Estimation of the upper limit of human butyrylcholinesterase dose required for protection against organophosphates toxicity: a mathematically based toxicokinetic model.
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Human butyrylcholinesterase (HuBChE) is a drug candidate for protection against organophosphates (OP) intoxication. A mathematically based model was validated and employed to better understand the role of the endogenous HuBChE in detoxification of OPs and to estimate the dose of exogenous HuBChE required for enhancing protection of humans from lethal exposure to OPs. The model addresses the relationship between the HuBChE dose needed to maintain a certain residual activity of human acetylcholinesterase (HuAChE) and the following parameters: (1) level and duration of exposure, (2) bimolecular rate constants of inhibition of HuAChE (kA) and HuBChE (kB) by OPs, and (3) time elapsed from enzyme load. The equation derived for the calculation of HuBChE dose requires the knowledge of kA/kB in human blood and the rate constant of HuBChE elimination. Predictions of HuBChE doses were validated by in vitro experiments and data of published human studies. These predictions highlight two parameters that are likely to decrease the calculated dose: (1) the rapid consumption of the less toxic isomers of OPs in human plasma, and (2) the volume of distribution of HuBChE that appears significantly greater than the volume of plasma. The first part of the analysis of the proposed model was focused on acute bolus exposures and suggests that upper limit doses of 134, 115, and 249 mg/70 kg are sufficient to protect RBC AChE above 30% of baseline activity following a challenge with 1 LD(50) VX, soman, and sarin, respectively. The principles of the validated model should be applicable for advanced predictions of HuBChE dose for protection against continuous exposures to OPs. (+info)
Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases.
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E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis. (+info)
Acetylcholinesterase and butyrylcholinesterase activities in cerebrospinal fluid from different levels of the neuraxis of patients with dementia of the Alzheimer type.
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Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities of cerebrospinal fluid (CSF) collected post mortem from the lateral ventricles, cisterna magna, and lumbar regions of the spinal cord of patients with a histologically confirmed diagnosis of Alzheimer's disease were compared with those of normal, age matched control patients, patients with dementia of non-Alzheimer aetiology, and patients with non-dementing neurological disorders. The AChE activity of the ventricular CSF of patients with Alzheimer's disease was 48% lower (p < 0.005) than that of age matched controls or patients with other types of dementia, and the AChE activity of CSF sampled from the basal cistern was 40% lower (p < 0.005) in patients with Alzheimer's disease. There were no significant differences between the AChE activity in Alzheimer's disease and control patients in CSF collected from the lumbar cistern. AChE activity was lower in CSF sampled from the basal and lumbar cistern of patients with dementia of non-Alzheimer aetiology, while ventricular activity was in the normal range. BuChE activity in ventricular CSF of Alzheimer's disease patients was 41% lower than normal (p < 0.05) and in the normal range in all other samples. The secretion of AChE from forebrain and hindbrain regions is reduced in Alzheimer's disease patients, leading to decreased ventricular and cisternal levels of the enzyme. Secretion from more caudal regions of the central nervous system seems to be unaffected by the disease, resulting in AChE in the lumbar CSF of patients with Alzheimer's disease being in the control range. Such altered secretion of AChE in the brain could have profound implications not only for cholinergic transmission in these patients but also for the proposed noncholinergic modulatory actions of AChE. (+info)
Organophosphates, serine esterase inhibition, and modeling of organophosphate toxicity.
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The highlighted article in this issue (Ashani and Pistinner, "Estimation of the Upper Limit of Human Butyrylcholinesterase Dose Required for Protection against Organophosphates toxicity: A Mathematically Based Toxicokinetic Model") is an innovative approach to modeling the amount of protective enzyme, human butyrylcholinesterase, that could be administered to humans to protect them from the lethal effects of organophosphate nerve agents. The threat of nerve agent exposures at lethal level regrettably remains a threat to military as well civilian populations, and the authors of this article have used their previous experimental data along with new in vitro data to devise and calibrate a mathematical model that could have practical utility in the prophylaxis of military personnel against chemical warfare agents. (+info)
Acetylcholinesterase/paraoxonase genotype and expression predict anxiety scores in Health, Risk Factors, Exercise Training, and Genetics study.
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Anxiety involves complex, incompletely understood interactions of genomic, environmental, and experience-derived factors, and is currently being measured by psychological criteria. Here, we report previously nonperceived interrelationships between expression variations and nucleotide polymorphisms of the chromosome 7q21-22 acetylcholinesterase-paraoxonase 1 (ACHE-PON1) locus with the trait- and state-anxiety measures of 461 healthy subjects from the Health, Risk Factors, Exercise Training, and Genetics Family Study. The AChE protein controls the termination of the stress-enhanced acetylcholine signaling, whereas the PON protein displays peroxidase-like activity, thus protecting blood proteins from oxidative stress damages. Serum AChE and PON enzyme activities were both found to be affected by demographic parameters, and showed inverse, reciprocal associations with anxiety measures. Moreover, the transient scores of state anxiety and the susceptibility score of trait anxiety both appeared to be linked to enzyme activities. This finding supported the notion of corresponding gene expression relationships. Parallel polymorphisms in the ACHE and PON1 genes displayed apparent associations with both trait- and state-anxiety scores. Our findings indicate that a significant source of anxiety feelings involves inherited and acquired parameters of acetylcholine regulation that can be readily quantified, which can help explaining part of the human variance for state and trait anxiety. (+info)
The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study.
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A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type. (+info)
Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals.
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Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples. (+info)
Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior.
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The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) << k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state. (+info)