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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 an aptamer binding organophosphorus pesticides [2], its three-dimensional model was constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy were calculated using MD trajectories and MM-PBSA approach [3]. On the basis of the energy values, the sequences that bind paraoxon most efficiently have been selected. Molecular docking of sixteen possible nucleotide pairs into PAS of AChE was performed and the pairs that bind with PAS most efficiently have been selected. The 5’-end of the aptamers for paraoxon was modified based on the results of molecular docking. The calculations have shown that the final aptamers interact with paraoxon and PAS of AChE more efficiently than AChE interacts with paraoxon.

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 with PROSS, a sequence- and structure-based algorithm for the soluble prokaryotic expression of difficult proteins. The protein is easily purified with two simple chromatographic steps. Despite 47 point mutations, the enzyme presents similar enzymatic parameters than the wild-type enzyme and its active site gorge structure is identical to that of the native enzyme produced in eukaryotic systems as determined by X-ray crystallography. These data validate the prokaryotic expression of human BChE which will greatly facilitate the screening of variants with nerve agent hydrolytic properties. We have initiated animal studies to assess the protein potency (immunogenicity, pharmacokinetic and bioscavenger efficiency) and will study the production of the tetramer form. On the other hand, we are currently developing high-throughput protocols for the prokaryotic expression, purification and screening of nerve agent hydrolysis.

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 hydrolyzes carboxylesters. However, if an OP is bound (organophosphorylation), its represents an enzymatic inhibition in some cases considered “the target” of toxicity or initial molecular event (IME) in their mode of action developing toxicity (adverse output pathway, AOP). The binding to proteins also represents a sequestration avoiding the OP interaction to other protein. However, there are protein binding OPs (non-catalytic bioscavengers) which can be slowly dephosphorylated, having a role as catalytic scavenger. A proportion of B-esterase activity in serum and brain shows reversible inhibition and their protective role just in situ in the target tissue of toxicity need to be investigated. Serum albumin is other example of B-esterase mainly thorough a tyrosine residue; its role in detoxication have been demonstrated and adducts applied as biomarker of exposure. Moreover, for a specific phosphoramidate family hydrolysis capacity may be enhanced by copper, probably by a mechanism not related with its B-esterase activity. Therefore, we have examples in the borderline between non-catalytic and catalytic scavengers.

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 in guinea pigs by administering the enzyme via a carotid catheter, followed 20 minutes later by a subcutaneous injection of increasing doses of the OP nerve agents GA, GB, GD, GF, VX, VR, or VM.  A stage-wise, adaptive dosing experimental design was used to determine the median lethal dose (LD₅₀) of each OP in the context of enzyme prophylaxis.  We report that a combination of two different OPH variants is capable of providing protection against at least 2 x LD₅₀s of all of the OPs tested.  The results indicate that broad spectrum prophylactic protection against OP intoxication can be provided with a cocktail of two different catalytic scavengers with appropriate catalytic activity.  Formulation of the enzymes to promote circulatory stability will be discussed.

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 of up to 1 to 2 mg/ml in mouse blood for up to 6 months. Mice containing 0.75 mg/ml or higher concentrations of I-F11 in their blood were afforded asymptomatic protection against multiple 5LD50 exposures of GD, GF, GA, and GB, a total of 9 exposures over a seven-week period. We also conducted studies showing that I-F11 is most efficacious in offering protection against GD followed by GF, GB and GA. Analysis of the mouse blood for serum chemistry and hematology parameters, and tissues by H&E staining, indicated no appreciable changes between control mice, mice overexpressing 1-F11 for 6 months, and mice surviving repeated G-agent exposures. These data suggest that AAV8-mediated catalytic bioscavenger gene therapy using 1-F11 is a safe, efficacious, and long-lasting pre-treatment strategy against G-agents.

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 of fusion proteins. To this end, we designed OPH to be expressed as a tethered monomer by joining two OPH subunits with a poly-glycine linker. We created the single polypeptide OPH with a tether 10 or 35 amino acids in length between the two halves, and named them T10 and T35 respectively. Western blot analysis and paraoxon hydrolysis assays revealed that T10 was being produced and retained some activity against paraoxon. This was a surprise as we expected T10 to have no enzymatic activity. T35 monomer (75 kDa) was also being produced and retained 71% of specific activity against paraoxon compared to untethered OPH. T10 and T35 showed no significant decrement in activity against the nerve agent sarin. Both constructs showed high molecular weight aggregates greater than 250 kDa in dynamic light scattering and native polyacrylamide gels. These tethered constructs are the first attempts known for producing OPH as a single polypeptide.

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). Structural predictions revealed strong similarities between planarian and human enzymes. Cholinesterase activities were detected in crude planarian homogenates after grinding and were inhibited after organophosphorus exposition. In situ Hybridization was further used to localize cholinesterases in planarians and showed two different patterns, Smed-AChE being mainly detected in cephalic ganglion and ventral nerve cords while Smed-BChE distribution was diffuse. Survival, behavior and regeneration were analyzed in whole planarian exposed to four OP [1]. The toxicity of OP degradation products generated by enzymatic hydrolysis with the robust phosphotriesterase enzyme SsoPox, from the archea Sulfolobus solfataricus [2], was further evaluated. OP were found to be highly toxic to planarians causing severe mortality and behavior disruption at sublethal concentrations as well as growth disruption during regeneration after cutting. Enzymatic decontamination drastically reduced toxicity and enhanced both mobility and development. These results underline that degradation products have a lower impact than initial organophosphorus substrates. A biotechnological application based on a filtration column incorporating detoxifying enzymes was developed to decontaminate wastewater with planarian as biosensor.

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 count and mean platelet volume (MPV) were determined by a cell counter. Flow cytometry was used to quantify platelet surface receptors, intracellular ROS and platelet function in diluted citrate-anticoagulated platelet-rich plasma. Platelet aggregation was analyzed by light transmission aggregometry in platelet-rich plasma. Results: Platelets from PON2-deficient mice displayed increased basal and agonist-induced ROS levels accompanied by decreased platelet count but increased MPV compared to wildtype platelets. PON2-deficient platelets showed increased surface expression of the von Willebrand receptor (vWF) GPIbα, vWF-binding, P-selectin surface expression, but no αIIbβ3 integrin/fibrinogen receptor activation ex vivo. Botrocetin induced enhanced binding of vWF to PON2-deficient platelets in vitro. However, agonist-induced αIIbβ3 integrin activation, P-selectin surface expression and platelet aggregation were impaired compared to wildtype platelets. Interestingly, addition of 0.5 mM Ca²⁺ to platelet-rich plasma normalized platelet hyporeactivity. Conclusion: Our data demonstrate that PON2 plays a crucial role in platelet ROS production, phenotype and function. Reactivity of platelets from PON2-deficient mice depends on extracellular Ca²⁺-concentration.

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 serum albumin (CSA 216 µg/mL) with 100 µM Cu²⁺, showed about 50% and 75% of R-(+)-HDCP hydrolysis after 60 and 120 min. In the same conditions other commercial serum metalloproteins with high affinity to Cu²⁺ (cuproproteins) as human serum ceruloplasmin or horse kidney metallothionein did not showed significant Cu²⁺-dependent hydrolysis. Moreover, other divalent cations (Zn²⁺, Fe²⁺, Ca²⁺, Mn²⁺ and Mg²⁺) did not showed this activation. The results confirm that the CSA is the protein responsible of "antagonistic stereoselectivity" that had been observed in the chicken serum. The effect of copper on the hydrolysis of HDCP by other animal albumins is shown in this work.

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 aspects may more efficiently interfere with the disease progression. Systematic integration of the data derived from different disciplines including computational modeling, X-ray crystallography, synthetic chemistry, in vitro / in vivo pharmacological tests, is mandatory for the selection of best-in-class compounds. In this context, we report on the key contribution of X-ray crystallography in highlighting peculiar mode of interaction of promising multi-target directed ligands, designed by combining the tacrine fragment to distinct pharmacophores i.e. juglone [2], benzofuran [3] and       L-tryptophan with a linker of a suitable length. Overall, the structural analysis highlights the molecular determinants responsible for the optimal binding of the multi-target ligands to AChE and pinpoints the utility of hybridization strategies in structure-based drug design programs. It also unveils the validity of X-ray crystallographic structures determination at certain milestones along the development of interacting inhibitory drugs based on molecular modeling studies.

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 a potent difluoromethyl ketone inhibitor, revealing the structural basis of insecticide resistance².  Although resistance-breaking inhibitors of G119S AgAChE exist, they also inhibit human acetylcholinesterase and thus lack the necessary species selectivity to be safely used as insecticides.  In our structures, we see specific features within the active site gorge, including an open “back door”, that are distinct from human acetylcholinesterase.  These differences provide a means for improving species-selectivity in the rational design of improved insecticides for malaria vector control.

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 processes during reactivation. Thus, we use room-temperature X-ray and neutron crystallography to obtain structures at physiological conditions and to visualize hydrogen atoms. Several X-ray structures of native and VX and POX-conjugated hAChE in complex with oxime reactivators, RS2-170B and RS-194B have been obtained. hAChE crystallized in a unit cell (a=124.3, c=129.1 Å; P3₁) amenable to neutron crystallography. For the first time we show how RS2-170B binds in the non-modified and OP-conjugated active site gorge at room temperature. RS-194B is observed with its oxime group pointing away from the catalytic Ser203 and the reactivator is pushed out to bind at the peripheral site in the VX-modified structure. Dynamics of hAChE was probed by neutron vibrational spectroscopy to look at harmonic vibrations. POX binding induces significant changes in the acyl pocket loop conformation expelling the weakly binding RS-194B from the active site gorge completely, and the loop becomes more dynamic. We hypothesize that increased dynamics of the acyl pocket loop contributes to the POX-conjugated hAChE resistance to reactivation.

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 form supramolecular structures by chelating Zinc. The nature of these molecules suggested that they could have affinity for the aromatic residues in the active site gorge of human cholinesterases. It was confirmed by determining their inhibition properties. A key question was whether these supramolecular ligands bind to human cholinesterases as their Zn-complex or monomeric form? The X-ray structures of two supramolecular complexes binding to the gorge of the hAChE and the hBChE reported herein showed that either cases are possible. These structural data on two new types of ligand can be used to design original cholinesterases inhibitors or reactivators.

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 of ChEs the only true antidotes of OP intoxicated individuals and therefore need to be considered in structure based design of improved oxime antidotes. We developed a novel, reference point based principle for overlay-independent pairwise comparison of liganded and non-liganded Cα conformations from respective PDB structures and encoded it in JAVA based computer algorithm for quick analysis. Comparisons are based on differences in distances between each Cα pair based on differences in the angle between center of mass, reference point and each of Cα in the comparison, revealing a subset of Cα in two structures that maintains their relative positions in the 3D space best and that can be used as tethering points for overlay of compared structures. Using NanoPro (Nanome Inc.) VR software, we visualized results of pairwise structure analyses creating .pdb format 3D graphs to identify interaction matrices between amino acids revealed upon ligand binding. Structure comparisons will be paralleled to OP inhibition and oxime reactivation parameters for some of analyzed ChE-OP-oxime systems to emphasize the importance for complete molecular target template characterization in the structure based antidotes design.

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 AChE⁺ cells revealed a close spatial localization of both proteins first within individual cells (nuclear ChAT, vs. extranuclear AChE), and later between adjacent cells, e.g., ACh-secreting and -degrading cells have the same cell lineage origin, and later remain in close apposition (4). Using our 3D stem cell organoid approach (retinal spheroids), we could show that ChAT⁺ cells were first to initiate IPL formation by establishing two synaptic sublaminae. Unexpectedly, the earliest ChAT⁺ cells co-expressed markers of Müller glial precursors (MCPs), indicating that a direct SAC precursor i) gives rise to neurons and glial cells, and ii) that these premature cholinergic cells drive earliest processes of network formation in vertebrate retinae, e.g. could function as IPL founder cells (5, cf. also 6,7). These findings could have profound relevance for a basic understanding of neuronal network formation.

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 hormone after i.v. ghrelin injection. In other words, high plasma BChE enhanced sensitivity of ghrelin’s target, the growth hormone secretagogue receptor, involved in emotional behaviors. That also fits BChE’s impact on feeding. BChE knockout mice have high ghrelin levels that drive overeating and obesity.  BChE-enhanced mice have low plasma ghrelin, they resist obesity on high-fat diet and show less rebound weight gain after a forced low-calorie diet. These findings suggest that BChE gene transfer could have substantial therapeutic impact on obesity and other conditions that involve ghrelin.

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 AChEIs are membrane permeable, and thus which could act as chemical chaperons in affecting the protein assembly of PRiMA-linked AChE in the endoplasmic reticulum (ER). In cultured neuroblastoma or cortical neuron, application of AChEIs, including tacrine (Cognex), rivastigmine (Exelon), but not donepezil (Aricept) and galantamine (Razadyne), caused an accumulation of the unfolded AChE being retained in ER fraction: the AChEI-bound enzyme was not able to transport to Golgi/plasma membrane fraction. As a result, the transcripts encoding AChE and PRiMA were decreased by 50% in the AChEI-treated cultures. In parallel, an increase of ubiquitin-associated enzyme degradation was revealed. The treatment of AChEI in the cultures induced the expression of apoptotic markers, e.g. cleaved caspase 3. In parallel, the apoptotic cell number and mitochondrial membrane potential (MMP) were increased in a dose-dependent manner. The AChEI-bound enzyme retained intracellularly could induce a result of ER stress, as indicated by increased expressions of BiP and CHOP in the treated cultures. The AChEI-induced ER stress resulted with an activation of cAMP signaling, which could regulate the expressions of miR132 and miR212. These findings provide guidance for the drug design and discovery in AD based on inhibition of AChE.

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) or also in postjunctional folds. Quantitative EM-analysis using nanogold labeled anti-AChE probes demonstrated that AChE sites are almost exclusively located on the BL rather than on pre- or postsynaptic membranes and are distributed in the PC and down the postjunctional folds, with a defined pattern. This localization pattern of AChE is suggested to ensure full hydrolysis of acetylcholine bouncing off receptors, thus eliminating its harmful re-binding. The methodology developed for normal NMJs provides a benchmark for studying other peripheral and central nervous system synapses under physiological or pathological conditions.

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 mice with paraoxon, physostigmine or pyridostigmine and recorded in great details the modifications of breathing in double chamber plethysmography. Physostigmine triggers very long end inspiration pauses (EIP) in WT whereas pyridostigmine provokes only short EIP. The duration of EIP was changed with physostigmine or pyridostigmine in PRiMA KO mice when the brain was adapted to a huge excess of ACh. Surprisingly, when AChE is absent at the NMJ, EIP were much shorter with physostigmine. If AChE in the respiratory center is a key target, we expected long EIP when AChE is normal in the brain and reduced in muscles. Altogether these observations do not support that the change of the synaptic transmission explains the central shutdown control of breathing when cholinesterases are inhibited. In addition, we observed that methacholine provokes similar alteration of breathing when injected subcutaneously to mice. I will discuss a novel model to reconciliate these observations.

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 miR-608 targets (2). Others reported that the rs4919510 SNP in the miR-608 gene reduces miR-608 levels in vitro and limits the risk of sepsis following head injury in vivo (3). To explore the combined effect of these two SNPs, we tested 444 healthy 30 years old US donors and 101 Israeli ex-prisoners of the 1973 war (EWP), 76 of whom returned with post-traumatic stress disorder(PTSD). Genotyping combined with R-statistics of the corresponding biomedical evidence demonstrated that the rare allele of the AChE SNP was more abundant among non-PTSD EWP donors compared to PTSD patients in this cohort (33 vs 19%, Chi-square 0.03). Moreover, we found in both of these cohorts interaction between the effect of the two SNPs on blood pressure, inflammation and anxiety-related parameters, with the miR-608 SNP stratifying the corresponding impact of the rare allele of the AChE SNP on these parameters. Our findings indicate an interaction between the SNPs in the AChE and miR-608 genes, possibly reflecting modified impact of this primate-specific miR on its numerous downstream targets.

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 both transcriptional and posttranscriptional regulations could be involved. Moreover,, muscular AChE expression was also disturbed by dioxin exposure. During myogenic differentiation of C2C12 cells, the mRNA expression of AChE T subunit and the enzymatic activity of AChE were significantly suppressed by dioxin exposure in parallel with the disturbances on the myotube formation. However, the addition of AhR antagonist was not able to reverse the suppressive effect of dioxin, suggesting a distinct role of AhR during the myogenic differentiation process. These results further support the notion that dioxin is a novel environmental AChE disruptor which acts on the biosynthesis processes via multiple molecular mechanisms.

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, played crucial role in Wnt3a-induced AChE expression in osteoblasts. This was confirmed by identification of Runx2-binding site in the ACHE gene promoter, over-expression of Runx2 and deletion of the Runx2-binding site in the ACHE promoter. Bone defect was observed in ACHE-/- mice. The non-enzymatic role of AChE in osteoblast was determined by over-expression system and application of AChE inhibitors. By transcriptomics, AChE was found to influence gene expressions of Wnt/β-catenin signaling components, and may participate in osteoblastic function, e.g. affecting osteoclastogenesis and cell adhesion of osteoblast. A notion of non-cholinergic role of AChE in osteoblast, as well as an insight for elucidating other possible mechanisms in regulation of bone formation was provided.

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 activity is decreased while BChE activity is unchanged or even increased, making both ChEs stimulating targets for the treatment of AD.  Current AD therapy is based on AChE inhibitors, although they have very modest clinical effects in treating the symptoms of the disease and are unable to halt disease progression. Oxidative stress (OS) and mitochondrial dysfunction are also considered critical factors in AD pathogenesis. As a result, targeting mitochondrial oxidative stress (OS) in the prodromal phase of AD to slow or prevent the neurodegenerative process and restore neuronal function is thus viewed as a valid therapeutic approach. As part of our drug discovery program focused in oxidative stress-related diseases, and following a multi-target strategy, new mitochondriotropic antioxidants based on natural scaffolds acting as dual and bifunctional cholinesterase inhibitors have been developed. The results will be reported in this communication.

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 driven a profound revolution in cholinesterase drug research [1-2]. We will present our journey from dual binding site AChE inhibitors as potent beta-amyloid modulators to the more recent serie of indolylpiperidines hybrids with an unexpected and very potent hBuChE inhibition. Experimental and computational studies have revealed the chameleon behavior of these molecules able to change their bioactive conformation depending on the cholinesterase binding site. Based on the potent activity of these compounds targeting BuChE, the low cellular toxicity and the in vivo target engagement, we can propose these indolylpiperidine derivatives as valuable tools for the study of the role of BuChE in AD and probably as potential drugs candidates for its future pharmacotherapy.

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 medicines, with disease-modifying potential. In this presentation, the multi-neuroprotective effects and the underlying mechanisms of those promising candidates will be comprehensively illustrated and discussed.

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, binding mechanisms and kinetic parameters is important to be considered. In particular, estimation of residence time has recently emerged as critical feature [2]. Therefore, accessing kinetic information on drug binding events at initial stages of the drug discovery process is gaining increasing interest among medicinal chemists. In the light of these considerations, the talk will present different approaches involving immobilized human cholinesterases (ChEs). Micro-immobilized enzyme reactors (IMERs) can be used in combination with HPLC systems while SPR biosensing technology can be exploited for binding and kinetic investigation. ChE-based IMERs and single or multiple sensing surface(s) can be used in combination as valuable screening tools, which allow to quickly retrieve a set of highly useful information which can assist scientists in the selection of new chemical entities to be further developed.

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 associated with cerebro-cardiovascular outcome. Recently we found that serum AChE activity increased with BMI in a dose-dependent manner until it reached a peak level at BMI of 30-35 kg/m², followed by a plateau (p<0.001, n=1,450). Similarly, AChE activity increased with waist circumference categories (p < 0.001 for men and P = 0.013 for women). The Obesity-related AChE resistance phenotype may be reversed following laparoscopic sleeve gastrectomy (LSG) surgery and correlates with metabolic outcomes (% excess weight loss, %fat, and delta Homeostasis Model Assessment (HOMA)). Further long-term studies will be needed to validate and evaluate the beneficial effect of AChE reduction post bariatric surgery and its possible relation to cognitive decline.

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 inhibition. Furthermore, studies in human neuroblastoma SH-SY5Y cells and in the lymphoblasts of patients with Alzheimer's disease have shown neuroprotective effects of this family of compounds, as well as their capacity to blunt the abnormal enhanced proliferative activity of AD lymphoblasts. Based on the in vitro and functional studies we performed in vivo studies of those best compounds employing transgenic mouse (TgAPP) model. The results of the in vivo study revealed that some of these compounds could be very promising candidates for the treatment of Alzheimer's disease.

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 (C), MCI or AD groups.  MRI, ¹⁸F-FDG and amyloid-PET were assessed.  ANCOVA compared main effects of i)diagnosis, ii)BCHE-K, iii)BCHE-A and iv)APOE4 status on each biomarker with age, education and sex as covariates.  Results: The allelic frequency was 20.8%, 4.6% and 26.5% for BCHE-K, BCHE-A and APOE4.  For MRI, main effects for diagnosis were significant (p<0.0001), with reduction in whole-brain and selected regional volumes (7-27%, p≤6x10^-6) in AD vs. C. For ¹⁸FDG-PET, the main effect for diagnosis was significant (p=5x10^-9), with 14% decrease in metabolism in AD vs. C (p=7x10^-10). For amyloid-PET, the main effects for diagnosis and APOE4 status were significant (p=0.034; p=3x10^-6), with 12% increase in retention in AD vs. C (p=0.023) and 16% increase among carriers of at least one APOE4 allele vs. non-carriers (p=8x10^-6).  No significant effects of these biomarkers were observed due to BCHE-K or BCHE-A status (p≥0.209).  Conclusions: These data suggest BCHE-K or BCHE-A may not significantly effect structural, metabolic or molecular AD biomarkers. Further ROI/voxel-wise analyses are warranted to uncover potential regional changes among AD BCHE variants.

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 that act with high potency and selectivity also at the histamine 3 receptor (hH₃R) [1], and the same could be achieved for cannabinoid 2 receptors (hCB₂R) [2, 3], both GPCRs represent important AD targets. Regarding dual-acting ChE inhibitors and hCB₂R agonists both covalently connected hybrids using the unselective ChE inhibitor tacrine as well as merged small molecules with high butyrylcholinesterase (BChE) selectivity have been obtained and pharmacologically characterized in vitro. Representative examples from all sets of compounds have been investigated in vivo in different AD mice models [3]. The case studies demonstrate that it is possible to obtain dual-acting compounds that a) act highly selectively and with high affinity at the respective targets, b) work in the same concentration range (“balanced affinity”), c) exhibit pronounced in vivo activity.