The aim of this study was to determine the ability of twenty novel substituted phenoxyalkyl pyridinium oximes to reactivate phosphylated acetylcholinesterase (AChE) in peripheral rat tissues, in vitro, inhibited by organophosphate anticholinesterase nerve agent surrogates. A sarin surrogate, phthalimidyl isopropyl methylphosphonate (PIMP), and a VX surrogate, 4-nitrophenyl ethyl methylphosphonate (NEMP), were used to inhibit AChE in skeletal muscle and serum samples. Reactivation of the widely used oxime 2-PAM was tested for comparison with the novel oximes. The novel oximes displayed a range of 23-102% reactivation of AChE in vitro across both tissue types. Most of the novel oximes tested in the present study demonstrated a higher percent reactivation of AChE, than 2-PAM. Therefore, these novel oximes have the potential to be effective antidotes used during the treatment of OP toxicity.

Phorate (O,O-diethyl S-ethylthiomethyl phosphorodithioate) is a toxic organophosphate anticholinesterase insecticide. Organophosphate insecticides can cause respiratory depression and seizures due to acetylcholinesterase inhibition. Inhibited acetylcholinesterase cannot break down the neurotransmitter, acetylcholine; thus, causing an overload of acetylcholine in synapses and neuromuscular junctions. Oxidative desulfuration, from metabolism by cytochrome P450 enzymes, converts the P=S phosphorothionate group on phorate to the P=O oxon group. Electrophilic oxon groups attack the active site on acetylcholinesterase, inducing the toxicity associated with organophosphate insecticides. Possible further bioactivation to phorate-oxon-sulfoxide and phorate-oxon-sulfone near the site of acetylcholinesterase in the brain may increase acetylcholinesterase inhibitory potency. Adult male Sprague-Dawley rat brain and liver microsomes were used to determine the proportions of the phorate metabolites formed through bioactivation. Phorate-sulfoxide was produced in much greater proportion than any other metabolite, which may contribute to the delay observed in phorate toxicity as it takes longer to produce phorate-oxon, phorate-oxon-sulfoxide, or phorate-oxon-sulfone metabolites.

The mechanism of toxic action for organophosphates (OPs), originally developed as insecticides, is the persistent inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and subsequent hyperstimulation of the nervous system. Many OPs require bioactivation via cytochromes P450 to oxon metabolites which are anticholinesterases. Organophosphates display a wide range of acute toxicities. Differences in the OPs’ chemistries results in differences in the compounds' metabolism and toxicity. Acute toxicities of OPs appear to be principally dependent on compound specific efficiencies of detoxication, and less dependent upon efficiencies of bioactivation and sensitivity of AChE. Esterases, such as carboxylesterase (CaE) and butyrylcholinesterase (BChE), play a prominent role in OP detoxication. Organophosphates can stoichiometrically inhibit these enzymes, removing OPs from circulation thus providing protection for the target enzyme, AChE. This in vitro study investigated: 1) age-related sensitivity of AChE, BChE and CaE to structurally different OPs in rat tissues; 2) interspecies and intraspecies differences in bioactivation and detoxication of the OP insecticide malathion in rat and human hepatic microsomes; and 3) interspecies and intraspecies differences in sensitivity of AChE from erythrocyte ghost preparations to malaoxon. Sensitivities of esterases to 12 OPs was assessed by IC50s. The OPs displayed a wide range of AChE IC50s (low nM-µM) with no differences among ages; however, the CaE IC50s generally increased with age (up to 100old) reflecting greater protection in adults. Kinetic analysis of the bioactivation of malathion to the anticholinesterase metabolite, malaoxon, was measured in hepatic microsomes from rats (adult) and humans (various ages) of both sexes. No statistical interspecies (rat and human) or intraspecies (among humans) differences were found. The CaE degradation of malathion and malaoxon was determined in the microsomal samples using indirect measurements. No interspecies or intraspecies differences were found; however, CaE activity in rat microsomes was significantly higher than in humans. Inhibition of AChE by malaoxon was analyzed kinetically in erythrocyte ghost preparations from rats (adults) and humans (three age groups) of both sexes. No statistical interspecies or intraspecies differences were found. These results suggest the age-related differences in acute toxicities of OPs in mammals is primarily a result of their detoxication capacity.

Lau Suk Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 128-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Table of content --- p.ii / List of Figures --- p.x / List of Tables --- p.xiv / Abbreviation --- p.xv / Abstract --- p.xviii / 論文摘要 --- p.xx / Chapter 1 --- Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Tetraodon nigroviridis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Genomic Sequencing Project --- p.3 / Chapter 1.1.3 --- Tetraodon nigroviridis as Study Model --- p.4 / Chapter 1.1.3.1 --- Genomic Comparison --- p.4 / Chapter 1.1.3.2 --- Gene Order and Structural Studies --- p.5 / Chapter 1.1.3.3 --- Genomic Evolution --- p.6 / Chapter 1.2 --- Transcriptional Regulation and Transcription Factors Binding Sites Prediction --- p.7 / Chapter 1.2.1 --- Transcriptional Regulation --- p.7 / Chapter 1.2.1.1 --- Chromatin Remodeling --- p.7 / Chapter 1.2.1.2 --- Locus Control Regions (LCR) and Boundary Elements --- p.8 / Chapter 1.2.1.3 --- Promoter Structure --- p.9 / Chapter 1.2.1.4 --- Transcriptional Machinery Assembly --- p.10 / Chapter 1.2.2 --- Transcription Factors and Their Binding Sites --- p.11 / Chapter 1.2.3 --- Transcription Factor Binding Site Prediction --- p.12 / Chapter 1.3 --- Acetylcholinesterase --- p.15 / Chapter 1.3.1 --- Background --- p.15 / Chapter 1.3.2 --- Regulation ofAChE --- p.17 / Chapter 1.3.2.1 --- Transcriptional Level --- p.17 / Chapter 1.3.2.2 --- Post-transcriptional Level --- p.19 / Chapter 1.3.2.3 --- Post-translational Level --- p.20 / Chapter 1.3.2.3.1 --- Oligomerization --- p.20 / Chapter 1.3.2.3.2 --- Glycosylation --- p.21 / Chapter 1.3.2.3.3 --- Phosphroylation --- p.22 / Chapter 1.3.3 --- Functions of AChE --- p.23 / Chapter 1.3.3.1 --- Hydrolysis Acetylcholine --- p.23 / Chapter 1.3.3.2 --- Embryonic Development --- p.23 / Chapter 1.3.3.3 --- Haemotopotesis and Thrombopsiesis --- p.24 / Chapter 1.3.3.4 --- Neuritogensis --- p.24 / Chapter 1.3.3.5 --- Amyloid Fibre Assembly --- p.24 / Chapter 1.3.3.6 --- Apoptosis --- p.25 / Chapter 1.3.4 --- AChE and Alzheimer's disease --- p.25 / Chapter 1.3.4.1 --- Treatment for AD Patients --- p.27 / Chapter 1.4 --- Inducible Cell Expression Systems --- p.28 / Chapter 1.5 --- Objectives --- p.32 / Chapter 2 --- Chapter 2 Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Primer Design --- p.34 / Chapter 2.2.2 --- Cell Culture --- p.34 / Chapter 2.2.3 --- Transformation --- p.35 / Chapter 2.2.4 --- Plasmids Preparation --- p.35 / Chapter 2.2.5 --- Plasmids Screening --- p.36 / Chapter 2.2.6 --- RNA Extraction --- p.36 / Chapter 2.2.7 --- Reverse Transcriptase Polymerase Chain Reaction and Construction tnAChE/pCR4 vector --- p.37 / Chapter 2.2.8 --- Genomic Analysis --- p.37 / Chapter 2.2.9 --- Protein Sequence Analysis --- p.38 / Chapter 2.2.10 --- Genomic DNA Extraction --- p.39 / Chapter 2.2.11 --- Construction of Reporter Vectors ptnAChE_565/pGL3 and ptnAChK1143/pGL3 --- p.39 / Chapter 2.2.12 --- Luciferase Assay --- p.40 / Chapter 2.2.13 --- Transcription Factors and Promoter Prediction --- p.40 / Chapter 2.2.14 --- Protein Assay --- p.41 / Chapter 2.2.15 --- AChE Activity Determined by Ellman's Method --- p.41 / Chapter 2.2.16 --- Histochemistry --- p.42 / Chapter 2.2.17 --- Protein Extraction from Tissues --- p.42 / Chapter 2.2.18 --- Construction of Bacterial Expression Vector His-MBP-tnAChEAC/pHISMAL --- p.43 / Chapter 2.2.19 --- Protein Expression in Bacterial Expression System --- p.43 / Chapter 2.2.20 --- Purification and Thrombin Cleavage of His-MBP- tnAChEAC --- p.44 / Chapter 2.2.21 --- SDS Electrophoresis --- p.44 / Chapter 2.2.22 --- Western Blotting --- p.45 / Chapter 2.2.23 --- Construction of Tet-Off Expression Vector --- p.45 / Chapter 2.2.24 --- Transient Expression of tnAChEAC --- p.46 / Chapter 2.2.25 --- Establishment of Stable Tet-Off CHO Cell Lines Overexpressing tnAChEAC --- p.47 / Chapter 2.2.26 --- MTT Assay --- p.47 / Chapter 2.2.27 --- Partial Purification of tnAChEΔC --- p.48 / Chapter 3 --- Chapter 3 Sequence Analysis of AChE Gene of Tetraodon nigroviridis --- p.49 / Chapter 3.1 --- Results --- p.49 / Chapter 3.1.1 --- Cloning of tnAChE from Tetraodon nigroviridis Brain --- p.49 / Chapter 3.1.2 --- "Comparative genomic analysis of tnAChE with Human, Rat, Mouse, Takifugu rubripes, ZebrafishAChE" --- p.49 / Chapter 3.1.3 --- Primary Sequence Analysis --- p.52 / Chapter 3.1.4 --- Promoter and Transcriptional Factors Predictedin tnAChE Promoter Region --- p.60 / Chapter 3.1.4.1 --- Promoter Region Analysis In Silico --- p.60 / Chapter 3.1.4.2 --- Promoter Activity Analysis --- p.76 / Chapter 3.2 --- Discussion --- p.78 / Chapter 4 --- Characterization of tnAChE in Prokaryotic and Eukaryotic Tet-Off Inducible Expression System --- p.91 / Chapter 4.1 --- Results --- p.91 / Chapter 4.1.1 --- AChE Expresses in Tetraodon nigroviridis --- p.91 / Chapter 4.1.2 --- Expression of recombinant tnAChE in Bacterial Expression System --- p.94 / Chapter 4.1.2.1 --- Construction of His-MBP-tnAChEΔC/pHISMAL Construct --- p.94 / Chapter 4.1.2.2 --- His-MBP-tnAChEAC Expression in E. coli Strains BL21 (DE) and C41 --- p.94 / Chapter 4.1.3 --- Expression of tnAChEAC in Mammalian Expression System --- p.99 / Chapter 4.1.3.1 --- Construction of tnAChEAC/pTRE2hgyo Mammalian Expression Vector --- p.99 / Chapter 4.1.3.2 --- Transient Expression of tnAChEAC --- p.99 / Chapter 4.1.3.3 --- Establishment of Tet-Off CHO Cells Stably Expressing the Inducible tnAChEAC --- p.101 / Chapter 4.1.3.4 --- Characterization of Tet-Off tnAChEAC Stably Transfected Cell Clones --- p.103 / Chapter 4.1.3.5 --- Effect of Over Expressed tnAChEAC on cell viability --- p.103 / Chapter 4.1.3.6 --- Partial Purification of tnAChEAC from Stably Transfected Cells --- p.107 / Chapter 4.1.3.7 --- tnAChE and tnAChEAC in Different pH Values --- p.112 / Chapter 4.1.3.8 --- Kinetic Study of tnAChEAC --- p.112 / Chapter 4.1.3.9 --- Inhibition of AChE Activity of Partial Purified tnAChEAC by Huperzine --- p.112 / Chapter 4.2 --- Discussion --- p.116 / Chapter 4.2.1 --- Bacterial Expression System --- p.116 / Chapter 4.2.2 --- Expression of tnAChEΔC in Mammalian System --- p.119 / Chapter 5 --- General Discussion --- p.124 / Chapter 5.1 --- Summaries --- p.124 / Chapter 5.2 --- Further works --- p.126 / Chapter 6 --- References --- p.128 / Appendix 1 internet software and database used in this project --- p.151 / Appendix 2 tnAChE mRNA sequence --- p.152 / Appendix 3 ptnAChE-1143 sequence --- p.154 / Appendix 4 Six open reading frame translation of ptnAChE-1143 --- p.156

L’acétylcholinestérase (AChE), une des enzymes les plus rapides dans la nature, est lacible d’un large nombre de toxiques, dont notamment les neurotoxiques organophosphorés.La première partie de ce manuscrit de thèse décrit le développement raisonné d’un nouveauréactivateur, qui présente des propriétés de réactivation supérieures aux moléculesactuellement sur le marché. Les interactions entre cette molécule, KM297, et l’AChE ontété étudiées par dynamique moléculaire, docking et cristallographie aux rayons X. La connaissancedes modes de liaison du KM297 dans l’AChE native ou inhibé par un OP ontpermis de développer la molécule JDS207, qui se lie de façon exclusive au site périphériquede l’AChE. La deuxième partie de la thèse est dédiée à l’analyse des simulations de laAChE par dynamique moléculaire. On observe que la combinaison de multiples trajectoiresgénérées avec des paramètres de vélocité initiale différents est une méthode fiablepour caractériser les conformations atteintes par les chaînes latérales des acides aminés. Encomparant la distribution des rotamères pour l’AChE humaine et celle du poisson Torpedocalifornica, on montre que des différences importantes existent entre les enzymes des deuxespèces. A partir de ces informations sur les conformations de résidus clés du site actif,une méthode a été développée pour générer des récepteurs utilisable pour des calcules dedocking flexible, de façon à prendre en compte la dynamique propre à chaque résidu del’enzyme. Cette méthode a été validé en comparent les résultats obtenues à des structurescristallographiques connues. / Acetylcholinesterase (AChE), one of nature fastest enzyme, is the target of multiple toxics,including organophosphate nerve agents (OP). In the first part of this thesis I present thestructure-based development of a new uncharged reactivator, which showed characteristicsbetter than any molecule commercially available to date. The molecule has been rationallydesigned to present both affinity to the inhibited enzyme and good reactivation capabilities.The interactions between the lead molecule KM297 and AChE has been characterizedby means of flexible docking, molecular dynamics simulations and X-ray protein crystallography.The deeper understanding of its binding modes to both native and OP-inhibitedAChE has helped in developing a derivative, JDS207, whose binding mode at the peripheralsite of AChE is optimized. This derivative has also been studied by flexible docking and Xraycrystallography. The design of this family of reactivators taught us that a deep insightof the AChE dynamics is necessary to optimize ligands. The second part of the thesis isdevoted to the analysis of molecular dynamics simulations of AChE. At first, we assessedthat combining multiple short simulations is a fast and reliable method to characterizethe dynamics of the amino-acids side-chains. By comparing dynamics of the side-chainsfrom hAChE and TcAChE, we confirm that some key dynamical differences exist betweenthe two enzyme. The knowledge of the rotamers issued of MD simulation has lead us todevelop a new method to generate flexible receptors for docking, which is specific to eachsingle residue in the enzyme. This method has been validated by comparing its outputstructures with the ones found on the PDB database.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Natália Kuzmiaková Supervisor: PharmDr. Marie Vopršalová, CSc . Consultant: mjr. PharmDr. Vendula Hepnarová, Ph.D. Title of diploma thesis: In vivo evaluation of the efficacy of the novel reactivator against tabun. This study tackles the problem of irreversible inhibition of acetylcholinesterase (AChE). This enzyme degrades neurotransmitter acetylcholine (ACh), wich ensures transmisson of nerve impulses in central nervous system and in periphery. Organophospates (OP) are substances that cause irreversible blocade of AChE and that susubsequently leads to accumulation of AChE in synapses and inducing of muscarinic and nicotinic effects for life threatening condition. Oximic nature reactivators shown to this day the gratest potencial in inhibiting OP bond with AChE. Because reactivation abilities of to date synthesided oxime are not sufficient, new reactivators are being researched. The doal of my work was to test the potencial to reactivate AChe one of them (precisely oxime K 870). The method i used was colorimetric Ellman method modified by Bajgar, where the activity of AChE after reactivation was measured by absorbance in brain, diaphragm and blood of modeled orgamisms. The...

One particular mechanism of resistance to organophosphate and carbamate insecticides is insensitive variants of the target enzyme acetylcholinesterase (AChE). The aims were to set up an expression system in Saccharomyces cerevisiae for insect AChE with the long term objective of studying mosquito AChE variants and to assess the potential of a yeast system for the large scale production of AChE. Initially work centred on the analysis of an expression construct pBM150-DAChE containing the Drosophila melanogaster AChE (DAChE) cDNA under the yeast GAL10 promoter. Preliminary analyses were conducted using the assay developed by Ellman et al (1961). High levels of interference were encountered on cell breakage making assessments of expression extremely difficult. This interference was found to be due to thiol groups within the yeast cell wall. Enzymatic digestion of the cell wall was found to reduce interference to a manageable level. Analysis of protoplasts indicated that active AChE was either not being expressed or being expressed at undetectable levels. AChE mRNA could not be detected by Northern blotting. The DAChE cDNA was ligated into the high copy number vector pG3 under the constitutive glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter to form the construct pG3-DAChE which was transformed into the BJ2168 strain of S. cerevisiae. Attempts were also made to subclone the DAChE cDNA into a steroid inducible vector (p2UG) and a secretion vector (pPIC9). Analysis of expression from construct pG3-DAChE by both the Sabine (1955) and Ellman methods revealed that biologically active AChE was being constitutively expressed by S. cerevisiae. The expressed DAME was further authenticated by inhibition with the insecticide Bendiocarb and the inhibitor phenylmethylsulfonyl fluoride. A Kc. t value f or the expressed DAChE was approximated at 6429.3 molecules s-1 but specific activities were found to be low (--0.05 Units). The percentage of total S. cerevisiae protein that was active DAME was estimated at 0.0009%. SDS-PAGE analysis indicated that this active percentage was likely to be commensurate with the total amount of enzyme protein translated. Northern blotting of total RNA detected low levels of a shortened transcript that may either translate to a truncated but enzymatically active DAME or may represent a transiently stabilised mRNA in the process of degradation. The presence of a small population of full length transcripts that remained undetected should not be discounted. The expressed DAChE was found to be located in the cell membrane of s. cerevisiae suggesting that it had passed through the secretory pathway and further that the active site was probably internal of the membrane rather than externally situated. Plasmid copy number estimations and growth rate analyses showed that the expressed DAChE did not have a deleterious effect on cellular metabolism. RT-PCR was used to generate a homologous cDNA probe that could in the future be used to screen a cDNA library from Culex molestus for a susceptible mosquito AChE gene.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 231-257). Also available in electronic version. Access restricted to campus users.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 128-152). Also available in electronic version.

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.