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Thyroid cancer is a rare type of malignancy. However, thyroid cancer constitutes more than 90 % of endocrine tumors. Metformin (N’, N’-dimethybiguanide) is the most commonly prescribed drug in the world, and the annual number of prescriptions for this drug exceeds 120 million. Metformin is the first-line oral treatment for patients with type II diabetes. Metformin has recently been investigated for potential anti-cancer activity in patients with thyroid cancer by stimulating the Adenosine Mono-Phosphate-Activated Protein Kinase (AMPK) pathway in some types of tumors. In general, the anti-cancer mechanism of metformin acts directly by blocking mitochondrial oxidative phosphorylation through down-regulation of mitochondrial complex I and mitochondrial glycerophosphate dehydrogenase. This leads to a state of metabolic stress that in turn stimulates the AMPK pathway due to ATP reduction, and leads to inhibition of the mechanical (mammalian) target of the rapamycin (mTOR) pathway, which subsequently inhibits cancer cell proliferation and stimulates apoptosis and autophagy with cell cycle perturbation. Metformin also acts in an independent manner, in addition to its indirect actions that target insulin resistance. In this review, we reviewed 21 studies on the use of metformin in thyroid cancer, which showed that administration of metformin in diabetic patients is associated with a reduced incidence of thyroid cancer. On the other hand, the use of metformin enhances the response to anticancer drugs in thyroid cancer. Overall, we need further prospective studies to elucidate the synergistic mechanism of metformin when it is used to treat thyroid cancer as adjuvant therapy with anticancer drugs.

The ventricular septal defect (VSD) is a congenital lesion characterized by the presence of an opening between cardiac chambers. The treatment might involve medical therapy to control symptoms or in certain cases, surgical resuscitation might be required.Objectives: The study was conducted to establish a database about the prevalence and pattern of VSD and their prognosis in children referred to by echocardiography in Ibn-Sena Teaching Hospital over the period of March 2019 to January 2020. Method: The present study is a prospective descriptive study conducted on all patients diagnosed with cardiac lesions revealed by echocardiography. The sample included in the study involves newborns (day 1) to 14-years-old children.Result: Out of 500 children included in the study; most of these cases were cyanotic congenital heart lesions and out of which two-third were perimembranous defects.Conclusion: The study concluded a higher prevalence of non-cyanotic lesions and peri-membranous type is the commonest VSD lesion.

Introduction: The novel corona virus (SARS-CoV-19) is mainly accountable for the disease outbreak infection, which began in Wuhan, China, in 2019. Numerous modest research has been carried so far to ascertain the risks of smoking on the magnitude, consequence, and morbidity of patients with COVID-19, but the findings have been incomplete.Aims: This study assesses the effects of current smokers on the magnitude and consequence in patients with COVID-19 infectious disease in Mosul, Iraq.Materials and Methods: A total of 160 patients (80 active smokers and 80 non-smokers) who were confirmed with COVID-19 infection using polymerase chain reaction (PCR) test were enrolled in this study. A detailed history was obtained from of the subjects, as well as a thorough clinical evaluation and laboratory tests. The intensity of illness, biomarkers, D-dimer, liver enzymes (LFT), oxygen consumption, hospitalization, and outcome were all documented and analyzed between a two groups.Results and conclusion: The symptoms of COVID-19, measured laboratory markers were significantly higher in the sample of smokers compared to non-smokers. There has been no significant difference in the use of oxygen, hospitalization, ICU admission, death, or post-recovery problems. Serious clinical COVID-19 infection was much more prevalent in current smokers, and inflammatory markers such as D-dimer and LFT appeared greater in non - smokers than in smokers. There was no statistically significant difference in O2 usage, hospitalization, ICU admittance, death, or persistent morbidity.

Introduction: “Ankylosing spondylitis (AS)” is an inflammatory disorder that affects the axial skeleton,   peripheral joints, as well as entheses, resulting in significant disability. “Tumor necrosis factor-α (TNF-α)” inhibitors are regarded to be a helpful treatment for patients with active “AS”. This study aimed to investigate the effectiveness and response rate of “etanercept” in a group of patients with “ankylosing spondylitis” in Mosul, Iraq.Methods: A prospective, “open-labeled”, non-randomized 12 weeks study was undertaken on 43 participants with “ankylosing spondylitis” in the “Rheumatology unit” of “Ibn Sina Teaching Hospital”, and the diagnosis was made using the “modified New York criteria”. Participants were assessed at the outset of the study, week 4, and week 12 after receiving etanercept 50mg subcutaneously once weekly. The “Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)” was utilized to assess disease activity, while the “Bath Ankylosing Spondylitis Function Index” was utilized to assess functional status (BASFI) at baseline, 4 weeks, and 12 weeks. BASDAI 50 was used to assess the response rate.Results: Mean patients’ age was “36.6±8.47” years; men accounted for “90.7 %” of the cases, with the mean disease length being “9.6±5.90” years. A marked decrease in BASFI and BASDAI was found four and twelve weeks after commencing treatment compared to baseline (p=0.000).  “BASDAI 50 %” response was fulfilled by 42.5 % of the participants after 4 weeks and by 65%% after 12 weeks of therapy with “etanercept”. There was a marked fall in the mean ESR and CRP after four and twelve weeks of “etanercept” therapy.Conclusion: In “AS” patients,  once weekly “etanercept” 50 mg given subcutaneously for twelve weeks was an effective therapy.

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.

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 and 50 % reactivation of inhibited enzymes (R₅₀) were evaluated by in vitro methods using human RBC-AChE and plasma BChE. In silico study revealed lower free binding energies, but novel oximes did not bind with catalytic anionic site of enzymes. In vitro studies showed higher intrinsic toxicity by K456 and K733 than K27 and pralidoxime. R₅₀ for human RBC-AChE were K456=203.59µM±66.96; K733= 405.55µM±67.36; K27=2.68µM ±0.98 and pralidoxime 30.71µM±5.10 (mean±SEM) respectively. No substantial reactivation in BChE was noted by tested concentration of novel oximes.  The study concludes that oximes with peripheral binding/far from catalytic anionic site are ineffective reactivators. K27 with central (inside the active gorge) binding was superior to all tested oximes.

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, iso-OMPA or PMSF inhibited both activities with similar kinetic constants for both activities. Moreover, the substrates acethylthiocholine and phenyl valerate showed competition in their activities. The results suggest that both activities are related to the same active center. This work studies in depth the kinetic interactions between phenyl valerate and acetylthiocholine in human butyrylcholinesterase, showing that the interactions are different to the competitive model of substrates according to the Michaelis-Menten reaction. The approach introduced in this work suggests that other site could be involved in the interaction with phenyl valerate. In addition, we have observed that human acetylcholinesterase has also phenyl valerate esterase activity, but with lower activity than human butyrylcholinesterase. The level of phenylvalerate esterase activity in cholinesterases depends on the species and the type of cholinesterase. Further evaluation of the molecular interactions is under study.

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, and hint at the molecular basis for the increased rate of OP hydrolysis. This work explores the potential for the development of αE7 as an enzyme therapeutic for OP poisoning, and worryingly for insecticide resistance, this work suggests that more efficient OP detoxification could be readily acquired by insect pests.

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.

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 melanogenesis was hypothesized to be mediated by two transcriptional factors: cAMP responsive element binding protein (CREB) and MITF. In cultured melanoma, application of cAMP suppressed the expression of AChE, as well as the promoter activity of human ACHE gene. This suppression was shown to be mediated by a cAMP responsive element (CRE) located on the ACHE promoter, and mutation of this site eliminated the suppression. In melanoma, over expression of MITF induced the transcription of ACHE gene, and mutation of E-box site of the promoter blocked the induction. In parallel, application of an AChE inhibitor in vitro greatly enhanced acetylcholine-mediated responses of melanogenic gene expressions; but the enhancement was not revealed in the present of agonists of muscarinic acetylcholine receptor. Therefore, our results indicated that AChE transcription is specifically regulated by cAMP-dependent signaling pathway during melanogenesis of B16F10 cells, suggesting a potential role of AChE being played in this differentiation process.

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.

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 AChE over mammalian one.^[3] In our contribution, we have developed novel, straightforward and facile synthesis for Cys-targeted insecticides containing either maleimide or succinimide scaffolds. Employment of Grubbs olefin metathesis allowed us to obtain the final compounds in multistep synthesis in relatively high yields. We propose that the described synthetic route might be used in large scale-up for further studies.

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 group for a series of carbamates with the same carbamoyl group.  For a given leaving group, when the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-dimethyl-, N-ethyl-N-methyl-, or N,N-diethyl-, the decarbamoylation rates decreased by 4-, 70-, and 1000-fold, respectively. Thus the larger the size of the alkyl groups, the slower the rate of decarbamoylation due to active site distortion.  Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.3 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change.

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 times. Docking studies made it clear that the high AChE affinity resulted from simultaneous interactions of the quinoline group with aromatic residues of both the catalytic active site and the peripheral site. In conclusion, the inhibition potency and selectivity classify several novel compounds as leads for further modification and optimization towards the development of new inhibitors of AChE and potential drugs for treatment of neurodegenerative diseases.

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 microseconds revealed atomic and dynamic details on the phosphtriesterase catalysis and its requirement of two cations, which offers insight into re-engineering of phosphotriesterase to develop an improved scavenger against phosphorous-containing inhibitors of acetylcholinesterase.

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 and substrate analogues have been determined. Although the 3D structure of the enzyme itself, and of its molecular dimer, are highly conserved, subtle structural differences are seen to occur upon the binding of certain inhibitors. These changes are well correlated with molecular dynamics data, and appear to be of functional significance. Unfortunately, upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human AChE is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. Using a novel computational strategy, we designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ~2,000-fold higher levels in E. coli compared to wild-type hAChE, and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography^2,3.

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.

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 bifunctional reactivators that comprise a peripheral site ligand (PSL) connected to a fluorinated reactivator function using a covalent linker. On the basis of our previous work on the synthesis of central hybrid reactivators bearing 6-alkanyl-3-hydroxy-2-pyridinadoxime moiety, and with the goal to develop reactivator with greater lipophilicity and enhanced blood brain barrier (BBB)  permeability, we decided to substitute the 3-hydroxy group, initially designed to decrease the oxime pka, with a more electronegative and electron-withdrawing group such as fluorine. Fluorine is known to modulate the pka of the proximal oxime, the conformational bias and the binding properties via molecular interactions. This structural change, compared to the known 6-substituted 3-hydroxy-2-pyridinadoxime scaffold, appeared valuable for both practical and fundamental reasons, eventually providing reactivators with increased reactivation potency and better pharmacological profiles.

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 been proposed to overcome insecticide resistance and to develop promising environmental-friendly insecticides. The eight cysteine-targeted insecticides (succimides or maleinimides) were prepared via optimised synthetic route. The inhibitory activity of novel compounds and standards (paraoxon, bendiocarb and carbofuran) towards human acetylcholinesterase, human butyrylcholinesterase and mosquito acetylcholinesterase from Anopheles gambiae were determined using the modified spectrophotometric Ellman’s method. The potentiometric titration using acetylcholine as a substrate was used for validation of Ellman’s method. All data showed that the IC₅₀ values obtained from both methods were almost similar. Human butyrylcholinesterase was used as common off-target for acetylcholinesterase inhibitors, and no inhibitory effect was determined. The binding mode of the inhibitors was determined using the rapid dilution assay. Pyridinium maleimides were found with excellent efficacy towards mosquito acetylcholinesterase in contrast to the human enzyme and with significantly improved selectivity index compared to paraoxon. Despite some limitations, we believe that specific optimisation of the structure of molecule connected to maleimide moiety may lead to the development of novel promising insecticides. This work was supported by Ministry of Health of the Czech Republic (no. 16-34390A) and University of Defense (Long-term organization development plan Medical Aspect of Weapons of Mass Destruction).

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 of human enzyme, but also its atypical form and the horse enzyme. On the other hand, 2-PAM shows a certain degree of activation with both human enzymes, but inhibits the horse BChE in a classical competitive manner. To avoid substrate activation, the experiments were performed at around 50 micromolar starting substrate concentrations and were followed by its completion in the presence of different modulator(s) concentrations. Subsequently, the effect of 2-PAM on the phosphorylation by DFP was studied, since the bottom of the active site does not differ in these three enzymes. It seems that the distinctive action of activating agents on the wild type, the atypical human and horse BChE is a consequence of differences in the dynamics of the acylation loop at the active site entrance, rather then the composition of the enzyme’s peripheral anionic site.

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.

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 anionic site (CAS) as well as peripheral anionic site (PAS) regions of AChE [1]. Recent research has been focused on studying the association between the intracellular amyloid beta (Aβ) cascade and the dysfunction of subcellular organelles, especially mitochondria. Mitochondrial enzyme amyloid beta binding alcohol dehydrogenase (ABAD) might contribute to the neuronal dysfunction associated with AD by interacting with intracellular Aβ [2]. These derivatives embodying 7-MEOTA and donepezil moieties [3] could be effective in the treatment of AD with the respect of their ability to interact with the multiple targets. Within our contribution, synthesis, in vitro biological evaluation including cholinesterase inhibitory activity and effects on mitochondrial function of 7-MEOTA-donepezil series will be reported.

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 library [2] guided by structure-based and fragment hotspot strategies. The identified compounds were also screened for in vitro inhibition of AChE and three of them were observed to be active. Further experimental (kinetic) and computational (molecular dynamics) studies have been performed to verify the allosteric activity. Thus, new compounds have been developed as allosteric modulators that may be valuable pharmacological tools in the study of non-cholinergic functions of AChE.

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 AChE in complex with three different azobenzene derived inhibitors, we confirmed AzoTHA-1 as the only photoactive compound and we determined its structure in its cis- and trans- isomeric forms bound to AChE. Three-dimensional structures, supported by online UV-Vis spectroscopy and kinetic data, explain why only AzoTHA-1 is an effective photoactive AChE inhibitor and suggest possible ways to improve photoactive drugs. We utilised S/WAXS to follow photo-isomerisation induced-changes in the wide-angle scattering region to demonstrate that photoisomerisation of the inhibitor induces its release from AChE’s active site.

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 of this project is to develop a new class of more efficient human nerve agent-inhibited acetylcholinesterase. We designed, synthesized and evaluated a new class of bifunctional uncharged hybrid reactivators composed of a 3-hydroxypyridinaldoxime linked to a tacrine derivative. The in vitro efficacy of this reactivators has been assessed. We show that this new class of reactivators outperform HI-6 in restoring the human AChE activity inhibited by VX, sarin, tabun and paraoxon. By X-ray crystallography, we have been able to observe some of these new hybrids inside of the catalytic site of hAChE and TcAChE.

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 oxime which acts via direct nucleophilic attack on the phosphorus to achieve reactivation of the enzyme. These compounds have limited effectiveness because the charged portion of the molecule does not allow them to cross into the central nervous system where acetylcholinesterase inhibition is most harmful. The results of studies that characterized a small molecule reactivator (4-amino-2-((diethylamino)methyl)phenol [ADOC]) that does not incorporate an oxime but is capable of reactivating nerve agent-inhibited enzyme as well as or better than current treatments have been used to inform the design of additional novel compounds. This study describes the in vitro characterization of these novel compounds as reactivators of phosphonylated human acetylcholinesterase.

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 were docked to hBChE with flexible residues within the active site. After manual inspection, over twenty molecules were chosen. Such molecules were modified (e.g. addition of oxime moiety, pK_a optimization) and redocked to hBChE with flexible residues. Finally, two novel compounds were recommended for synthesis. The newly designed compounds will be further synthesized and evaluated on the model of OP-inhibited hBChE and hAChE. They could be used for development of new series of hBChE reactivators.

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 enzymes, and combinational strategies. Rational design of novel ChE-based catalytic bioscavengers requires a better understanding of chemical mechanisms of inhibition, aging of conjugate, and spontaneous reactivation. Kinetic studies, X-ray crystallography and molecular modeling, in particular QM/MM calculations, present valuable insights into specific reaction routes, role of specific amino acids and obstacles against effective reactivation of phosphylated ChEs. Introducing new functional groups surrounding the phosphylated serine should create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states, and intermediates. Direction of nucleophilic attack of water molecule on phosphorus atom may determine whether dephosphylation is favored over aging. Mutations of key residues surrounding human BChE active site, creating new reaction pathways, have been considered. QM/MM calculations suggest that introduction of a histidine, directing attack of water molecule from apical position competes with the aging reaction, while axial direction of water attack does not. Secondary mutations for stabilizing imidazolium upon activation of water molecule lead to lower energy barrier of reactivation reaction [1].

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 that resides at the end of β-strand 8.  Upon binding to the active site, substrates are further activated for hydrolysis by a direct interaction of the phosphoryl oxygen with the β-metal ion.  The chemical reaction is initiated via the direct attack of the bridging water/hydroxide at the phosphorus center of the substrate and proceeds with an inversion of stereochemistry.  Wild-type PTE is stereoselective for the hydrolysis of chiral substrates.  However, the catalytic preferences for the hydrolysis of chiral substrates can be enhanced, relaxed, or inverted by selective mutation of key residues in the active site that dictate the size and shape of the substrate-binding cavity.  The extreme toxicity and persistence of the G-type (sarin and soman) and V-type (VX and VR) organophosphorus nerve agents makes the detoxification of these compounds of significant interest.  A rational and random mutagenesis strategy has been developed and implemented for the evolution of mutant forms of PTE that are more fully optimized for the catalytic destruction and detoxification of the most toxic organophosphorus nerve agents.

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.