeton-S

Butyrylcholinesterase-encapsulating bioadhesive liposomes are investigated as prophylactic scavengers of organophosphates for local administration to skin, eyes, airways, and lungs-gates through which organophosphates penetrate living systems. The systems were optimized with respect to: encapsulation efficiency; type of bioadhesive ligand bound to liposomes (collagen or hyaluronan); ligand density at the liposomal surface; retention of encapsulated-enzyme activity; protection of encapsulated enzyme from proteolysis; and scavenging the model organophosphate Demeton-S (DS). Monolayers of PC-12 cells were selected for feasibility testing based on: high affinity binding of the bioadhesive liposomes-DeltaG0 release upon binding ranged from -9 to -12 kcal/mol ligand; ability to mimic an organophosphate attack upon intact cells and measuring its impact on intracellular acetylcholinesterase. Under attack, unprotected cells lost 80-90% of intracellular enzyme activity. The loss was reduced to 20-30% for protected cells (pre-treated with the formulations), at the expense of liposomal Butyrylcholinesterase. These results support our prophylactic approach.     

Pharmacological effects of a novel isosorbide-based butyrylcholinesterase inhibitor

Isosorbide-2-benzylcarbamate-5-benzoate, a novel butyrylcholinesterase inhibitor, shows interspecies variation in its inhibitory activity (IC(50) of 4.3 nM for human plasma butyrylcholinesterase, but 1.09 microM for mouse plasma butyrylcholinesterase). Stability studies revealed that this drug is resistant to hydrolysis by human plasma (no degradation in 1 h). However, it was found to undergo rapid degradation when incubated with mouse plasma or mouse liver homogenate, yielding benzyl carbamate and benzoic acid. The addition of the carboxylesterase inhibitor bis-(4-nitrophenyl) phosphate (BNPP) inhibited the degradation of the novel drug, indicating that it may be a substrate for both butyrylcholinesterase and carboxylesterase. The absence of carboxylesterase from human plasma explains the drug's stability in this medium. In vivo, pharmacodynamic studies on single doses of 1 mg/kg to na?ve male C57BL/6 mice revealed maximal plasma butyrylcholinesterase inhibition 20 min after intraperitoneal administration (approximately 60% inhibition) and 1 h after administration by gavage (approximately 45% inhibition). While this plasma butyrylcholinesterase inhibition was short-lived, the drug also penetrated the blood-brain barrier resulting in a slight (10-15%) but persistent (> or =72 h) reduction in brain butyrylcholinesterase activity.     

Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002–250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine ‘slides down’ the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π–π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme–substrate, enzyme–inhibitor and enzyme–substrate–inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.     

Human serum butyrylcholinesterase interactions with cisplatin and cyclophosphamide

Human serum Butyrylcholinesterase (BChE) is an important enzyme in detoxification with its capacity for hydrolyzing esters. The inhibitory effect of cisplatin (CDDP) and cyclophosphamide (CY) on BChE is characterized. Time dependent inhibition of BChE with both chemotherapeutics was rapid, reversible. CY was found as non-competitive inhibitor with Ki of 503.6 ± 50.4 μM. Time dependent CDDP studies displayed progressive inhibition. The constants for apparent dissociation (Ka), first order constant for the break down of the Michaelis complex (k + 2), and bimolecular rate (ka) were calculated as 6.38 × 10⁻⁵ M⁻¹ min⁻¹, 0.063 min⁻¹, and 9.83 × 10⁻⁴ M, respectively. Enzyme protection could be achieved with moderate butyrylthiocholine concentrations (0.3 mM) but higher concentrations increased CDDP inhibition. Apparent Ki value for CDDP was 191.8 ± 71.2 μM. These results suggest that used in combination therapy, CY and CDDP cause considerable BChE inhibition and may aggravate conditions observed during chemotherapy.     

Dialkyl phenyl phosphates as novel selective inhibitors of butyrylcholinesterase

A series of dialkyl phenyl phosphates (DAPPs) were synthesized and evaluated in silico and in vitro for inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Among the compounds examined, several DAPPs were shown to be potent inhibitors of butyrylcholinesterase, while having little activity against acetylcholinesterase. The most potent and selective inhibitors were di-n-butyl phenyl phosphate (K(i)=43 microM), di-n-pentyl phenyl phosphate (K(i)=6 microM), and di-cyclohexyl phenyl phosphate (K(i)=7 microM), the first which was shown to be a competitive inhibitor while the latter two being partial competitive inhibitors. Flexible docking simulations suggested that relative binding affinities generally increased as a function of alkyl chain length, while the strength and nature of inhibitory activity depended on whether the compound bound deeply or midway in the active site gorge, or in the proposed peripheral site.     

Five-membered heteroaromatic ring fused-pyrimidine derivatives: Design,synthesis, and hedgehog signaling pathway inhibition study

A series of novel five-membered heteroaromatic ring fused-pyrimidine derivatives including purines, pyrrolo[2,3-d]pyrimidines, pyrrolo[3,2-d]pyrimidines, thieno[2,3-d]pyrimidines, thieno[3,2-d]pyrimidines and furo[3,2-d]pyrimidines have been identified to be potent inhibitors of hedgehog signaling pathway. The synthesis and SAR of these compounds are described. Among this new series of hedgehog signaling pathway inhibitors, most compounds exhibited significant inhibitory activity compared to vismodegib, indicating that the five-membered heteroaromatic ring fused-pyrimidines stand out as encouraging scaffolds among the currently reported structural skeletons for hedgehog signaling pathway inhibitors, deserving more exploration and further investigation.     

Design and synthesis of new vancomycin derivatives

A set of vancomycin derivatives with lipid chain attached via a glyceric acid linker was designed and synthesized. A concise synthesis towards these derivatives was developed and the IC₅₀s of these new lipoglycopeptides were tested. Some of them showed very potent activity against both vancomycin sensitive and resistant strains.     

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes

Abstract

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer’s disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.     

Acetylcholinesterase/butyrylcholinesterase inhibition activity of some new carbacylamidophosphate derivatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2 with R = pCl-C6H4 1a, pBr-C6H4 2a, C6H5 3a, and pMe-C6H4 4a and RC(O)NHP(O)(NC4H8O)2 R = pCl-C6H4 1b, pBr-C6H4 2b, C6H5 3b, pMe-C6H4 4b, were selected to compare the inhibition kinetic parameters, IC50, Ki, kp and KD, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.     

The peptidase activity of human serum butyrylcholinesterase: Studies using monoclonal antibodies and characterization of the peptidase

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90–95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyryl-cholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. ThepH optimum of the peptidase was in the range of 7.5–9.5 and the divalent cations Co²⁺, Mn²⁺, and Zn²⁺ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co²⁺, Mn²⁺, and Zn²⁺ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited -chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.