MMSL, 2018 (vol. 87), Suppl.1

CHRONIC ILLNESS FROM ORGANOPHOSPHORUS TOXICANT EXPOSUREMeeting abstracts

Oksana Lockridge, Lawrence M. Schopfer

MMSL 2018, 87(88):1  

The mechanism of toxicity from acute exposure to organophosphorus toxicants (OP) is understood.  Thousands of publications have confirmed that AChE inhibition results in muscle weakness and respiratory failure.  AChE activity returns to normal levels within one month, but symptoms can persist for a lifetime.  For example, people exposed to sarin in the 1995 Tokyo subway attack still have adverse symptoms 23 years later.  Farmers and sheep dippers exposed to OP pesticides have an elevated risk of psychiatric disorders and suicidal behavior.  Epidemiology studies show an association between OP exposure and Alzheimer’s disease and Parkinson’s...

THE CATALYTIC POWER OF PHOSPHOTRIESTERASES FOR THE HYDROLYSIS AND DESTRUCTION OF ORGANOPHOSPHORUS NERVE AGENTSMeeting abstracts

Frank M. Raushel

MMSL 2018, 87(88):2  

Phosphotriesterase (PTE), an enzyme originally isolated from Pseudomonas diminuta, is capable of catalyzing the hydrolysis of many organophosphorus nerve agents.  The turnover number for the enzymatic hydrolysis of paraoxon (diethyl p-nitrophenyl phosphate) by PTE is ~500,000 min^-1.  The protein adopts a distorted (β/α)₈-barrel structural fold and the active site is perched at the C-terminal end of the β-barrel.  The water used for nucleophilic attack of the substrate bridges two divalent metal ions in the active site and is further activated by the side chain carboxylate from an aspartate residue...

RECENT BREAKTHROUGHS IN THE STRUCTURE/FUNCTION STUDIES OF ACETYLCHOLINESTERASEMeeting abstracts

J.L. Sussman, I. Silman

MMSL 2018, 87(88):3  

The synaptic enzyme acetylcholinesterase (AChE) terminates transmission at cholinergic synapses by rapidly hydrolysing acetylcholine. Examination of the 3D structure of AChE¹ shows that the active site is located at the bottom of a deep and narrow gorge, lined largely by aromatic residues, with its peripheral anionic site located at the top, near the entrance to of the gorge. 3D structures of AChE have been determined for the Torpedo, Electrophorus, mouse, Drosophila and human enzymes. Overall, more than a hundred crystal structures of AChEs, and of covalent conjugates and reversible complexes with various inhibitors...

COUPLING OF ACETYLCHOLINESTERASE TO THE INTERFACIAL PHASE STATEMeeting abstracts

Bernhard Fichtl, Stefan Nuschele, Konrad Kaufmann, Israel Silman, Matthias F Schneider

MMSL 2018, 87(88):4  

The state of the lipid interface is known to influence activity of membrane-bound enzymes. Indeed, many enzymes exhibit changes in activity at phase transitions in the membrane to which they are attached. We utilized a Langmuir trough in which detergent-soluble Torpedo californica acetylcholinesterase (DS-TcAChE)¹ was anchored to the solvent face of a phospholipid monolayer in order to study this phenomenon. A peak in activity was observed at the compressibility maximum accompanying the transition between the ordered and fluid phases. Neither molecular nor physical alterations affected this correlation qualitatively, as shown...

THE OTHER SIDE OF AChE: ALLOSTERIC SITES AND MODULATORSMeeting abstracts

Carlos Roca, Carlos Requena, Víctor Sebastián-Pérez, Sony Malhotra, Chris Radoux, Concepción Pérez, Ana Martinez, Juan Antonio Páez, Tom L. Blundell, Nuria E. Campillo

MMSL 2018, 87(88):5  

The best-known function of acetylcholinesterase (AChE) is the hydrolysis of the neurotransmitter acetylcholine, however we are increasingly aware of the multifunctionality of this enzyme [1]. The non-hydrolytic functions of AChE are driven by allosteric sites as the peripheral allosteric site (PAS) responsible for amyloidosis in Alzheimer’s disease through interaction with β-amyloid peptide. We would like to show our work about the identification and characterization of new allosteric sites in AChE, using computational tools. This study has allowed us to identify allosteric inhibitors by virtual screening using our in-house MBC chemical...

PARTIAL UNFOLDING OF INSECT ACETYLCHOLINESTERASE: STEPS TOWARD CYSTEINE-TARGETING INSECTICIDESMeeting abstracts

Yuan-Ping Pang

MMSL 2018, 87(88):6  

To obtain insight into the development of thermally stable insect acetylcholinesterases, 200 distinct, independent, unrestricted, unbiased, isobaric–isothermal, 316-ns molecular dynamics simulations of a substrate-bound mosquito acetylcholinesterase responsible for cholinergic functions (AP-agAChE)¹ were performed using forcefield FF12MC² and PMEMD of AMBER 11 with a periodic boundary condition at 1 atm and 340 K. In-depth conformational analysis of these simulations with an aggregated simulation time of 63.2 microseconds revealed partially unfolded regions of AP-agAChE that could be stabilized with mutations for developing...

THE PROTONATION STATE OF Glu197 AND ITS IMPORTANT ROLE IN STABILIZING CATALYTIC TRIAD OF BUTYRYLCHOLINESTERASEMeeting abstracts

Junjun Liu, Xiao Wan

MMSL 2018, 87(88):7  

The Glu197 of butyrylcholinesterase (BChE) has been long considered as deprotonated in various studies, e.g. discovering the dynamical characters, interpreting the binding properties of inhibitors, and proposing hypotheses for BChE-catalyzed reaction mechanism. By performing a series of 100 ns molecular dynamics simulations, we accidently discovered that Glu197 needed to be protonated to have the structures simulated appropriately, whereas the deprotonated Glu197 eventually caused the collapse of catalytic triad with long enough simulation time.[1] we found that a highly conserved water molecule required Glu197 to be protonated in order to form an...

RECIPES TO DESIGN SPECIFIC LIGANDS OF HUMAN BUTYRYLCHOLINESTERASEMeeting abstracts

Florian Nachon, Jacques-Philippe Colletier, Nicolas Coquelle, Xavier Brazzolotto

MMSL 2018, 87(88):8  

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze the neurotransmitter acetylcholine and function thereby as regulators of cholinergic neurotransmission. Recently, interest has greatly increased in BChE. Firstly, BChE is a good broad spectrum bioscavenger of nerve agent and its efficiency could be significantly increased by the mean of specific reactivators. Secondly, BChE activity in the brain increases with the progression of Alzheimer’s disease, thus classifying BChE as a promising drug target in the advanced phase of the disease. AChE and BChE display specificities for substrates and ligands that only partially overlap....

EXPLORING THE EVOLUTIONARY POTENTIAL OF THE αE7 CARBOXYLESTERASEMeeting abstracts

Galen J. Correy, Colin J. Jackson

MMSL 2018, 87(88):9  

The evolution of insecticide resistance is a model system for studying enzyme evolution. Three insect species have independently evolved catalytic organophosphate (OP) detoxification through a single active-site mutation in the αE7 carboxylesterase. To explore the evolutionary potential of αE7, we subjected αE7 from the sheep blowfly to nine rounds of mutation and screening. The final variant contained 11 mutations which increased the rate of OP-hydrolysis more than 1000-fold. Atomic resolution X-ray crystal structures of the evolutionary intermediates reveal the changes in structure and dynamics at each step in the evolutionary trajectory,...

PROTEIN DYNAMICS OF PHOSPHOTRIESTERASE: TWO CATIONS REQUIRED FOR ENZYME CATALYSISMeeting abstracts

Yuan-Ping Pang

MMSL 2018, 87(88):10  

To investigate how protein dynamics facilitates substrate entering and product exiting the phosphotriesterase active site, over 60 distinct, independent, unrestricted, unbiased, isobaric–isothermal, microsecond molecular dynamics simulations of zinc-containing phosphotriesterase in complex with a substrate analog¹ were performed using the second-generation cationic dummy atom model for the zinc divalent cation, forcefield FF12MC², and PMEMD of AMBER 16 with a periodic boundary condition at 1 atm and 277 K, 300 K, and 340 K. In-depth conformational analysis of these simulations with an aggregated simulation time of over 76...

NEW PROGRESS IN DRUG DESIGN, DISCOVERY AND DEVELOPMENT INVOLVING CHOLINESTERASESMeeting abstracts

Fang Zheng, Chang-Guo Zhan

MMSL 2018, 87(88):11  

This talk will briefly discuss our newest progress in drug design, discovery and development involving cholinesterases, particularly in three major therapeutic areas. (1) On the basis of our previous design and discovery of cocaine hydrolases (CocHs) engineered from human butyrylcholinesterase (BChE), we have further developed a novel, long-acting CocH form, and demonstrated the promising clinical potential of CocHs for therapeutic treatment of cocaine overdose and addiction in clinically relevant animal models. One of the long-acting CocHs is currently in the large-scale protein drug manufacturing process development. (2) It has been demonstrated...

IN SEARCHING FOR THE MECHANISM OF BUTYRYLCHOLINESTERASE ACTIVATORSMeeting abstracts

Jure Stojan

MMSL 2018, 87(88):12  

It is known that cholinesterases show homotropic pseudocooperative effects: their activity at millimolar substrate concentrations is higher than expected by simple saturation kinetics and they are strongly inhibited at the submolar concentrations. However, we have reported that the anionic site directed inhibitors tetramethylammonium and tetraethylamonium too, increase the activity of human butyrylcholinesterase. At that time, the same phenomenon could not be shown for the horse counterpart. Here, it was searched for other putative activators among often used compounds in cholinesterase research. Indeed, imidazole significantly  increase the activity...

COMPUTATIONAL ANALYSIS OF REACTION MECHANISMS FOR OPTIMIZATION OF BUTYRYLCHOLINESTERASE-BASED CATALYTIC BIOSCAVENGERS AGAINST ORGANOPHOSPHORUS AGENTSMeeting abstracts

Sofya Lushchekina, Bella Grigorenko, Alexander Nemukhin, Sergei Varfolomeev, Patrick Masson

MMSL 2018, 87(88):13  

Catalytic bioscavengers are second generation bioscavengers. These biopharmaceuticals can be used to degrade toxic organophosphorus agents (OPs) on the skin for decontamination or in the bloodstream for pre-treatment and post-exposure treatment of OP poisoning. Because degradation has to be fast, their catalytic efficiency has be as high as possible (k_cat/K_m>10⁶ M^-1min^-1). To be of interest, the catalytic activity of certain enzymes, in particular self-reactivating ChEs, has to be increased by several orders of magnitude. This can be reached by computer-redesign, directed evolution of existing...

ENHANCEMENT IN PYRIDINIUM OXIME-ASSISTED REACTIVATION OF TABUN-INHIBITED ACETYLCHOLINESTERASE ACHIEVED BY ACTIVE SITE MUTATIONSMeeting abstracts

Zrinka Kovarik, Maja Katalinić, Nikolina Maček Hrvat, Goran Šinko, Tamara Zorbaz, Anita Bosak

MMSL 2018, 87(88):14  

Tabun represents a phosphoramide class of organophosphosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. The currently used therapy in excessive cholinergic stimulation consists of the muscarinic antagonist of acetylcholine stimulation, an anti-seizure drug when indicated and an oxime as the reactivator of inhibited AChE. Since common oximes are particularly ineffective in tabun exposure, we probed the reactivation of phosphoramidate conjugates in more depth by using mutants of AChE and pyridinium oximes to reveal the structural subtleties and yield more information on the architecture of...

THE SEARCH FOR RESISTANCE-BREAKING AND SPECIES-SELECTIVE MOSQUITOCIDAL INHIBITORS OF Anopheles gambiae AChEMeeting abstracts

Paul R. Carlier, Jeffrey R. Bloomquist, Jonah Cheung, Jianyong Li, Max Totrov

MMSL 2018, 87(88):15  

The widespread deployment of insecticide-treated bednets (ITNs) in sub-Saharan Africa has led to a dramatic decline in malaria mortality. However, wide-spread and growing resistance of Anopheles gambiae mosquitoes to the pyrethroid class of voltage-gated Na⁺ channel modulators used on these nets jeopardizes this achievement, and has prompted the search for suitable insecticidal AChE inhibitors to replace pyrethroids. Such compounds would have three favorable characteristics:  excellent contact toxicity towards susceptible adult An. gambiae, good contact toxicity to those that bear the G119S resistance mutation of AChE, and...

STERIC EFFECTS IN THE DECARBAMOYLATION OF CARBAMOYLATED ACETYLCHOLINESTERASEMeeting abstracts

Kunisi S.Venkatasubban, Joseph L. Johnson, Jamie L. Thomas, Abdul Fauq, Bernadette Cusack, Terrone L. Rosenberry

MMSL 2018, 87(88):16  

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) in a two-step process, with initial transfer of the carbamoyl acyl group to a serine residue of AChE accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme.  This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days.  Since carbamates are poor, slowly reversible AChE substrates, they are effective AChE inhibitors that have been developed as insecticides and therapeutic agents.  We show that decarbamoylation rates are independent of the leaving...

ASSESSMENT OF SCORING FUNCTIONS FOR AChE-LIGAND INTERACTIONSMeeting abstracts

Goran Šinko

MMSL 2018, 87(88):17  

Computer-aided drug design is based on molecular modelling which includes two steps; molecular docking accompanied by scoring docked poses. Molecular docking fits the right molecular “key” to a known receptor “lock” by optimizing the atomic coordinates of a ligand to adapt its 3D structure in such a way to accommodate the binding into the receptor. The second step is the determination of a good fit between the ligand “key” and receptor “lock” using a function that correctly prioritizes the docked ligand poses and predicts their binding affinities by taking into account molecular interactions between the...

PHENYL VALERATE ESTERASE ACTIVITY OF HUMAN CHOLINESTERASESMeeting abstracts

Jorge Estévez, María Romo, Marina Terol, Iris Mangas, Miguel Ángel Sogorb, and Eugenio Vilanova

MMSL 2018, 87(88):18  

The toxicity of organophosphorus compounds (OPs) cannot be explained only by action on acetylcholinesterase or neuropathy target esterase (NTE). A fraction of the membrane bound phenylvalerate esterase activity (PVase) is associated to NTE, the key initiating molecular event in the OP-induced delayed neuropathy (OPIDN). An enzymatic fraction in chicken brain soluble PVase has been reported to be due to a butyrylcholinesterase protein, and we suggested that this enzymatic fraction could be related to the mode of action of the potentiation/promotion phenomenon of the OPIDN. We showed that human butyrylcholinesterase (hBuChE) shows PVase activity. Mipafox,...

EFFECTS OF MEMANTINE AND ITS METABOLITE Mrz 2/373 ON SOMAN-INDUCED INHIBITION OF BOVINE ERYTHROCYTE ACETYLCHOLINESTERASE IN VITROMeeting abstracts

Miloš P. Stojiljković, Ranko Škrbić, Milan Jokanović

MMSL 2018, 87(88):19  

Background: Memantine is the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, used in the treatment of Alzheimer’s disease. Memantine pretreatment assured protection of skeletal muscles from poisoning with nerve agents and an interaction between memantine and AChE was proposed [1].  Aim: Memantine and its main metabolite (1-amino-3-hydroxymethyl-5-methyl adamantine, Mrz 2/373) were used to ascertain their interaction with erythrocyte acetylcholinesterase (AChE) in vitro. The effect of these two compounds on the kinetics of the soman-induced AChE inhibition and on the aging of the soman-AChE complex was also investigated....

OXIMES WITH ORTHO-POSITIONED CHLORINE MOIETY EXHIBIT IMPROVED PHYSICAL-CHEMICAL PROPERTIES, EFFICIENT REACTIVATION OF INHIBITED HUMAN ACETYLCHOLINESTERASE AND REDUCED IN VIVO TOXICITYMeeting abstracts

David Malinak, Tamara Zorbaz, Adam Skarka, Martina Hrabinova, Nikola Marakovic, Jana Janockova, Ondrej Soukup, Jan Misik, Daniel Jun, Kamil Kuca, Zrinka Kovarik, Kamil Musilek

MMSL 2018, 87(88):20  

The series of bisquaternary oximes with ortho-positioned chlorine moiety was designed, prepared and evaluated. The novel compounds exhibited valuable pK_a properties [1] with improved in vitro reactivation ability of sarin, cyclosarin, VX, paraoxon- and dichlorvos-inhibited human AChE exceeding the standard monoquaternary or bisquaternary reactivators (pralidoxime, methoxime, trimedoxime, obidoxime and asoxime syn. HI-6). Additionally, some chlorinated compounds presented in vitro reactivation ability of tabun-inhibited human AChE similar to the efficiency of trimedoxime. The in vitro results were further explained...

DESIGN AND SYNTHESIS OF BIFUNCTIONAL FLUOROPYRIDINALDOXIME REACTIVATORS FOR NERVE AGENT-INHIBITED HUMAN ACETYLCHOLINESTERASEMeeting abstracts

Jagadeesh Yerri, José Dias, Florian Nachon, Rachid Baati

MMSL 2018, 87(88):21  

Acetylcholinesterase (AChE) is a key enzyme of the Central Nervous System (CNS), which hydrolyzes the neurotransmitter acetylcholine.¹ By targeting AChE, organophosphorus nerve agents (OPNA) and organophosphorus pesticides irreversibly inhibit the cholinergic transmission, which is leading to death if untreated.² Over several years, our group and colleagues have been concentrating on the development a new class of non-permanently charged bifunctional reactivators, that display higher affinity for AChE and high in vitro and in vivo efficiencies compared to 2-PAM and Hi6.³ By analogy, recently, we designed...

COMBINATION OF OXIMES WITH OVERLAPPING REACTIVATION SPECTRA: OBIDOXIME AND HI-6Meeting abstracts

Timo Wille, Horst Thiermann, Franz Worek

MMSL 2018, 87(88):22  

Despite extensive oxime research in the last 60 years pralidoxime is still the standard oxime in e.g. United States, British and French forces and obidoxime standard therapy for OP poisoning in several European countries. Oxime research focusses on highly potent oximes with activity against selected nerve agents, broad-spectrum oximes with activity against relevant nerve agents and centrally active (non-)oximes but virtually no compound brought significant improvements compared to the established obidoxime and pralidoxime. In the US MMB-4 is sought to replace pralidoxime and in Germany, France, UK, Canada and other European countries HI-6 is in advanced...

DESIGN OF BROAD SPECTRUM ANTIDOTESMeeting abstracts

Cecilia Lindgren, Nina Forsgren, Christine Akfur, Lotta Berg, David Andersson, Franz Worek, Anna Linusson, Fredrik Ekström

MMSL 2018, 87(88):23  

The design of reactive molecules such as nerve agent antidotes is inherently challenging due to two intertwined processes imperative for their efficiency: The reversible binding of the initial non-covalent complex in a low energy conformation and the chemical reaction that proceeds via a transition state of high(er) energy. Furthermore, a structural and chemical diversity among different nerve agents and their corresponding complex with AChE complicates the design of broad-spectrum antidotes. The development of broad spectrum antidotes has proven challenging and although progress has been made, no new drugs with improved properties have been...

DEMONSTRATION OF THE FIRST SMALL MOLECULE THERAPEUTICS FOR RESURRECTION OF THE AGED FORM OF ACETYLCHOLINESTERASE AFTER EXPOSURE TO ORGANOPHOSPHORUS CHEMICAL NERVE AGENTS AND PESTICIDESMeeting abstracts

Andrew J. Franjesevic, Qinggeng Zhuang, Ola, Nosseir, William H. Coldren, Christopher S. Callam, Christopher M. Hadad

MMSL 2018, 87(88):24  

Organophosphorus (OP) compounds are potent acetylcholinesterase (AChE) inhibitors that have found use as both chemical warfare agents (CWAs) and as pesticides. Following inhibition of AChE by OP compounds, a competitive dealkylation reaction of the phosphylated serine residue occurs – a process referred to as aging. Current therapeutic reactivators of OP-inhibited AChE, mainly oximes, are not effective once aging has occurred. For the first time, we have demonstrated in vitro conversion of the aged AChE to the native form using small drug-like molecular therapeutics.  As part of this effort, a diverse library of small molecule therapeutics...

NANOTECHNOLOGY STRATEGIES USING OXIMES-LOADED LIPID NANOPARTICLES FOR BRAIN PROTECTION AGAINST ORGANOPHOSPHORUS POISONINGMeeting abstracts

Tatiana N. Pashirova, Irina V. Zueva, Anissa Braïki, Konstantin A. Petrov, Vasily M. Babaev, Evgenia A. Burilova, Darya A. Samarkina, Ildar Kh. Rizvanov, Eliana B. Souto, Ludovic Jean, Pierre-Yves Renard, Patrick Masson, Lucia Ya. Zakharova, Oleg G. Sinyashin

MMSL 2018, 87(88):25  

Nanotechnological "two-in-one" approach using nanoparticles for packaging two oximes in single carriers and nose-to-brain delivery for brain protection against poisoning by organophosphorus agents have been developed. Strategies for designing nanocarriers for drug delivery to the CNS and crossing the BBB showed that nanoparticles based on natural and biodegradable materials are promising. Solid lipid nanoparticles (SLNs) are biocompatible, biodegradable and have very low toxicity, thereby fulfilling the requirements of preclinical safety [1]. 2-PAM and a novel reactivator of VX-, paraoxon-, and tabun-phosphylated AChE [2] a poorly water soluble 6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxypicolinaldehyde...

UTILIZING STRUCTURE-ACTIVITY RELATIONSHIPS AND MECHANISTIC INSIGHTS TO DESIGN NONOXIME REACTIVATORSMeeting abstracts

C. Linn Cadieux, Zachary Canter, Kevin Martin, Keith Morgan, Michael Hepperle

MMSL 2018, 87(88):26  

Organophosphorus nerve agents are highly toxic compounds which pose a threat worldwide. These compounds induce toxicity by covalently binding to the active site serine of acetylcholinesterase, which results in inhibition of the enzyme. Without functional acetylcholinesterase, the levels of the neurotransmitter acetylcholine in neuromuscular junctions rise quickly, causing overstimulation of the nervous system, which will culminate in death if not treated. Current treatments rely on small molecules to interact with inhibited enzyme to disrupt the covalently bound phosphorus moiety at the active site. The most effective molecules incorporate a pyridinium...

NEW NON-OXIME REACTIVATORS OF ORGANOPHOSPHATE INHIBITED ACETYLCHOLINESTERASE WITH PROMISING REACTIVATION POTENCYMeeting abstracts

Martijn de Koning, Franz Worek, Gabriele Horn, Marco van Grol

MMSL 2018, 87(88):27  

Organophosphate (OP) compounds inhibit the enzyme acetylcholinesterase (AChE) resulting in severe symptoms and ultimately death. OP intoxications are currently treated by administration of atropine and certain oxime compounds (Obidoxime, HI-6 or 2-PAM). The latter compounds contain nucleophilic oximes that reactivate OP-inhibited AChE by liberating the phosphylated serine. However, these oximes have several drawbacks such as their intrinsic toxicity, their permanent charge which thwarts penetration of brain tissues and their inability to effectively reactivate all types of nerve agent inhibited AChEs. Therefore, the search for new (non-ionic) antidotes...

IDENTIFYING AXONAL TRANSPORT-RELATED TARGETS FOR REVERSING THE ADVERSE EFFECTS OF ORGANOPHOSPHATE EXPOSUREMeeting abstracts

Sean X. Naughton, Alvin V. Terry, Jr

MMSL 2018, 87(88):28  

The chemicals known as the organophosphates (OPs) are found in hundreds of useful agricultural, industrial, and commercial products; however, they have also been associated with a variety of adverse health effects in humans and other non-target organisms.  The acute toxicity of OPs is attributed to the inhibition of the enzyme acetylcholinesterase; however, this mechanism is inadequate to explain all of the long-term adverse effects of OPs.   In both live imaging studies in primary neuronal culture as well as in manganese-enhanced magnetic resonance imaging (MEMRI) studies of the brains of living rats, we have observed impairments in axonal transport...

DIAGNOSIS OF POISONING WITH О-ISOBUTYL-S-[2-(DIETHYLAMINO) ETHYL]METHYLPHOSPHONOTHIOATE (VR) UNDER ANTIDOTAL THERAPY WITH CARBOXIMMeeting abstracts

Nadezhda L. Koryagina, Elena I. Savelieva, Anton I. Ukolov, Darya S. Prokofieva, Nataliia S. Khlebnikova, Tatiana I. Aliushina, Elena S. Ukolova, Andrey S. Radilov, Nikolay V. Goncharov

MMSL 2018, 87(88):29  

The choice of biomarkers for establishment of exposure to organophosphorus compounds (OPs) is made based on the results of assessment of the real situation with account for such factors as the required timeframe for providing the results of expert examination, nature and volume of biosamples, available equipment, and the degree of confidence of information on the influencing factor (substance, dose, way of entry, use of antidote). We estimated the efficiency of express methods of diagnosis of exposure to OPs, specifically, Ellman’s cholinesterase activity assay, as well as GC-MS/MS and HPLC/MS/MS determination of OPs fluoride-regenerated from...

COPPER-DEPENDENT HYDROLYSIS OF TRICHLORONATE BY TURKEY SERUM AND ALBUMINMeeting abstracts

Damianys Almenares-López, Antonio Monroy-Noyola

MMSL 2018, 87(88):30  

Trichloronate is a racemic organophosphatioate insecticide. It induced delayed neuropathic in hens and human. The avian are species with greater susceptibility to organophosphorus poisoning due to their low levels of A-esterases. However, a copper-dependent hydrolyzing activity of hexyl dichlorophenyl phosphoramidate (HDCP), known as “antogonistic stereoselectivity” was recently identified in chicken serum. This study shows the activating effect of copper on the hydrolysis of trichloronate enantiomers by turkey serum and albumin (TSA) using chiral chromatography with CHIRALCEL OD column and heptane HPLC as mobile phase. The trichloronate...

MASS SPECTRAL DETECTION OF DIETHOXYPHOSPHOTYROSINE ADDUCTS ON PROTEINS FROM HEK293 CELLS USING MONOCLONAL ANTIBODY DEPY FOR ENRICHMENTMeeting abstracts

Seda Onder, Lawrence M. Schopfer, Ozden Tacal, Thomas A. Blake, Rudolph C. Johnson, Oksana Lockridge

MMSL 2018, 87(88):31  

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins.  Tyrosine readily reacts with organophosphorus toxicants in proteins that have no active site serine.  We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence ¹. Our goal was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon.  Cell lysates treated with chlorpyrifos oxon were examined by ELISA and capillary electrophoresis Western blot.  Tryptic peptides...

INNOVATIVE BIOCATALYSTS AS TOOLS TO DETECT AND INACTIVATE NERVE AGENTSMeeting abstracts

Elena Porzio, Francesca Bettazzi, Luigi Mandrich, Immacolata Del Giudice, Odile F. Restaino, Serena Laschi, Ferdinando Febbraio, Valentina De Luca, Maria G. Borzacchiello, Teresa M. Carusone, Franz Worek, Antonio Pisanti, Piero Porcaro, Chiara Schiraldi, Mario De Rosa, Ilaria Palchetti, Giuseppe Manco

MMSL 2018, 87(88):32  

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development...

IONIZABLE, ZWITTERIONIC OXIMES AS COUNTERMEASURES TO VOLATILE ORGANOPHOSPHATE (OP) EXPOSUREMeeting abstracts

Palmer Taylor, William C. Hou, Jeremiah Momper, Yan-Jye Shyong, Zoran Radic, John McDonough, Zrinka Kovarik, Yvonne Rosenberg, K. Barry Sharpless

MMSL 2018, 87(88):33  

Small ionizable, zwitterionic oximes of limited toxicity show successful outcomes in non-human primates upon intramuscular post-treatment of exposures to OP’s, that enter via the respiratory tract.  Along with their inherent limitations, we consider the bases for success in post-exposure treatment of OP toxicity and reversal of OP-induced sequelae of symptoms¹.  (1) High vapor pressure OPs carry the largest acute exposure risk in mass terrorism.  Toxic OPs released from explosive devices or into controlled ventilation environments are governed by partial pressure and Fick’s Second Law of Diffusion (inverse square of the distance); ...

DEVELOPMENT OF PRE- AND POST-COUNTERMEASURES AGAINST OP TOXINS IN MACAQUESMeeting abstracts

Yvonne Rosenberg, James Fink, Lingjun Mao, Xiaoming Jiang, Jonathan Lees, Jerry Wang, Tara Ooms, Narayanan Rajendra, Zoran Radic, Palmer Taylor

MMSL 2018, 87(88):34  

DDeliberate sarin releases in Syria with large numbers of fatalities emphasize the need for OP countermeasures for both military and civilian populations. Therapeutic countermeasures  involve several strategies: (i) preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets in the brain and in the periphery (ii) post-exposure oxime that can rapidly reactivate OP-inhibited AChE or (iii) a combination of both. In terms of a pretreatment, our recent studies have demonstrated that administration of an aerosolized (aer)-rHuBChE employing a user friendly nebulizer, forms a...

HUMAN PLASMA-DERIVED BUTYRYLCHOLINESTERASE IS BEHAVIORALLY SAFE AND EFFECTIVE IN CYNOMOLGUS MACAQUES (Macaca Fascicularis) CHALLENGED WITH SOMANMeeting abstracts

Todd M. Myers

MMSL 2018, 87(88):35  

Organophosphorus compounds (OP) pose a significant threat. Administration of human butyrylcholinesterase (Hu BChE) may reduce or prevent OP toxicity. Thus, we evaluated the safety and efficacy of Hu BChE in monkeys using sensitive neurobehavioral tests while concurrently characterizing absorption and elimination in the presence and absence of high-dose soman exposure to predict time course and degree of protection. Eight young adult male cynomolgus macaques were trained on two distinct automated tests of neurobehavioral functioning. Hu BChE purified under current-Good-Manufacturing Practices (CGMP) was injected intramuscularly at 13.1 mg/kg, producing...

DESIGN OF A COMBINED APTAMER FOR PARAOXON AND ACETYLCHOLINESTERASE BY IN SILICO APPROACHMeeting abstracts

Daria A. Belinskaia, Pavel A. Avdonin, Nikolay V. Goncharov

MMSL 2018, 87(88):36  

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of poisoning could be achieved with the help of aptamers, which are able to bind poisons in the bloodstream [1]. The effectiveness of the aptamers for OPs could be strengthened by their possibility to bind non-covalently with the peripheral anionic site (PAS) of acetylcholinesterase (AChE) defending the active site gorge from OPs molecules. In the present work, we have applied for the first time the in silico design of a combined aptamer for paraoxon and PAS of AChE. Based on the published sequence of...

PROKARYOTIC EXPRESSION OF HUMAN BUTYRYLCHOLINESTERASE AS A TOOL FOR CATALYTIC BIOSCAVENGER DEVELOPMENTMeeting abstracts

Xavier Brazzolotto, Alexandre Igert, Virginia Guillon, Gianluca Santoni, Florian Nachon

MMSL 2018, 87(88):37  

Currently, the best bioscavenger candidate against nerve agent intoxication is human butyrylcholinesterase (BChE). However, the effective dose cost, estimated to about 200 milligrams of pure enzyme, remains challenging despite the production and purification progresses realized these last years. A strategy for reducing dosage and cost would be to turn this scavenging protein into a nerve agent hydrolyzing enzyme, a catalytic bioscavenger. Up to now, screening of large mutant libraries has been hindered by the restricted eukaryotic expression of active BChE. Here we present the successful prokaryotic expression of an active human BChE variant designed...

BIOSCAVENGERS AND THE MEDICAL MANAGEMENT CHAINMeeting abstracts

T. M. Mann, H. Rice

MMSL 2018, 87(88):38  

Survival and recovery from nerve agent poisoning requires a continuum of medical care, starting with a rapid initial response followed by continued support though the medical chain. In a military context, research into countermeasures to nerve agent poisoning has traditionally focussed on first-aid, pretreatment and prophylaxis; however, there are many opportunities to optimise the management of nerve agent-poisoned casualties. We have previously demonstrated the efficacy of bioscavenger as a post-exposure, pre-symptomatic therapy in guinea-pigs poisoned by VX via the dermal route. Data will be presented on the efficacy of bioscavenger before, on and...

BORDERLINE BETWEEN CATALYTIC AND NON-CATALYTIC BIO SCAVENGERS: THE EXAMPLE OF ALBUMIN AND REVERSIBLE B-ESTERASESMeeting abstracts

Eugenio Vilanova, Jorge Estévez, Miguel Ángel Sogorb, Iris Mangas, Antonio Monroy

MMSL 2018, 87(88):39  

Protective mechanism against organophosphorus compounds (OPs) toxicity are mainly based in molecular processes frequently divided conceptually in (A) catalytic and (B) non-catalytic bio-scavengers. Modified natural proteins and small molecules have been developed for applying in therapy and protection. The catalytic ones are mainly associate to the classical concept of A-esterases (phosphotriesterases, PTEs, i.e. paraoxonase); they hydrolyze carboxylesters and OPs by a divalent cation dependent mechanism.  The non-catalytic scavengers are mainly associated to covalent binding to proteins, especially B-esterases with a serine or tyrosine residue, which...

CATALYTIC SCAVENGERS PROVIDE BROAD-SPECTRUM PROTECTION AGAINST ORGANOPHOSPHORUS NERVE AGENTSMeeting abstracts

Shane A. Kasten, Sandra J. DeBus, Thuy L. Dao, Michael V. Boeri, Zachary A. Canter, Sean M. Hodgins, Robyn B. Lee, Douglas M. Cerasoli, Tamara C. Otto

MMSL 2018, 87(88):40  

Efforts to develop a single enzyme capable of catalyzing the hydrolysis of a broad spectrum of organophosphorus (OP) compounds into non-toxic products have produced multiple candidate enzymes on different structural scaffolds.  While protection against multiple OPs from a single enzyme has been obtained, no single enzyme has been identified that can provide protection against all G- and V-type OP nerve agents.  The most promising candidate enzyme platform is the bacterially produced recombinant variant of organophosphorus hydrolase (OPH) from B. diminuta.  In vivo protective efficacy of candidate OPH scavengers as prophylactics was tested...

PARAOXONASE 1 VARIANT I-F11 GENE THERAPY USING ADENO-ASSOCIATED VIRUS8 (AAV8) OFFERS LONG-TERM PROTECTION AGAINST G-TYPE CHEMICAL WARFARE NERVE AGENTSMeeting abstracts

Venkaiah Betapudi, Deborah M. Doctor, Nageswararao Chilukuri

MMSL 2018, 87(88):41  

The paraoxonase 1 variant I-F11 affords asymptomatic protection against the lethal effects of G-type chemical warfare nerve agents (CWNA). Here, we tested whether adeno-associated virus8 (AAV8) is able to deliver I-F11 for extended periods of time and at levels affording asymptomatic protection against 2-5LD50 doses of G-type CWNA in mice. I-F11 gene expression levels in mouse blood were assessed under the influence of three different promoters and found to be significantly higher with TBG compared to CMV and CASI. A single tail vein or intramuscular injection of AAV8-TBG-I-F11 resulted in robust production of the enzyme, which reached concentrations...

ORGANOPHOSPHATE HYDROLASE (OPH) DESIGNED AS A TETHERED MONOMERMeeting abstracts

Jaffet Santiago Garcia, Cetara Baker, Richard Sweeney, Stephen Kirby

MMSL 2018, 87(88):42  

Organophosphate hydrolase (OPH) mutants have shown potential use as a medical countermeasure against organophosphorus compounds (OPs). OPH is typically expressed in bacteria as a homodimer. Two separate subunits (35 kDa each) self-assemble through non-covalent bonding at the enzyme face close to the putative active site. OPH homodimers do not secrete expediently from mammalian cells. This causes potential problems when trying to express the protein from a heterologous plasmid or viral delivery system. To enhance secretion of OPH from mammalian cells, we sought to increase protein solubility without catastrophic detriment to activity and without addition...

A NEW ANIMAL MODEL TO INVESTIGATE ORGANOPHOSPHORUS POISONING AND ENZYMATIC DECONTAMINATIONMeeting abstracts

Laetitia Poirier, Pauline Jacquet, Laure Plener, Cédric Torre, Eric Ghigo, David Daudé, Eric Chabrière

MMSL 2018, 87(88):43  

Freshwater planarians from Platyhelminthes, harboring a mammal-like cholinergic nervous system, have emerged as a promising in vivo model for investigating neurotoxicity. Moreover a large proportion of stem cells provide planarian an unconventional capacity of regeneration allowing for developmental disruption studies. Schmidtea mediterranea (Smed) was used as model for organophosphorus (OP) poisoning and for evaluating the efficacy of detoxifying enzymes. Acetylcholinesterase and butyrylcholinesterase from planarian (Smed-AChE and Smed-BChE) share 35% identity with their human counterpart (Hs-AChE and Hs-BChE)....

PARAOXONASE-2 DEPENDENT REDOX CONTROL OF PLATELET PHYSIOLOGYMeeting abstracts

V.Petermann, H. Kleinert, K. Jurk

MMSL 2018, 87(88):44  

Background and Objective: Platelets are not only central players in hemostasis and thrombosis but also important modulators of immune responses, inflammation and cancer. Activated platelets generate reactive oxygen species (ROS) that modulate platelet function through redox signaling and oxidative stress. The anti-oxidative enzyme paraoxonase-2 (PON2) is known to counteract inflammation and atherosclerosis. Recently, we showed that PON2-deficient mice exhibit tissue factor-dependent hypercoagulability¹. Here, we investigated the role of PON2 in ROS production, phenotype and activation of platelets from PON2-deficient mice. Methods: Platelet...

COPPER WITH CHICKEN SERUM ALBUMIN SHOW STEREOSELECTIVE HYDROLYSIS OF CHIRAL PHOSPHORAMIDATESMeeting abstracts

Antonio Monroy-Noyola, Miguel Angel Sogorb, Eugenio Vilanova

MMSL 2018, 87(88):45  

Chiral analogous compound of methamidophos insecticide are only poorly hydrolyzed by Ca²⁺-dependent phosphotriesterases in mammals tissues including the human serum. We reported the hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) in chicken serum. The hydrolysis of the R-(+)-HDCP isomer is strongly increased in vitro in the presence of 30-250 µM copper.  It is the opposite estereoselectivity of that showed by liver Ca²⁺-dependent activity. We name it as "antagonistic stereoselectivity". Diluted chicken serum (10 µL in 1 mL solution of 400 µM HDCP) or the equivalent amount of commercial chicken...

INSIGHTS INTO THE YIN AND THE YANG OF ACETYLCHOLINESTERASE INHIBITION BY MECHANISTIC X-RAY CRYSTALLOGRAPHYMeeting abstracts

M. Bartolini, M.L. Bolognesi, J. Korábečný, K. Kuca, D. Lamba, A. Pesaresi, X. Zha

MMSL 2018, 87(88):46  

Drug discovery and development is a complex and expensive process. Thanks to the exponential growth of molecular data and advancement in technologies, efforts have been tremendously amplified. Among new approaches multipotent compounds are emerging as the next paradigm in drug discovery [1] and includes: (i) single drug acting on multiple targets of a unique disease pathway, or (ii) single drug acting on multiple targets pertaining to multiple disease pathways. These compounds are thought to have best beneficial effects in the treatment of complex diseases, like Alzheimer’s Disease, in which the simultaneous regulation of various pathological...

PHOTO-INDUCED RELEASE OF AN ACETYLCHOLINESTERASE INHIBITORMeeting abstracts

Eugenio de la Mora, Johannes Broichhagen, Peter Mayer, Elisabet Artursson, Fredrik Ekström, Joel Sussman, Israel Silman, Dirk Trauner, Giorgio Schirò, Martin Weik

MMSL 2018, 87(88):47  

Light–induced isomerization of enzyme ligands allows controlling specific biological processes in time and space. Photoisomerisable azobenzene-based inhibitors allow photo-control of acetylcholine (ACh) signalling by regulating acetylcholinesterase (AChE), the enzyme that catalyses ACh hydrolysis in the central and peripheral nervous system. By regulating AChE, this family of inhibitors would allow spatial and temporal regulation of ACh levels in the synaptic cleft. Adequate regulation of ACh levels is an essential part of Alzheimer’s disease (AD) treatment and other common pathologies. Win this work we present the crystal structures of...

STRUCTURAL STUDIES OF Anopheles gambiae ACETYLCHOLINESTERASE PROVIDE INSIGHT TOWARDS IMPROVED INSECTICIDES FOR MALARIA VECTOR CONTROLMeeting abstracts

Jonah Cheung, Arshad Mahmood, Ravi Kalathur, Lixuan Liu, Max Totrov, Paul Carlier

MMSL 2018, 87(88):48  

Malaria is transmitted by the Anopheles gambiae mosquito in sub-Saharan Africa and tropical regions where the disease is prevalent.  Indoor spraying with anticholinesterase insecticides is a proven method to control populations of the mosquito and to reduce spread of the disease; however,  widespread use of insecticides has led to the rise of an insecticide-resistant G119S mutant acetylcholinesterase in the mosquito which threatens ongoing disease-control efforts.  We have solved high resolution X-ray structures of the G119S mutant acetylcholinesterase of An. gambiae (G119S AgAChE), in the ligand-free state and in complex with...

ROOM-TEMPERATURE CRYSTALLOGRAPHY AND NEUTRON SCATTERING STUDIES OF HUMAN ACETYLCHOLINESTERASE TO INFORM THE DESIGN OF OXIME REACTIVATORSMeeting abstracts

Oksana Gerlits, Mikolai Fajer, Xiaolin Cheng, Donald Blumenthal, Palmer Taylor, Zoran Radić, Andrey Kovalevsky

MMSL 2018, 87(88):49  

Human acetylcholinesterase (hAChE) is responsible for degrading neurotransmitter acetylcholine at synapses of the nervous system. Organophosphate (OP) nerve agents and pesticides inactivate hAChE through chemical modifications of the catalytic serine. The current generation of oxime antidotes is not highly efficient. Insights into the molecular structures of AChEs from various species reveal possible limitations in enhancing reactivation rates, but provide only limited information, because the structures have been obtained at cryo-temperatures. Moreover, X-ray crystallography usually cannot resolve positions of hydrogen atoms involved in proton transfer...

CRYSTAL STRUCTURES OF HUMAN CHOLINESTERASES IN COMPLEX WITH SUPRAMOLECULAR LIGANDSMeeting abstracts

José Dias, Xavier Brazzolotto, Xiao-Yu Cao, Artur Stefankiewicz, Jean-Marie Lehn, Florian Nachon

MMSL 2018, 87(88):50  

Human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) are related enzymes. hAChE plays a key role in neurotransmission and is the target of organophosphorus nerve agents. hBChE is good a natural stoichiometric scavenger of nerve agents, preventing their diffusion to the central and peripheral nervous system where they inhibit hAChE. hAChE and hBChE display different specificities for substrates and ligands due to differences in the number of aromatic residues lining the active site gorge. These aromatic residues are essential for the binding of quaternary and aromatic ligands. Some molecules containing quaternary and/or aromatic moieties...

MODIFICATIONS OF CHOLINESTERASE STRUCTURE AND FUNCTION IN COVALENT ORGANOPHOSPHATE CONJUGATES VISUALIZED IN 2D, 3D AND VRMeeting abstracts

Zihan Zheng, Wanlu Yu, Jacqueline Rohrer, Alexandria Tran, Zoran Radić

MMSL 2018, 87(88):51  

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093-0650, USA Backbone conformations in hundreds of PDB deposited cholinesterase (ChE) X-ray structures show surprising similarity with typical variability of ~1Å or less among native and liganded acetylcholinesterases (AChEs; 3.1.1.7) and as low as ~2 Å between AChEs and butyrylcholinesterases (BChEs; 3.1.1.8). The largest backbone deviations are observed in their covalent conjugates with organophosphate (OP) inhibitors. Those deviations are likely to influence approach, binding and reaction efficacy of nucleophilic oxime reactivators...

CHOLINERGIC MECHANISMS AT THE CORE OF SKELETAL AND RETINAL HISTOGENESISMeeting abstracts

Gesine Bachmann, Afrim Bytyqi, Florian Frohns, Matthias Rieke, Gopenath Thangaraj, Paul G. Layer

MMSL 2018, 87(88):52  

Recently we could establish major cholinergic impact on vertebrate in vivo and in vitro skeletogenesis (1,2). Cholinergic mechanisms are also at the core of formation of the vertebrate retina. Retinal histogenesis of a so-called inner plexiform layer (IPL) was disturbed in an AChE KO mouse (3). Characterized best by their ChAT expression, the only cholinergic cells in all vertebrate retinae are so-called starburst amacrine cells (SACs), which send processes into synaptic IPL sublaminae. We documented that SACs are derived from a larger pool of postmitotic AChE⁺ cells. A developmental comparison of ChAT⁺ and...

BUTYRYLCHOLINESTERASE AS A GHRELIN MODULATOR IMPACTING ANXIETY, STRESS, OBESITY, AND DRUG CRAVINGSMeeting abstracts

S. Brimijoin, Y. Gao, L.Y. Geng, V.P. Chen

MMSL 2018, 87(88):53  

Our recent studies on butyrylcholinesterase (BChE) have led us to conclude that this enzyme has a major physiological role in regulating levels and impact of ghrelin, the “hunger hormone.” A key step toward this realization was finding that, over time, group-housed mice given AAV8-BChE expression vector showed a sharp drop in fighting. Eventually we linked this reaction to a large decrease in plasma ghrelin, which is involved in food-seeking and stress. At first, we assumed that lowered ghrelin was reducing stimulation of growth hormone secretagogue receptors in brain.  Instead, treated mice showed larger pulses of circulating growth...

ASSEMBLY OF PRIMA-LINKED FORM OF ACETYLCHOLINESTERASE IN NEURONS: THE ROLE OF ENZYME INHIBITOR ACTING AS CHEMICAL CHAPERONMeeting abstracts

Karl W. K. Tsim, Etta Y. L. Liu, Miranda L. Xu, Xiang P. Kong, Qiyun Wu, Ran Duan, Tina T. X. Dong

MMSL 2018, 87(88):54  

Acetylcholinesterase (AChE) is anchored onto cell membranes by a transmembrane protein PRiMA (Proline-Rich Membrane Anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. Several lines of evidence suggest that the dimer formation probably represents an intermediate in the assembly of the tetramer. In addition, the assembly of AChE tetramers with PRiMA requires the presence of a C-terminal “t-peptide” in the AChE catalytic subunit (AChE_T). This protein assembly could be affected by chaperons. AChE inhibitors (AChEIs) are the most established treatment strategy for Alzheimer's disease (AD). Many...

EVOLUTION OF THE FIRST DISULFIDE BOND IN THE CHOLINESTERASE-CARBOXYLESTERASE (COESTERASE) FAMILY: POSSIBLE CONSEQUENCES FOR CHOLINESTERASE EXPRESSION IN PROKARYOTESMeeting abstracts

Arnaud Chatonnet, Xavier Brazzolotto, Thierry Hotelier, Nicolas Lenfant, Pascale Marchot

MMSL 2018, 87(88):55  

Within the alpha/beta hydrolase fold superfamily of proteins, the COesterase group (carboxylesterase type B, block C, cholinesterases…) diverged from the other groups through addition of an N-terminal disulfide bond and simultaneous increase in the mean size of the protein (1). This disulfide bond creates a large loop, which is essential for the high catalytic activity of cholinesterases through formation of the upper part of the active center gorge. In some non-catalytic members of the family, the loop may be necessary for heterologous partner recognition. The shuffling of this portion of protein occurred at the time of emergence of the fungi/metazoan...

ACETYLCHOLINESTERASE IN NEUROMUSCULAR SYNAPTIC CLEFTS OF VERTEBRATESMeeting abstracts

Edna Blotnick-Rubin, Lili Anglister

MMSL 2018, 87(88):56  

Precise positioning and density of acetylcholinesterase (AChE) in the synaptic cleft is required to correctly control the duration of transmitter action in cholinergic synapses according to the particular functional demands of the synapse. We had previously evaluated the densities of AChE at neuromuscular junctions (NMJs) by EM-autoradiography, using radiolabeled probes. The current study addressed fundamental issues concerning the precise location and distribution of the enzyme in the cleft, i.e., whether it is associated with pre- or postsynaptic membranes, or with synaptic basal lamina (BL), and whether it is present only in the primary cleft (PC)...

RESPIRATION DURING ORGANOPHOSPHATE AND CARBAMATE INTOXICATION WHEN ACETYLCHOLINESTERASE IS NOT ANCHORED AT CHOLINERGIC SYNAPSESMeeting abstracts

Eric Krejci, Aurélie Nervo, Imene Kellout, Anne Sophie Hanak, Guilhem Calas, Florian Nachon

MMSL 2018, 87(88):57  

Intoxications with organophosphate or carbamate shut down control of breathing in minutes. These central apneas are reversed by atropine the well-known antidote of acetylcholinesterase (AChE) inhibitors. But how the excess of ACh triggers the crisis remains unclear. If the buildup of ACh on the post-synaptic receptors at cholinergic synapses is critical, we expected that mice in which the synaptic transmission is adapted to the deficit of AChE should resist to intoxication with carbamates. AChE is specifically anchored in the synapses by ColQ at the neuromuscular junction (NMJ) and by PRiMA in central nervous system (CNS). We have thus intoxicated...

SINGLE NUCLEOTIDE POLYMORPHISMS IN THE GENES ENCODING AChE AND ITS miR-608 REGULATOR CO-MODULATE ANXIETY AND BLOOD PRESSUREMeeting abstracts

Alon Simchovitz, Nimrod Madrer, Rotem Haviv, Geula Hanin, Shani Shenhar-Tsarfaty, Einor Ben Assayag, Shlomo Berliner, Zehava Solomon, Hermona Soreq

MMSL 2018, 87(88):58  

Cholinergic-regulated phenotypes including anxiety, cardiac and immune-related properties show inter-individual variability which might be affected by genomic Single Nucleotide Polymorphisms (SNPs) in the corresponding protein coding genes and their targeting microRNAs (miRs), but the combined impact of such SNP pairs is unknown. We have recently shown that the rs17228616 SNP in the Acetylcholinesterase (AChE) gene reduces the affinity of AChE mRNA to the primate-specific miR-608 and elevates both AChE levels in brain and blood as well as trait anxiety and blood pressure (1) while affecting PTSD-related neural circuits and downregulating numerous brain...

DIOXIN SUPPRESSES AChE EXPRESSION IN NEURON AND MUSCLEMeeting abstracts

Heidi Qunhui Xie, Yingjie Xia, Tuan Xu, Yangsheng Chen, Yali Luo, Rui Sha, Yiyun Liu, Li Xu, Bin Zhao

MMSL 2018, 87(88):59  

Acetylcholinesterase (AChE, EC3.1.1.7) plays an important role in the cholinergic neurotransmission in central and peripheral nervous systems, which has been widely recognized as a biomarker for monitoring pollution of organophosphate and carbamate pesticides. Recently, a broad spectrum of environmental toxic substances has been found to decrease AChE activity in various species. Dioxin is one of the emerging environmental AChE disruptors, which is a typical persistent organic pollutant with multiple toxic effects on the nervous system. We have reported that dioxin suppresses the expression of neuronal AChE via aryl hydrocarbon receptor (AhR), in which...

Wnt3a INDUCES THE TRANSCRIPTION OF ACETYLCHOLINESTERASE: AN ENZYME PLAYING A ROLE IN OSTEOBLASTIC DIFFERENTIATIONMeeting abstracts

Miranda L. Xu, Etta Y. L. Liu, Qiyun Wu, Duan Ran, Tina T. X. Dong, Karl W. K. Tsim

MMSL 2018, 87(88):60  

Acetylcholinesterase (AChE) plays hydrolytic role to terminate cholinergic transmission in vertebrate. AChE is intensively reported to exist in different tissues, and may participate in differentiation process. Here, AChE was demonstrated to participate in osteoblastic differentiation. In rat-derived bone tissues and primary cultured osteoblasts, the expression of AChE was increased in parallel with bone development, as well as osteoblastic differentiation. Transcriptional expression and protein of AChE in differentiating osteoblast could be enhanced by application of Wnt3a. Runx2, a downstream transcription factor in Wnt/β-catenin signaling pathway,...

RESTORING MITOCHONDRIA (DYS)FUNCTION AND ACETYLCHOLINE LEVELS AS A PROSPECTIVE THERAPEUTIC STRATEGY FOR ALZHEIMER’S DISEASEMeeting abstracts

Fernanda Borges

MMSL 2018, 87(88):61  

Alzheimer´s disease (AD) is a progressive and degenerative neurological disorder resulting in memory loss and cognitive decline. The severity of AD dementia was found to correlate with the extent of the cholinergic loss and acetylcholine (ACh) depletion. In brain synapses ACh can be hydrolyzed by two cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which were found in neurons and glial cells as well as in AD neuritic plaques and tangles. AChE is the prevalent enzyme in the healthy brain, while BChE is considered to play a minor role in the regulation of synaptic ACh levels. However, in AD advanced stages, AChE...

FROM DUAL BINDING SITE AChE INHIBITORS TO CHAMELEON MOLECULES: DISCOVERY OF POTENT BuChE INHIBITORSMeeting abstracts

Carlos Roca, Talita P.C. Chierrito, Concepción Perez, Loreto Martinez, Nuria Campillo, Ana Martinez

MMSL 2018, 87(88):62  

Current pharmacotherapy for Alzheimer's disease (AD) involves compounds aimed at increasing the levels of acetylcholine in the brain through inhibition of AChE. These drugs, known as acetylcholinesterase inhibitors, have been shown to improve cognition and global functions but have little impact on improving the eventual progression of the disease. However, there are evidences that other cholinesterases such as butyrylcholinesterase (BuChE) can play an important role in cholinergic function in the brain, and the long-suspected non-cholinergic actions of acetylcholinesterase, mainly the interference with the beta-amyloid protein cascade, have recently...

DISCOVERY AND DEVELOPMENT OF NEUROPROTECTIVE AND DISEASE-MODIFYING ANTI-AD DRUG LEADS FROM THE CHINESE MEDICINEMeeting abstracts

Marvin Mak, Wei Cui, Yifan Han

MMSL 2018, 87(88):63  

Alzheimer's disease (AD) represents a chronic and progressive brain disorder, and has now become the most common neurodegenerative disorders among the older population. Although the disease is now seen as major public health problems, the currently available therapeutics only offer temporary symptomatic relieves. Therefore, research and development of more effective and disease-modifying agents for the prevention and/or treatment of AD will have tremendous value from both scientific and economic standpoints.  Over the past few years, our series of studies have identified some highly promising anti-AD drug leads, including those derived from the Chinese...

FIFTY SHADES OF CHOLINESTERASE IMMOBILIZATION AND THEIR APPLICATION TO DRUG DISCOVERYMeeting abstracts

Anna Tramarin, Edoardo Fabini, Piotr Drączkowski, Marina Naldi, Daniele Tedesco, Krzysztof Jóźwiak, Vincenza Andrisano

MMSL 2018, 87(88):64  

New screening methodologies capable of identifying new enzyme inhibitors in a faster, more reproducible and automated way may help early drug discovery. Indeed high throughput screening methodologies for the identification of new cholinesterase inhibitors can reduce screening time and screening costs. In this frame, “immobilized enzymes” [1] can serve as handy and efficient alternatives to conventional in-solution methods. On the other hand, other than massive screening, highly informative approaches may provide decisive information in the selection of best-in-class compounds. Hence, combination of several parameters spanning from inhibition,...

SERUM CHOLINESTERASE ACTIVITY AND ALZHEIMER DISEASE COMORBIDITIES - CAN BARIATRIC SURGERY HANGE YOUR SYMPATHETIC PRONE STATE?Meeting abstracts

Shani Shenhar-Tsarfaty, Shiri Sherf-Dagan, Galia Berman, Shira Zelber-Sagi, Oren Shibolet, Itzhak Shapira, David Zeltser, Shlomo Berliner, Ori Rogowski

MMSL 2018, 87(88):65  

Alzheimer disease comorbidities, such as hypertension, obesity, metabolic syndrome, diabetes mellitus and inflammation are all associated with impaired sympathetic/parasympathetic response. Inherited and/or acquired sympathetic prone state, expressed by elevated serum Acetylcholinesterase (AChE) can lead to excessive inflammatory load and cognitive decline. To evaluate the sympathetic/parasympathetic balance we measured serum cholinesterase activities in stroke, myocardial infarction, diabetes mellitus, morbid obese patients and apparently healthy control. Our findings identify the potential value cholinesterases as possible biomarkers in diseases...

INDAZOLYLKETONES: HIT TO LEAD OPTIMIZATION OF A MULTITARGET DRUGSMeeting abstracts

Pedro González-Naranjo, Natalia Pérez, Concepción Pérez, Carlos Roca, Rocio Girón, Eva Sánchez-Robles, Ángeles Martín Requero, Maria L. de Ceballos, Nuria E. Campillo, Juan Antonio Páez

MMSL 2018, 87(88):66  

A new family of indazolylketones with a multitarget profile as modulators of cholinergic and BACE-1 enzymes and  cannabinoids receptors [1] was  designed based on our previous results [2]. We present the synthesis, computational studies and biological evaluation and of a new family of heterocyclic compounds. Pharmacological evaluation include in vitro inhibitory assays in AChE/BuChE enzymes and BACE-1. In addition, functional activity for cannabinoid receptors has been carried out. The results of the pharmacological tests have revealed that some of these derivatives behave as CB2 cannabinoid agonists and simultaneously show BuChE and/or BACE-1...

BUTYRYLCHOLINESTERASE GENETIC POLYMORPHISM AND NEUROIMAGING BIOMARKERS IN ALZHEIMER’S DISEASEMeeting abstracts

DeBay, Drew R., Maxwell, Selena, Luke, David, Fisk John D., Burrell, Steve, Bowen Chris V., Song, Xiaowei, Black, Sandra E., Darvesh, Sultan

MMSL 2018, 87(88):67  

Objective: The influence of butyrylcholinesterase (BChE) genetic polymorphism in Alzheimer’s (AD) remains controversial.  BCHE-K and BCHE-A genetic variants cause reduction of BChE, an enzyme implicated in AD. Some studies have reported a protective effect of BCHE-K, others suggest increased AD risk, particularly when associated with APOE4. We utilized a candidate gene-driven analyses to determine the effects of BCHE-K and BCHE-A on AD biomarkers using ADNI data (http://adni.loni.ucla.edu/).  Methods: Participants were genotyped for BCHE-K (615) and BCHE-A (785), each stratified into control...

CASE STUDIES FOR SUCCESSFUL COMBINATION OF ChE INHIBITORS AND GPCR LIGANDS (CANNABINOID 2 AND HISTAMINE 3 RECEPTORS)Meeting abstracts

Dominik Dolles, Fouad H. Darras, Antonios Drakopoulos, Andrea Strasser, Hans-Joachim Wittmann, Christoph A. Sotriffer, Steffen Pockes, Bassem Sadek, Tangui Maurice, Michael Decker

MMSL 2018, 87(88):68  

The combination of cholinesterase inhibitors with GPCR ligands in hybrid molecules seems highly promising for Alzheimer’s disease (AD) therapy, since two very different molecular targets can be addressed at the same time. Nevertheless, significant challenges come with this rationale: a) hybrids might possess too high molecular weights to be orally bioavailable and/or pass the blood-brain-barrier, b) the compounds might act in different concentration ranges, c) and selectivity and affinity has to be optimized for several very distinct targets. We have designed – applying computational methods - and synthesized dual-acting ChE-inhibitors...

FROM ACETYLCHOLINESTERASE INHIBITORS TO MULTI-TARGET-DIRECTED LIGANDS (MTDLs): A STEP FORWARD IN ALZHEIMER'S DISEASE DRUG DISCOVERYMeeting abstracts

Maria-Laura Bolognesi

MMSL 2018, 87(88):69  

Notwithstanding clinical effectiveness evidences continue to suggest benefit from the acetylcholinesterase inhibitors (AChEIs) in alleviating Alzheimer’s disease (AD) symptoms, these drugs do not appear to delay or prevent the underlying neurodegeneration. In this context, novel prospects are offered by the strategy of developing single chemical entities able to modulate multiple targets, i.e. the multi-target-directed ligands (MTDLs). On this basis, several multifunctional AChEIs have been rationally designed with the deliberate aim of enlarging their biological profiles, beyond the ability to inhibit cholinesterases. This is because it has...

FROM SELECTIVE BUTYRYLCHOLINESTERASE INHIBITORS TO MULTI-TARGET-DIRECTED LIGANDS AS LEAD COMPOUNDS FOR ALZHEIMER’S DISEASEMeeting abstracts

Urban Košak, Damijan Knez, Boris Brus, Stanislav Gobec

MMSL 2018, 87(88):70  

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. First, a hierarchical virtual screening was performed followed by biochemical evaluation of highest scoring hit compounds. Three compounds showed...

DISCOVERY AND CHARACTERIZATION OF TACRINE/HUPRINE-TRYPTOPHAN HETERODIMERS AS NOVEL MULTIPOTENT COMPOUNDS AGAINST ALZHEIMER'S DISEASEMeeting abstracts

Jan Korabecny, Katarina Spilovska, Manuela Bartolini, Barbara Monti, Doriano Lamba, Rosanna Caliandro, Alessandro Pesaresi, Vendula Hepnarova, Daniel Jun, Martina Hrabinova, Rafael Dolezal, Jana Zdarova Karasova, Ondrej Soukup, Eva Mezeiova, Eugenie Nepovimova, Maria Laura Bolognesi, Kamil Kuca

MMSL 2018, 87(88):71  

Combination of tacrine/huprine, connected through a different linker tether length, with tryptophan led to the generation of a novel, highly-potent family of multi-target directed ligands targeting key molecular mechanisms of Alzheimer’s disease. Based on in vitro biological profile, the 6-chloro-tacrine-(CH₂)₆-L-tryptophan heterodimer S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE) within the series, with nanomolar IC₅₀ values (6.31 and 9.07 nM, respectively). Moreover, S‑K1035...

NOVEL CONJUGATES BASED ON γ-CARBOLINES, CARBAZOLES, PHENOTHIAZINES, AND AMINOADAMANTANES AS MULTIFUNCTIONAL AGENTS FOR ALZHEIMER’S DISEASE TREATMENTMeeting abstracts

G.F. Makhaeva, N.P. Boltneva, N.V. Kovaleva, S.V. Lushchekina, E.V. Rudakova, R.J. Richardson, S.O. Bachurin

MMSL 2018, 87(88):72  

Neurodegenerative diseases are multifactorial. Therefore, their treatment requires drugs that can act simultaneously on multiple pathogenic targets. We synthesized several series of hybrid structures combining certain pharmacophores essential for neurodegenerative disease treatment: γ-carbolines, carbazoles, phenothiazines, and aminoadamantanes [1-3]. Inhibitory activity of these conjugates against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE) was studied along with their ability to competitively displace propidium iodide from the peripheral anionic site of electric eel AChE to assess their potential effect...

PLEIOTROPIC PRODRUGS: A NOVEL POLYPHARMACOLOGY APPROACH TO TREAT NEURODEGENERATIVE DISEASESMeeting abstracts

Christophe Rochais, Patrick Dallemagne

MMSL 2018, 87(88):73  

Today, treatment of Alzheimer's Disease (AD) mainly involves acetylcholinesterase inhibitors (AChEIs). AChEIs display solely a symptomatic benefit, alleviating the cognitive disorders associated to AD through a temporary restoration of the cholinergic neurotransmission impaired by the neurodegeneration. The gradual loss of efficiency for AChEIs led to associate them to drugs exhibiting potential disease-modifying properties. The “Multi-Target-Directed Ligands” (MTDLs) was used in the recent years with a great potential benefit towards multiple targets implicated in the complex AD,¹ as well as other neurodegenerative syndromes,...

TOWARD AN INNOVATIVE TREATMENT OF ALZHEIMER’S DISEASE: DESIGN OF MULTI-TARGET DIRECTED LIGANDS (MTDLs) TARGETING ACETYLCHOLINESTERASE (AChE) AND alpha-7 NICOTINIC RECEPTORS (alpha-7 nAChRs)Meeting abstracts

Mégane Pons, Buron Frédéric, Ludovic Jean, Sylvie Chalon, Sylvain Routier, Pierre-Yves Renard

MMSL 2018, 87(88):74  

Alzheimer’s disease (AD) is a complex and progressive neurodegenerative disorder. The available therapy is limited to the symptomatic treatment and its efficacy remains unsatisfactory ^[1]. In view of the prevalence and expected increase in the incidence of AD, the development of an effective therapy is crucial for public health. Due to the multifactorial etiology of this disease, the multi-target-directed ligand (MTDL) approach is a promising method in search for new drugs for AD. Aiming at developing new MTDLs, this project consists on the development of new multifunctional agents, which will act simultaneously on the different players...

MOLECULAR MODELING IN SEARCH OF NEW, MULTI-TARGET LIGANDS AGAINST ALZHEIMER'S DISEASE. EXPLORING THE BIOCHEMICAL MULTIVERSE.Meeting abstracts

Jakub Jończyk, Dawid Panek, Anna Więckowska, Justyna Godyń, Marek Bajda, Tomasz Wichur, Anna Pasieka, Damijan Knez, Anja Pišlar, Jan Korabecny, Ondrej Soukup, Vendula Sepsova, Raimon Sabaté, Janko Kos, Stanislav Gobec, Barbara Malawska

MMSL 2018, 87(88):75  

In response to the complex and still not fully understood pathomechanism of Alzheimer's disease, many researchers have turned towards the promising paradigm of designing ligands with a multi-target nature¹. One of the possible benefits of this approach in Alzheimer's disease is an opportunity to merge activity against cholinesterases, which are used in the current symptomatic therapies, with disease-modifying targets associated with β-amyloid and tau protein pathways². Optimization of ligand with respect to several biological targets while maintaining good physicochemical parameters is not an easy task.  Computer modeling...

DESIGN OF A BUTYRYLCHOLINESTERASE MUTANT FOR DETOXIFYING COCAINE AND ITS TOXIC METABOLITES IN CONCURRENT USE OF COCAINE AND ALCOHOLMeeting abstracts

Fang Zheng, Xirong Zheng, Ting Zhang, Xiabin Chen, Chang-Guo Zhan

MMSL 2018, 87(88):76  

Cocaine abuse is a major medical and health problem. There is no FDA-approved medication for treatment of cocaine overdose and addiction. Statistical data show that 92% of cocaine users also consume alcohol. The risk of immediate death is 18 - 25 times greater for cocaine co-ingested with alcohol than for cocaine alone. Alcohol can react with cocaine to get a series of toxious compounds in body including cocaine, cocaethylene, norcocaine, norcocaethylene and benzoylecgonine. In combination of our “virtual screening of transition states” computational protocol and artificial intelligence, a novel approach was used to design BChE mutants...

7-METHOXYDERIVATIVE OF TACRINE IS A ‘FOOT-IN-THEDOOR’ BLOCKER OF GluN1/GluN2 AND GluN1/GluN3 NMDA RECEPTORSMeeting abstracts

Martina Kaniakova, Lenka Kleteckova, Katarina Lichnerova, Kristina Holubova, Kristyna Skrenkova, Miloslav Korinek, Jan Krusek, Tereza Smejkalova, Jan Korabecny, Karel Vales, Ondrej Soukup, Martin Horak

MMSL 2018, 87(88):77  

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the mammalian central nervous system (CNS), but their dysregulation results in the aetiology of many human CNS disorders. Several NMDAR modulators including memantine have been used successfully in clinical trials. Indeed, 1,2,3,4-tetrahydro-9-aminoacridine (tacrine; THA) was the first approved drug for Alzheimer’s disease (AD) treatment. 7-methoxyderivative of THA (7-MEOTA) is less toxic and showed promising results in patients with tardive dyskinesia. Here, we employed electrophysiological recordings in HEK293 cells and rat...

THE Caenorhabditis elegans PHARYNX AS A MODEL SYSTEM TO INVESTIGATE AND MITIGATE AGAINST THE EFFECTS OF ANTI-CHOLINESTERASE DRUGSMeeting abstracts

Patricia Gonzalez, Christopher Green, John Tattersall, Lindy Holden-Dye, Vincent O'Connor

MMSL 2018, 87(88):78  

C. elegans is a free-living worm widely used as model to study neurotoxicology. Despite its simplicity, C. elegans has a high level of genetic and molecular conservation with vertebrates. Similar to mammals, intoxication with anti-cholinestereses triggers the accumulation of synaptic acetylcholine causing continuous stimulation of both nicotinic and muscarinic receptors, hypercontracting the muscles of the worm¹. The pharynx, the nematode feeding organ, depends on cholinergic function. Pharyngeal movements, readily observed in whole organism, are disrupted by impairments in cholinergic transmission. Therefore, quantitative...

PHARMACOKINETICS OF BIS-PYRIDINIUM MONO-ALDOXIMESMeeting abstracts

Huba Kalász, Kamil Kuca, Kamil Musilek, Gellért Karvaly, Syed Nurulain, Kornélia Tekes

MMSL 2018, 87(88):79  

Bis-pyridinium mono-aldoxime (BPMA) compounds are potential antidotes against organophosphorus inhibitors of either acetylcholinesterase or these of butyrylcholinesterase. From the points of drug distribution and pharmacokinetics essential characteristics were determined (concentration versus time curves). Experimental results of pharmacokinetics of BPMA will be detailed with special focus on drug distribution and HPLC analysis of oxime K117. The concentration of BPMAs decreases fast in the body of rats, and thus they fulfil the basic requirement for antidotes: elimination should be as fast as possible. Their elimination curve should be characterized...

3D STRUCTURE OF NATURAL TETRAMERIC FORM OF HUMAN BUTYRYLCHOLINESTERASE OBTAINED BY CRYO-ELECTRON MICROSCOPYMeeting abstracts

Konstantin M. Boyko, Timur N. Baimukhametov, Yury M. Chesnokov, Michael Hons, Sofya V. Lushchekina, Peter Konarev, Alexey Lipkin, Alexandre L. Vasiliev, Patrick Masson, Vladimir Popov

MMSL 2018, 87(88):80  

Human butyrylcholinesterase (BChE) is a stoichiometric bioscavenger of toxic organophosphates. It can be used as an antidote to protect acetylcholinesterase, and is a protein of choice for development of detoxification biocatalysts for clinical applications. Despite the number of different monomeric structures of recombinant human BChE obtained to date, all attempts to obtain an atomic structure of the natural glycosylated tetrameric BChE were unsuccessful. Here, we present for the first time the 3D structure of the natural tetrameric form of human butyrylcholinesterase, obtained by Cryo-EM technique at a final resolution of 8.8Å. The tetramer...

BUTYRYLCHOLINESTERASE-PROTEINS INTERACTIONS IN HUMAN BLOOD SERUMMeeting abstracts

Jacek Jasiecki, Krzysztof Waleron, Bartosz Wasąg

MMSL 2018, 87(88):81  

Blood serum proteins serve various functions, including transport of lipids, hormones, vitamins. They are responsible for maintaining acid-base balance, oncotic pressure, plasma viscosity, and functioning of the immune system. There are several hundred different proteins in the blood serum, which total concentration varies within the limits of 6.6-8.7 g/dl, but only a small amount is determined for laboratory diagnostics. One of the serum protein is butyrylcholinesterase  (BChE, EC 3.1.1.8), which exists predominantly in the form of a glycosylated tetramer (G4) with a mass of 340 kDa. Four identical subunits assemble into a tetramer by the interaction...

DIMERIZATION INTERFACE OF CHOLINESTERASES: ANALYSIS OF CRYSTAL STRUCTURES, FREE ENERGY MOLECULAR DYNAMICS CALCULATIONS,AND IN SILICO ALANINE SCREENINGMeeting abstracts

Novichkova D. A., Lushchekina S. V., Sussman, Joel L.

MMSL 2018, 87(88):82  

For acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), several oligomeric forms are known. In vivo, the monomers in dimers are covalently bound by a C-terminal disulfide bond formed after association of the monomers. Available crystal structures were obtained for truncated forms without disulfide bonds and serve as good models for describing the role of non-covalent interactions in the dimerization of cholinesterases before linking by disulfide bonds. Here, we analyze the formation of the four-alpha-helix bundle in cholinesterases and differences between AChE and BChE dimers. To identify interactions stabilizing the four-alpha-helix...

4-AMINOQUINIOLINES AS REVERSIBLE INHIBITORS OF HUMAN CHOLINESTERASE ACTIVITYMeeting abstracts

Anita Bosak, Dejan M. Opsenica, Goran Šinko, Matija Zlatar, Zrinka Kovarik

MMSL 2018, 87(88):83  

We synthesised eight derivatives of 4-aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) carbon of 4-aminoquinoline, and tested their potency to inhibit human AChE and BChE. All of the compounds reversibly inhibited both enzymes with dissociation inhibition (K_i) constants from 0.50 to 50 µM exhibiting selectivity. In other words, for all compounds, AChE exhibited higher affinity than BChE. The most potent inhibitors of AChE were compounds with an octyl chain or adamantane, regardless of the group in position C(7). The shortening of the chain length caused the AChE inhibition decrease by 5-20...

SYNTHESIS OF NERVE AGENTS’ SURROGATES FROM DIALKYL ALKYLPHOSPHONATES FOR ANTIDOTE SCREENING AND TOXICOLOGICAL STUDIESMeeting abstracts

Samir F. de A. Cavalcante, Leandro B. Bernardo, Kamil Kuča, Alessandro B. C. Simas

MMSL 2018, 87(88):84  

Nerve Agents are toxic organophosphorus compounds which inhibit cholinesterases, pivotal enzyme in Parasympathetic Neurotransmission. As they are Schedule 1 compounds in accordance to Chemical Weapons Convention, strict controls are applied and some research groups may have their work hampered due to requirements for synthesis and manipulation. Nerve Agents’ surrogates have emerged as affordable substitutes for more realistic approach for development of antidotes and biochemical and toxicity studies, as they are structurally related to Nerve Agents and considered as CWC Schedule 2 compounds, yielding similar enzyme adducts. As Laboratório de...

BIOLOGICAL EVALUATION OF CYSTEINE TARGETED INSECTICIDESMeeting abstracts

Hrabinova M., Schmidt M., Gorecki L., Kucera T., Psotka M., Svobodova B., Hrabcova V., Hepnarova V., Jun D., Kuca K., Musilek K., Korabecny J.

MMSL 2018, 87(88):85  

According to the World Malaria Report, there were 216 million cases of malaria with 445000 causalties in 2016. Current anticholinesterase insecticides, such as carbamates and organophosphates, act via covalent modification of serine at the bottom of the active site. Traditional chemical insecticides are highly toxic to insect but similarly to mammals. The cysteine-targeting concept of new insecticides is focused on cysteine 447 located in the peripheral site of mosquito acetylcholinesterase. In mammalian enzyme, the cysteine residue is replaced by phenylalanine, whereas honeybees or bumble-bees have this cysteine residue protected. This approach has...

AN ALTERNATIVE SUBSTRATE FOR HUMAN ERYTHROCYTE ACETYLCHOLINESTERASE ACTIVITY DETECTIONMeeting abstracts

Sheemona Chowdhary, Rajasri Bhattacharyya, Dibyajyoti Banerjee

MMSL 2018, 87(88):86  

Acetylcholinesterase (AChE) is the target of pesticides like organophosphates (OP). OP exert their toxic effect by irreversible phosphorylation of the AChE leading to cholinergic crisis and neurotoxicity. Erythrocyte AChE is the surrogate biomarker for the detection of inhibition by OP. There are numerous methods for the detection of AChE activity.¹ Unfortunately, the method popularly used for AChE detection has inherent limitations.¹ To overcome such a problem, we have explored 1-Naphthyl acetate (1-NA) as an alternative substrate for the assessment of AChE activity using in silico tools and in vitro experiments. The in silico...

ACETYLCHOLINESTERASE REACTIVATORS BASED ON OXIME-FUNCTIONALIZED BIODEGRADABLE IONIC LIQUIDSMeeting abstracts

Yevgen Karpichev, Illia Kapitanov, Nicholas Gathergood, Ondřej Soukup, Vendula Hepnarova, Daniel Jun, Kamil Kuča

MMSL 2018, 87(88):87  

Progress in the development of biodegradable ionic liquids (ILs) [1] allowed finding sustainable fragments to assist the synthesis of sustainable molecules by means of “benign by design” approach. Based on our recent experience in creating micellar catalytic systems for decomposition of organophosphates [2, 3] we have elaborated the following oxime-functionalized low-toxic biodegradable ILs as potential AcChE reactivators: amide/ester linked (amino acid free) IL (I) as well as L-alanine (II) and L-phenylalanine (III) containing compounds with pyridinium aldoxime moiety in cationic part. Variation of amino acid variation (e.g. Me for I and...

IN SILICO SCREENING OF NOVEL BChE-REACTIVATORSMeeting abstracts

Tomas Kucera, Rafael Dolezal, Kamil Musilek

MMSL 2018, 87(88):88  

Several years, there are ideas how to use reactivators of BChE in prophylaxis of OP-poisoning. They could be applied in combination with human BChE as a pseudo-catalytic scavenging system. However, the effective hBChE reactivator is still missing. The aim of this project is to find highly active and plausibly universal reactivator of hBChE. In the first phase, a database of about 6 mil. structures (ZINC Lead Like) was screened by rigid molecular docking. The receptor (hBChE) was found in the PDB database (pdb code 3DJY, hBChE inhibited by tabun) and prepared for docking. For the second phase, over one hundred molecules were selected. These structures...

PHENYLTETRAHYDROISOQUINOLINE-BASED TRIAZOLE COMPOUNDS ARE HIGH-AFFINITY POTENTIAL REACTIVATORS OF NERVE AGENT-INHIBITED HUMAN ACETYLCHOLINESTERASEMeeting abstracts

Nikolina Maček Hrvat, Jarosław Kalisiak, Antonio Zandona, Goran Šinko, Zoran Radić, K. Barry Sharpless, Palmer Taylor, Zrinka Kovarik

MMSL 2018, 87(88):89  

Ten phenyltetrahydroisoquinoline-based compounds synthesized using alkyne+azide [3+2] building block cycloaddition were tested as potential reactivators of human acetylcholinesterase (hAChE) inhibited by different organophosphates. Computational docking indicated molecule phenyltetrahydroisoquinoline moiety association with the hAChE peripheral anionic binding site (Trp286, Tyr337 and Tyr341). Therefore, stabilization near the gorge opening seemed to control the general orientation of the pyridinium ring with its attached aldoxime group inserted into the internal gorge of the hAChE active center. All of the oximes were tested in vitro as potential...

IN SILICO AND IN VITRO EVALUATION OF TWO NOVEL OXIMES K456 AND K733 AGAINST PARAOXON INHIBITED HUMAN ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASEMeeting abstracts

Syed M Nurulain, M. Qaiser Fatmi, Amna Iqbal, Shahrukh Malik, Huba Kalasz, Kamil Musilek, Kamil Kuca, Georg Petroianu

MMSL 2018, 87(88):90  

Organophosphorus compounds (OPs) irreversibly inhibit cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). There is wide variety of applications of OP compounds including warfare chemicals and pesticides. Oxime-type reactivators are used to reactivate the OP inhibited AChE and BChE. Present study was aimed to evaluate the reactivation potency of two novel oximes K456 and K733 against organophosphate inhibited AChE and BChE. Efficacy was compared with K27 and pralidoxime (2-PAM). Molecular mechanism of reactivation by the oximes is predicted by In silico method. Intrinsic toxicity of novel oximes in term of IC₅₀...

FACILE SYNTHESIS OF CYSTEINE-ACETYLCHOLINESTERASE TARGETED INSECTICIDESMeeting abstracts

Miroslav Psotka, Lukas Gorecki, Barbora Svobodova, Kamil Musilek, Daniel Jun, Jan Korabecny, Kamil Kuca

MMSL 2018, 87(88):91  

Malaria is annually responsible for more than 400 thousands causalties. The disease is transmitted via infected female Anopheles mosquitoes. Spread of the malaria can be prevented by using either chemical compounds known as insecticides or by genetically engineered plants.^[1,2] Mechanism of action of currently deployed insecticide involves inactivating acetylcholinesterase (AChE, EC 3.1.1.7) enzyme by binding to Ser360 (Anopheles gambiae numbering). More recently, Cys447 located close to active site entrance was emerged as an alternative target to overcome insecticide resistance and also improving selectivity towards insect...

INHIBITION OF HUMAN ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE BY METHYLENE VIOLET 3RAXMeeting abstracts

Seda Onder, Kevser Biberoglu, Ozden Tacal

MMSL 2018, 87(88):92  

Cholinesterases are divided into two classes according to differences in substrate specificity, behaviour in high substrate concentrations, inhibitor sensitivity and tissue distribution: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The both enzymes are sensitive to broad spectrum of molecules and may be inhibited by several compounds, such as organophosphate and carbamate pesticides or nerve agents. In a previous study, a phenazine-derived natural product, geranyl-phenazine-diol was shown to inhibit human AChE with IC50 value of 2.62 mM. Phenazines which are naturally produced by bacteria and archaeal Methanosarcina species...

MOLECULAR MODELING STUDIES ON THE INTERACTIONS OF AFLATOXIN B1 AND ITS METABOLITES WITH PHERIPHERAL AND CATALYTIC ANIONIC SITES OF HUMAN ACETYLCHOLINESTERASEMeeting abstracts

Joyce S. F. D. de Almeida, Rafael Dolezal, Samir F. de A. Cavalcante, Kamil Kuca, Kamil Musilek, Daniel Jun, Tanos C. C. França

MMSL 2018, 87(88):93  

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus. Among them Aflatoxin B1 (AFB1) is the most frequent type in nature and the most carcinogenic and hepatotoxic for mammals. AFB1 is also inhibitor of the enzyme acetylcholinesterase (AChE) and, therefore, a potential chemical and biological warfare agent, as well as its metabolites. In order to investigate this, we performed inedited theoretical studies on the interactions of AFB1 and its metabolites inside the catalytic and the peripheral anionic sites (CAS and PAS) of human acetylcholinesterase (HssAChE), to verify their stability,...

CHLORINATED PYRIDINIUM OXIMES ARE POTENT REACTIVATORS OF ACETYLCHOLINESTERASE INHIBITED BY NERVE AGENTSMeeting abstracts

Tamara Zorbaz, Nikola Maraković, Kamil Musilek, Zrinka Kovarik

MMSL 2018, 87(88):94  

Chlorinated bispyridinium aldoximes (Cl-oximes) analogous to previously reported efficient reactivators of inhibited AChE (K027, K048, K203) were designed and synthesized with the premise that the addition of a chlorine atom increases their lipophilicity in comparison to the reference oximes and that they could therefore achieve higher brain concentrations than the ones reported for non-chlorinated analogues. The affinity of hAChE for Cl-oximes was moderate, but higher than for analogous non-chlorinated oximes, as well as higher than the affinity of hBChE for Cl-oximes. Their reactivation efficacy for nerve agent-inhibited AChE was in the following...

BISTABLE DYNAMIC BEHAVIOR OF ENDOGENOUS BUTYRYLCHOLINESTERASE EXPRESSED IN Expi293 CELLSMeeting abstracts

Irina Zueva, Sofya Lushchekina, Oksana Lockridge, Lawrence M. Schopfer, Patrick Masson

MMSL 2018, 87(88):95  

An endogenous tetrameric wild-type human BChE expressed in Expi293 cells hydrolyzes the neutral substrate N-methylindoxyl acetate (NMIA) with the same K_m as wild-type huBChE (0.14 mM) [1]. For this enzyme, the steady state is preceded by a pre-steady state phase of several minutes in 10 mM Bis-Tris, pH 7 at 25°C. Thermal inactivation of this BChE is biphasic. Kinetic constants (k₁ and k₂) for thermal inactivation shows differences between this mutant and plasma derived wtBChE: the Expi293 is more stable at 55°C and less stable at 60°C than natural wtBChE [2]. At 55°C half-life times of the first and the second phases...

Expi 293 CELLS EXPRESSING AN ENDOGENOUS WILD-TYPE BUTYRYLCHOLINESTERASE, AND A VARIETY OF ESTERASES THAT SELF-REACTIVATES AFTER PHOSPHYLATION BY ALL TYPES OF ORGANOPHOSPHORUS AGENTSMeeting abstracts

Irina Zueva, Oksana Lockridge, Lawrence M. Schopfer, Patrick Masson

MMSL 2018, 87(88):96  

A human embryonic kidney cell line (Expi293), adapted for suspension growth in serum-free medium, secretes a tetrameric butyrylcholinesterase (BChE). Expression levels are very low, but are increased 10-fold upon treatment with polyethylenimine. DNA sequencing shows that this enzyme is wild-type BCHE. This endogenous BChE displays catalytic properties very close to that of natural huBChE with butyrylthiocholine and N-methylindoxyl acetate as substrates [1]. Several endogenous co-secreted esterases self-reactivate after inhibition by echothiophate, paraoxon, cresyl saligenin phosphate (CBDP), racemic coumarin(CM)-soman, CM-tabun and CM-VX. Overall...

REACTIVATING EFFICACY OF OXIMES K203 AND K027 AGAINST A DIRECT ACETYLCHOLINESTERASE INHIBITOR IN RAT DIAPHRAGM: DOSE-RESPONSE MODELINGMeeting abstracts

Evica Antonijevic, Kamil Musilek, Kamil Kuca, Danijela Djukic-Cosic, Marijana Curcic, Zorica Bulat, Biljana Antonijevic

MMSL 2018, 87(88):97  

In efficacy testing of experimental oximes, traditionally reactivation of OP-inhibited acethylcholinesterase (AChE) has been analysed by comparing the obtained effects of the single dose with the control [1]. However, quantitative analysis of in vivo dose-response data by benchmark dose (BMD) approach would improve both identification and quantification of the effect and it will allow more rigorous comparison of different oximes efficacies [2]. Thus, we have evaluated in vivo dose-response relationship for two promising experimental oximes, K203 and K027, concerning reactivation of diaphragmal AChE inhibited by dichlorvos (DDVP). To compare...

INHIBITION OF CHOLINESTERASES FOLLOWING PERCUTANEOUS INTOXICATION WITH V AGENTS IN RATSMeeting abstracts

^*Jiri Bajgar, Kamil Kuca, Jiri Kassa

MMSL 2018, 87(88):98  

Female Wistar rats were percutaneously (p.c.) intoxicated (1xLD₅₀) with VX and its two derivatives differing in their substitution on nitrogen (diethyl- and dibutyl- derivatives). Blood cholinesterase activity was continuously monitored; 100 min after the intoxication (or after death), acetylcholinesterase (AChE)  activity was determined in diaphragm and brain parts (pontomedullar area - PM, frontal cortex - FC and basal ganglia – BG). Blood ChE activity remains unchanged at very short interval (5 min) after VX administration; this interval was prolonged for diethyl- and dibutyl derivatives. AChE activity was decreased to 20-30% of...

NEAR ATTACK CONFORMATION APPROACH FOR MOLECULAR MODELING STUDIES UPON THE PROPHYLACTIC AGENT 7-METHOXYTACRINE-4-PYRIDINEALDOXIME HYBRID COMPARED WITH OTHER REACTIVATORS OF VX-INHIBITED HssAChEMeeting abstracts

Jorge Alberto Valle da Silva, Eugenie Nepovimova, Kamil Kuča, Teodorico Castro Ramalho, Tanos Celmar Costa França

MMSL 2018, 87(88):99  

The novel 7-methoxytacrine-4-pyridinealdoxime agent, named hybrid 5C, is a hybrid compound comprised of a linkage between 7-methoxytacrine (7-MEOTA-4-PA) and reactivator 4-pyridinealdoxime (4-PA) moieties through a 5-carbon length-spacer. This compound was formerly designed as a prophylactic agent for intoxication by organophosphates (OP), able to form a complex with acetylcholinesterase (AChE) and reactivate this enzyme in case of OP inhibition. In order to check if the 5 carbons spacer is the ideal to maximize the interactions of this compound inside AChE, we performed in this work docking, molecular dynamics and mmpbsa studies on a series of analogues...

DESIGN, SYNTHESIS AND IN VITRO EVALUATION OF A PROMISING NEW CLASS OF BIFUNCTIONAL UNCHARGED HYBRID REACTIVATORS FOR NERVE AGENT-INHIBITED HUMAN ACETYLCHOLINESTERASEMeeting abstracts

José Dias, Julien De Sousa, Yerri Jagadeesh, Charlotte Courageux, Anne-Julie Gastellier, Christopher Timperley, Richard Brown, Gianluca Santoni, Martin Weik, Rachid Baati, Florian Nachon

MMSL 2018, 87(88):100  

Acetylcholinesterase (AChE) is a key enzyme of the Central Nervous System (CNS) hydrolyzing the neurotransmitter acetylcholine. By targeting AChE, OPNA and organophosphorus pesticides irreversibly inhibit the cholinergic transmission leading to a certain death if untreated. The current treatment available in the French army consists of an auto-injector containing a methanesulfonate salt of 2-PAM for AChE reactivation, an anticholinergic drug, atropine and avizafone, a prodrug of diazepam for limiting convulsions. However, this treatment displays major drawbacks in terms of CNS bioavailability, restricted spectrum action and effectiveness. The aim...

THE EARLY TISSUE ALTERATION INDUCED BY DIFFERENT OXIMES IN RATSMeeting abstracts

Vesna Jaćević, Eugenie Nepovimova, Kamil Kuča

MMSL 2018, 87(88):101  

Newly developed oximes, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the different organs. In this work, we focused our attention on an investigation of morphological lesions produced by increasing doses of asoxime, obidoxime, K027, K048, and K075 were selected as experimental reactivators. The whole experiment was conducted on Wistar rats. All rats were sacrificed 24 hrs and 7 days after single im application of 0.1 LD₅₀, 0.5 LD₅₀ and 1.0 LD₅₀ of each reactivator. Tissue alterations were carefully quantified by semiquantitative grading scales - cardiac, diaphragm,...

CYTOTOXICITY STUDY OF OXIME@CB7 COMPLEXES FOR CENTRAL NERVOUS SYSTEM PENETRATION OF QUATERNARY ACETYLCHOLINESTERASE REACTIVATORSMeeting abstracts

Petr Jost, Lubica Muckova, Jana Zdarova Karasova

MMSL 2018, 87(88):102  

Acetylcholinesterase (AChE, E.C. 3.1.1.7) reactivators (also known as oximes) represent a class of antidotes that are used as therapeutics in cases of organophosphate (pesticide or nerve agent) poisoning. The AChE reactivators are highly hydrophilic compounds due to their quaternary nitrogen/s and hydrophilic oxime groups included in the structure. The absorption and distribution of such antidotes is limited by these structural factors. Their delivery may be improved through their encapsulation into macrocycles. Use of these vehicles may provide some retention effect or better targeting into the central nervous system via enhanced biological barriers´...

BRAIN EXPOSURE OF BIS-PYRIDINIUM OXIME KR-26256Meeting abstracts

Hyun Myung Lee, Sunjoo Ahn, Sung Hee Cho, Soo Bong Han, Hyejin Kim, Sang Ho Lee, Gyeunghaeng Hur, Young-Sik Jung

MMSL 2018, 87(88):103  

A number of strategies through structural modification of pyridinium oximes have been developed to circumvent the Blood-Brain Barrier (BBB). Some of the attempted examples are (1) enhancement of lipophilicity by introduction of a fluorine atom into pyridinium ring, (2) facilitation of glucose transporters introduction of glucose moiety on the pyridinium nitrogen, (3) use of a prodrug by uncharged dihydropyridyl moiety, etc One of the strategies that our group tried was the introduction of fluorine atoms into the heterocyclic ring of pyridinium oximes to increase their lipophilicity.¹ In our continuing effort towards the development of new...

ANALYSIS OF BIS-PYRIDINIUM MONO-ALDOXIMES IN SERUM AND ORGANS USING A HIGH-THROUGHPUT HIGH PERFORMANCE LIQUID CHROMATOGRAPHY APPROACHMeeting abstracts

Gellert Karvaly, Kornélia Tekes, Huba Kalász

MMSL 2018, 87(88):104  

Bis-pyridinium mono-aldoximes (BPMA) are established first-line antidotes against anticholinergic toxicants. Several novel BPMA substances are even more promising candidates, yet their pharmacological properties have not been elucidated. The most prominent candidate compounds are K-117, K-127  and K-269. A bioanalytical method employing high-performance liquid chromatography with ultraviolet absorbance detection is presented for the quantitative assay of K-117, K-127 and K-269. Following brief sample preparation consisting of extraction or dilution with 0.3 mol/L perchloric acid, the substances were determined in serum, cerebrospinal fluid, kidney,...

A COMPARISON OF THE REACTIVATING AND THERAPEUTIC EFFICACY OF A NOVEL BISPYRIDINIUM OXIME K870 WITH COMMONLY USED PRALIDOXIME AND THE OXIME HI-6 IN SARIN-POISONED RATS AND MICEMeeting abstracts

Jiri Kassav, Vendula Hepnarova, Kamil Musilek, Daniel Jun

MMSL 2018, 87(88):105  

The ability of a novel bispyridinium oxime K870 and currently available oximes (pralidoxime, HI-6) to reactivate sarin-inhibited acetylcholinesterase and to reduce acute toxicity of sarin was evaluated. In vivo determined percentage of reactivation of sarin-inhibited rat blood, diaphragm and brain acetylcholinesterase showed that the potency of newly developed oxime K870 to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to the reactivating efficacy of pralidoxime with the exception of diaphragm where the oxime K870 was more effective than pralidoxime. However, the oxime HI-6 was found to be the most efficient reactivator...

THE MONOQUARTERNARY REACTIVATORS FOR THE TREATMENT OF ORGANOPHOSHOROUS INTOXICATIONMeeting abstracts

Gorecki L., Korabecny J., Svobodova B., Kucera T., Malinak D., Junova L., Hepnarova V., Hrabinova M., Soukup O., Jun D., Musilek K., Kobrlova T., Konecny J., Psotka M., Dolezal R., Honegr J., Kuca K.

MMSL 2018, 87(88):106  

Mono- and bis-pyridinium aldoximes are the only causal antidotes that are designated for the treatment of organophosphate (OP) poisoning. Intoxication by OPs is caused either by pesticides or by the nerve agents, the latter belong to group of chemical warfare agents. These compounds irreversibly inhibit enzyme acetylcholinesterase (AChE) that is no more able to fulfill its physiological function. Mono- and bis-pyridinium aldoximes are able to restore catalytic function of AChE. The reactivating ability of aldoximes is limited by several drawbacks like low blood-brain barrier permeation, low reactivation potency against specific nerve agents etc. In...

DECONTAMINATION OF WARFARE AGENTMeeting abstracts

Jan Marek, Marketa Benkova, Jan Misik, Ondrej Soukup, Marek Matula, Daniel Jun

MMSL 2018, 87(88):107  

Project is aimed at the development of new combined micellar decontamination systems based on quaternary nitrogen compounds having detergent and active decontamination properties, which will cause faster hydrolysis of chemical warfare agents. In the case of biological agents, these molecules are strong disinfectants, able to destabilize pathogen membrane structures. Several decontamination mixtures will be prepared and tested both in vitro and in vivo for their decontamination and disinfection properties against selected chemical and biological agents. The expected result of the project is efficient decontamination solution for personal skin decontamination...

IN VITRO CHARACTERIZATION OF THE STANDARD ACETYLCHOLINESTERASE REACTIVATORSMeeting abstracts

Muckova L., Hepnarova V., Misik J, Jun D, Soukup O

MMSL 2018, 87(88):108  

Acetylcholinesterase (AChE; 3.1.1.7) reactivators play a key role in the treatment of organophosphate poisoning. The main mechanism of reactivators is disruption of the covalent bond between organophosphorus compounds and AChE and restore the physiological function of this enzyme. On the other hand, there are some evidence, other mechanisms not related to reactivation, which may lead to survival. Thus, their effect on muscarinic (M₁ subtype), nicotinic (α₇ subtype) and N-methyl-D-aspartat (NMDA; 2B subtype) receptor was studied. They are able to significantly modulate the receptors at higher concentration (100 µM) and...

UNCHARGED REACTIVATORS OF CHOLINESTERASES INHIBITED BY ORGANOPHOSPHORUS NERVE AGENTSMeeting abstracts

N. Probst, A. Braïki, P. Warnault, J. Renou, C. Gomez, G. Mercey, T. Verdelet, R. Baati, J. Dias, G. Calas, F. Nachon, M. Weik, L. Jean, P. Y. Renard

MMSL 2018, 87(88):109  

The acute toxicity of OPNA results from irreversible inhibition of AChE (EC 3.1.1.7), a key enzyme in neurotransmission, via the formation of a covalent P–O bond at the catalytic serine. Inhibition of AChE leads to the accumulation of acetylcholine neurotransmitter (ACh) in the synaptic cleft causing among other symptoms, seizures and respiratory arrest leading to death.  The current urgency treatment of OPNA poisoning is based on the administration of a cocktail of three components: an antimuscarinic agent (e.g. atropine), an anticonvulsant drug (e.g. diazepam) and mono or bispyridinium AChE reactivator (e.g. pralidoxime, obidoxime, trimedoxime)....

IN VITRO DETERMINATION OF OXIDATIVE STRESS INDUCED BY OXIME REACTIVATORS USING CHROMATOGRAPHIC METHODSMeeting abstracts

Váňova, N., Mišík, J., Múčková, L., Herman, D., Pejchal, J.

MMSL 2018, 87(88):110  

Even though reactive oxygen/nitrogen species (ROS/RNS) are physiologically generated in biological systems, their excessive production may cause severe damage of cellular components. Excessive production of ROS/RNS can occur in response to various stressors such as xenobiotics, radiation or pathological processes. Oxidative stress has also been reported to cause adverse effects of some therapeutic drugs including acetylcholinesterase (AChE) oxime reactivators which are used in therapy of organophosphate poisoning.  In this study, we determined the effect of obidoxime, methoxime, asoxime, pralidoxime and trimedoxime on redox homeostasis in cultured...

IN VITRO EVALUATION OF QUINUCLIDINIUM OXIMES AS REACTIVATORS OF HUMAN CHOLINESTERASES INHIBITED BY ORGANOPHOSPHORUS COMPOUNDSMeeting abstracts

Antonio Zandona, Ines Primožič, Maja Katalinić, Zrinka Kovarik

MMSL 2018, 87(88):111  

This study focused on the evaluation of the use of quinuclidinium oximes as potential antidotes in organophosphorus compound (OPs) poisoning. We determined the reversible inhibition of human red blood cell acetylcholinesterase (AChE) and human plasma butyrylcholinesterase (BChE) by 14 quinuclidinium oximes as well as the reactivation of tabun-, VX-, paraoxon-, sarin- and cyclosarin-inhibited enzymes. Reversible inhibition constants were within 3 μM to 4 mM, depending on the oxime structure. The highest inhibition was observed for Q5, which has a long aliphatic chain on the quinuclidinium ring quaternary nitrogen. It seems that AChE is selective...

DETERMINATION OF BChE ACTIVITY BY MASS SPECTROMETRIC ANALYSIS OF ITS ADDUCT WITH RUSSIAN VxMeeting abstracts

Murashko E.A., Dubrovskii Ya. A., Beltyukov P.P., Radilov A.S., Babakov V.N.

MMSL 2018, 87(88):112  

Phosphonylated butyrylcholiesterase (BChE) is a marker of exposure to organophosphorus compounds, including nerve agents and pesticides. In cases of poisoning with nerve agents, it is important not only to establish the fact of poisoning, but also to give a quantitative estimate. The most common quantitative characteristic is BChE inhibition. We developed a highly sensitive method for the quantification of BChE inhibition by Russian Vx (VR) by mass spectrometry. For model experiments we used donor human blood plasma exposed to VR at concentrations of 1‒100 ng/ml. Butyrylcholinesterase was selectively isolated from plasma by immunoprecipitation...

AN IN-VITRO INDUCTION OF PARAOXONASE 3 ACTIVITY IN HEPATOCYTES BY RESVERATROLMeeting abstracts

Kumari Priyanka, Kiran Dip Gill, Surjit Singh, Indu Verma

MMSL 2018, 87(88):113  

BACKGROUND: Managing burden of Coronary Artery Disease (CAD) is a battle for researchers over the globe as disease seems to be multifactorial. Duet concert of genetics and environmental factors over oxidative stress and inflammation accounts for disease progression. Human Paraoxonase 3 an HDL associated endogenous antioxidant enzyme, has been identified as antiatherogenic entity. Modifiable risk factors like diet and lifestyle play a supreme role in regulating paraoxonase activity. RATIONALE: To understand how the activity of Paraoxonase 3 can be modulated by using various pharmacological agents to derive the therapeutic benefit in CAD patients....

SMART & INNOVATIVE TOOLS FOR CHOLINESTERASES RELATED APPLICATIONSMeeting abstracts

Emilie David, Benoît Roubinet

MMSL 2018, 87(88):114  

CHEMFORASE¹ is a french innovative biotechnological start-up, built in 2016, and specialized in the manufacturing and marketing of affinity resins for purification of cholinesterases. The lead product, Hupresin®, makes it possible to purify efficiently both plasmatic and recombinant human butyrylcholinesterase (BChE). This innovative Hupresin® technology has perfect characteristics to specifically bind cholinesterases, providing the best performances on the market. CHEMFORASE also developed a fast flow affinity resin, Hupresin^® AC, efficient for the purification of plasmatic BChE. A new affinity resin with better...

IN VITRO EVALUATION OF STANDARD ACETYLCHOLINESTERASE REACTIVATORS AS REACTIVATORS OF HUMAN PLASMA BUTYRYLCHOLINESTERASEMeeting abstracts

Lucie Junova, Vendula Sepsova, Kamil Musilek, Daniel Jun

MMSL 2018, 87(88):115  

Bioscavengers are considered to be a promising approach in the prophylaxis or treatment of poisoning by organophosphorus inhibitors (OPI; nerve agents and organophosphate pesticides). They can efficiently neutralize diverse OPIs in the bloodstream before they reach their natural targets - cholinesterases. Antidotal efficacy of administered butyrylcholinesterase (BChE; EC 3.1.1.8), one of the possible bioscavengers, could be further increased when it is co-administered with an oxime reactivator of a sufficient reactivation potency. Therefore, the activity of BChE, inhibited by OPI, could be continuously renewed (pseudo-catalytic bioscavenger). In this...

NOVEL BISQUATERNARY HETEROAROMATIC COMPOUNDS AS POTENTIAL REACTIVATORS OF HUMAN BUTYRYLCHOLINESTERASEMeeting abstracts

David Malinak, Eugenie Nepovimova, Marketa Neugebauerova, Miroslava Hozova, Vendula Hepnarova, Daniel Jun, Rafael Dolezal, Kamil Musilek, Kamil Kuca

MMSL 2018, 87(88):116  

Human butyrylcholinesterase (hBChE) is well-known stoichiometric scavenger in case of organophosphorus (OP) intoxication. However, its major limitation lies in binding of only one OP moiety per hBChE molecule and thus necessity of its very high dosage prior or post intoxication. This issue might be resolved by use of hBChE reactivators that could cleave irreversibly bound OP moiety from the enzyme active site and restore its scavenging function. This concept has been called pseudo-catalytic scavenger. Within our contribution, we would like to present bisquaternary heteroaromatic compounds that are butyrylcholinesterase reactivators...

BUTYRYLCHOLINESTERASE AND ITS VARIANTS (rs3495 & rs1803274) ASSOCIATION WITH MAJOR DEPRESSIVE DISORDERMeeting abstracts

Sliha Awan, Syed M Nurulain, Sadaf Munir, Sania Ghafoor, Rabia Habib, Maleeha Azam

MMSL 2018, 87(88):117  

Major Depressive Disorder (MDD) is a psychiatric condition. Globally, it is known to be the fourth leading source of ill health. Butyrylcholinesterase is a cholinergic enzyme with diversified reported functions. Objectives of the present study was to find the status of BChE in depressive individuals and to investigate the association of two SNPs of BCHE (rs3495; c.*189G<A) and (rs1803274; c.*1699G>A). Study was conducted with the approval from Ethical Review Board of the Department of Biosciences and consents from participants. Seventy six MDD patients and fifty four healthy controls were recruited for the study. Depressive individuals...

ALKALOIDS DERIVED FROM TRADITIONAL CHINESE MEDICINE ARE INHIBITORS FOR INFLAMMATION AND ACETYLCHOLINESTERASEMeeting abstracts

Xiang P. Kong, Miranda L. Xu, Etta Y. L. Liu, Qiyun Wu, Tina T. X. Dong, David C. C. Wan, Karl W. K. Tsim

MMSL 2018, 87(88):118  

The inhibitors for acetylcholinesterase (AChE), an enzyme hydrolyzing acetylcholine in cholinergic synapses, have been used for the treatment of Alzheimer's disease (AD). Alkaloids inhibiting AChE activity are commonly found in traditional Chinese medicine (TCM), e.g. gelantamine from Lycoris radiata, berberine from Coptis chinensis, huperzine A from Huperzia serrata. Many of these alkaloids also show regulatory role on inflammation, including the suppression on neuro-inflammation. Here, we aimed to reveal the possible relationship of these alkaloids in having both anti-inflammation and anti-AChE properties, in particular the role...

ACETYLCHOLINESTERASE REGULATES INFLAMMATORY RESPONSES IN CULTURED MACROPHAGES: A PLAYER IN CHOLINERGIC ANTI-INFLAMMATORY PATHWAYMeeting abstracts

Etta Y. L. Liu, Miranda L. Xu, Xiang P. Kong, Qiyun Wu, Tina T. X. Dong, Karl W. K. Tsim

MMSL 2018, 87(88):119  

Acetylcholine (ACh), the primary neurotransmitter released by vagus nerve, suppresses the levels of pro-inflammatory cytokines and tissue damage via the α7-nicotinic ACh receptor (α7-nAChR); this connection is being known as “cholinergic anti-inflammatory pathway (CAP)”, a communication between immune and nervous systems. Acetylcholinesterase (AChE) is responsible for rapid elimination of ACh in vertebrate. In the treatment of Alzheimer's disease (AD), AChE inhibitors are commonly employed. The modulatory role of AChE inhibitors in inflammation have been reported. Here, the expression profile of AChE was determined in cultured...

GENISTEIN, A PHYTOESTROGEN IN SOYBEAN, INDUCES THE EXPRESSION OF ACETYLCHOLINESTERASE VIA G PROTEIN-COUPLED RECEPTOR 30 IN PC12 CELLSMeeting abstracts

Etta Y.L. Liu, Miranda L. Xu, Qiyun Wu, Tina T.X. Dong, Sibao Chen, Karl W. K. Tsim

MMSL 2018, 87(88):120  

Several flavonoids have been identified to induce the expression of AChE in PC12 cells, e.g. daidzin, irisflorentin, cardamonin and genistein. Among them, genistein is the most robust inducer for AChE activity. Genistein, 4',5,7-trihydroxyisoflavone, is a major isoflavone in soybean, which is known as phytoestrogen having known benefit to brain functions. Being a common phytoestrogen, the possible role of genistein in the brain protection needs to be further explored. In PC12 cells, application of genistein significantly induced the expression of neurofilaments, markers for neuronal differentiation. In parallel, the expression of tetrameric form of...

STATUS OF CHOLINESTERASES IN HEROIN, HASHISH (CANNABIS) AND POLYDRUG ADDICTSMeeting abstracts

Syed M Nurulain, Sliha Awan, Sadaf Munir, Tahira Javed, Sania Ghafoor, Rabia Habib

MMSL 2018, 87(88):121  

Drug addiction is strongly influenced by biochemical, neuromodulator and genetics. It has been established that cholinergic system acts as neuromodulator with dopaminergic system, a major player in addiction. Putative role of cholinergic enzymes other than cocaine is hardly addressed. Present study was designed to evaluate the status of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in heroin, hashish (cannabis) and polydrug users. Study was conducted with the approval from Ethical Review Board of the Department of Biosciences and consent from participants. Twenty healthy non-addict age and sex matched individuals and eighteen male substance...

INDIRECT EFFECTS OF DIOXIN ON NEURONAL AChE EXPRESSION VIA ASTROCYTESMeeting abstracts

Rui Sha, Yangsheng Chen, Yiyun Liu, Li Xu, Heidi Qunhui Xie, Bin Zhao

MMSL 2018, 87(88):122  

Acetylcholinesterase (EC3.1.1.7; AChE) is one of the most important enzymes in the cholinergic system. Our previous works showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a notorious persistent organic pollutants, suppressed neuronal AChE activity by both transcriptional and post-transcriptional regulations via aryl hydrocarbon receptor pathway in SK-N-SH human neuroblastoma cells [1, 2]. In the nervous system, the most abundant cell type, astrocyte is regarded to play vital roles in protecting neurons from various kinds of insults, including environmental pollutants. Astrocytes have been considered as one of the target cells of dioxin in the...

NON-NEURONAL CHOLINERGIC SYSTEM IN RAT AORTAMeeting abstracts

Szmicseková, K., Kiliánová, Z., Krajčovičová, K., Krejci E., Paul Hrabovská, A.

MMSL 2018, 87(88):123  

We have showed before that both cholinesterases are present in rat aorta, while inhibition of butyrylcholinesterase impairs physiology of the isolated organ. The endothelium-dependent vasodilatory effect of acetylcholine (ACh) on vessels is well known, but physiological or pathological importance of this effect in live animals is questionable, and origin of possibly acting ACh unclear. Hypothesizing that aorta is a non-neuronal cholinergic tissue, the main aim of this project was to examine the presence of the proteins involved in the synthesis, storage, release, and degradation of ACh. Target-specific primers were used in RT-qPCR for determination...

MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION FACTOR REGULATES ACETYLCHOLINESTERASE EXPRESSION DURING MELANOGENESIS OF B16F10 CELLS: A CHOLINERGIC REGULATOR IN PIGMENTATIONMeeting abstracts

Qiyun Wu, Aster H. Y. Fung, Miranda L. Xu, Etta Y. L. Liu, Ran Duan, Ping Yao, Tina T. X. Dong, Karl W. K. Tsim

MMSL 2018, 87(88):124  

Acetylcholinesterase (AChE) hydrolyses acetylcholine that functions as a neurotransmitter in neurons. The non-neuronal functions of AChE have been proposed in different cell types. Here, we revealed the expression of AChE in melanocyte and melanoma, in which the tetrameric (G4) form was the major isoform. In the melanogenesis of cultured B16F10 murine melanoma, the amount of AChE was markedly decreased. The differentiation of melanoma led to: (i) increase of melanin and its synthesis enzyme tyrosinase; (ii) change of intracellular cAMP level; and (iii) decrease of microphthalmia-associated transcription factor (MITF). The regulation of AChE during...

DUAL BINDING SITE INHIBITORS OF ACETYLCHOLINESTERASE
AS THERAPEUTIC TREATMENTS FOR ALZHEIMER’S DISEASE: ANY NEED FOR AN UPDATE?Meeting abstracts

K. Petrov, I. Zueva, J. Dias, S. Lushchekina, V. Semenov, F. Nachon, E. Nikolsky, P. Masson

MMSL 2018, 87(88):125  

Alzheimer’s disease (AD) is a broadly spread neurodegenerative disorder of ageing population manifesting itself in progressing loss of cognitive functions down to total demolition of intellect and disability. Profound synaptic dysfunction contributes to early loss of short-term memory in Alzheimer’s disease. Here we show the protective effects against amyloid-induced synaptic toxicity of C-35, a potent reversible inhibitor of acetylcholinesterase (AChE). Crystal structure of the complex between human AChE and C-35 revealed tight contacts of ligand along the enzyme active site gorge. Molecular dynamics simulations indicated that the external...

DETECTION OF ALZHEIMER’S DRUG CANDIDATE BY SURFACE-ENHANCED RAMAN SPECTROSCOPYMeeting abstracts

Jan Proska, Lucie Maresova, Marek Prochazka, Eugenie Nepovimova, Kamil Kuca

MMSL 2018, 87(88):126  

Drug candidate 1-EN-142 was designed and synthesized as a multipotent therapeutic agent to treat Alzheimer´s disease. In its molecule it combines tacrine moiety with naphthoquinone scaffold. For the study of centrally-active molecules in biological samples it is necessary to develop appropriate detection methodology that would determine such compounds in low concentration. Spectroscopy based on Surface-Enhanced Raman Scattering (SERS) was chosen as a comparative method for the electronic detection of compound 1-EN-142 by interdigitated impedance sensor decorated with gold nanoparticles. Since spectroscopic data were not available for this new drug...

BUTYRYLCHOLINESTERASE INHIBITORS GRAFTED WITH ANTIOXIDANT AND NEUROPROTECTIVE ACTIVITIES: NOVEL MULTIFUNCTIONAL LIGANDS FOR ALZHEIMER’S DISEASEMeeting abstracts

Damijan Knez, Nicolas Coquelle, Anja Pišlar, Simon Žakelj, Marko Jukič, Matej Sova, Janez Mravljak, Florian Nachon, Xavier Brazzolotto, Janko Kos, Jacques-Philippe Colletier, Stanislav Gobec

MMSL 2018, 87(88):127  

Current symptomatic treatment has only limited clinical efficacy and minute effect on progression of Alzheimer’s disease. The research focus has thus shifted from single targets towards multifunctional ligands targeting several pathological processes of the disease [1, 2]. A potent picomolar selective inhibitor of human butyrylcholinesterase [3] was used as the starting point to develop a new series of multifunctional ligands. A focused library of derivatives was designed and synthesized that showed both butyrylcholinesterase inhibition and good antioxidant activity comparable to natural antioxidants. The crystal structure of compound 11 in...

7-MEOTA-DONEPEZIL HYBRIDS: POTENTIAL CHOLINESTERASE INHIBITORS FOR THE TREATMENT OF ALZHEIMER’S DISEASEMeeting abstracts

Katarina Spilovska, Eva Mezeiova, Jan Korabecny, Jana Hroudova, Vendula Hepnarova, Martina Hrabinova, Ondrej Soukup, Kamil Musilek, Daniel Jun, Kamil Kuca

MMSL 2018, 87(88):128  

Alzheimerʼs disease (AD) is a devastating neurodegenerative disorder characterized by a severe, progressive loss of memory. Currently, AD therapy is limited on the administration of cholinesterase inhibitors (ChEIs) and the N-methyl-D-aspartate (NMDA) antagonist, memantine. Tacrine as the first registered acetylcholinesterase (AChE, E.C. 3.1.1.7) inhibitor was withdraw due to its adverse effects. 7-Methoxytacrine (7-MEOTA) was prepared as a pharmacologically equal active compound with lower toxicity compared to THA. Donepezil as a highly selective inhibitor for AChE was connected with 7-MEOTA scaffold due to the ability to interact within calatytic...