Structural features of thyroglobulin linked to protein trafficking.

Thyroglobulin must pass endoplasmic reticulum (ER) quality control to become secreted for thyroid hormone synthesis. Defective thyroglobulin, blocked in trafficking, can cause hypothyroidism. Thyroglobulin is a large protein (~2750 residues) spanning regions I-II-III plus a C-terminal cholinesterase-like domain. The cholinesterase-like domain functions as an intramolecular chaperone for regions I-II-III, but the folding pathway leading to successful thyroglobulin trafficking remains largely unknown. Here, informed by the recent three-dimensional structure of thyroglobulin as determined by cryo-electron microscopy, we have bioengineered three novel classes of mutants yielding three entirely distinct quality control phenotypes. Specifically, upon expressing recombinant thyroglobulin, we find that first, mutations eliminating a disulfide bond enclosing a 200-amino acid loop in region I have surprisingly little impact on the ability of thyroglobulin to fold to a secretion-competent state. Next, we have identified a mutation on the surface of the cholinesterase-like domain that has no discernible effect on regional folding yet affects contact between distinct regions and thereby triggers impairment in the trafficking of full-length thyroglobulin. Finally, we have probed a conserved disulfide in the cholinesterase-like domain that interferes dramatically with local folding, and this defect then impacts on global folding, blocking the entire thyroglobulin in the ER. These data highlight variants with distinct effects on ER quality control, inhibiting domain-specific folding; folding via regional contact; neither; or both.

Steady-state kinetic analysis of human cholinesterases over wide concentration ranges of competing substrates.

Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate. Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate. For both enzymes, progress curves for hydrolysis of PhA at very low concentration (≪Km) in the presence of increasing concentration of ATC showed that: a) the competing substrate and the reporter substrate are hydrolyzed at the same time, b) complete hydrolysis of PhA cannot be reached above 1 mM competing substrate. This likely results from accumulation of hydrolysis products (P) of competing substrate and/or accumulation of acetylated enzyme·P complex that inhibit hydrolysis of the reporter substrate.

Effect of Morus alba leaf extract dose on lipid oxidation, microbiological stability, and sensory evaluation of functional liver pâtés during refrigerated storage.

Mulberry (Morus alba L.), and above all the extract from the leaves of this plant, is a natural medicine that has been used in traditional medicine for hundreds of years. Mulberry leaves contains polyphenol compounds: flavonoids, coumarins, numerous phenolic acids, as well as terpenes and steroids. The antioxidant effect of these compounds may be beneficial to the fat fraction of meat products, thereby increasing their functional qualities. The aim of the study was to evaluate the effectiveness of the use of mulberry water leaf extract, as an additive limiting adverse fat changes and affecting the functionality in model liver pâtés. Pork pâtés were prepared by replacing 20% of animal fat with rapeseed oil (RO), and water extract of mulberry leaves was added in the proportion of 0.2%, 0.6% and 1.0%. It has been shown that the addition of mulberry leaf extract delayed the appearance of primary and secondary fat oxidation products. The most effective antioxidant effect during 15-day storage was observed in the sample with the addition of 0.6% and 1.0% water mulberry leaf extract. These samples also showed inhibiting activity against angiotensin-converting enzymes and cholinesterase's. During storage, the tested pâtés had a high sensory quality with unchanged microbiological quality. Mulberry leaf extract can be an interesting addition to the production of fat meat products, delaying adverse changes in the lipid fraction and increasing the functionality of products.

Histamine H3 receptor and cholinesterases as synergistic targets for cognitive decline: Strategies to the rational design of multitarget ligands.

The role of histamine and acetylcholine in cognitive functions suggests that compounds able to increase both histaminergic and cholinergic neurotransmissions in the brain should be considered as promising therapeutic options. For this purpose, dual inhibitors of histamine H3 receptors (H3 R) and cholinesterases (ChEs) have been designed and assessed. In this context, this paper reviews the strategies used to obtain dual H3 R/ChEs ligands using multitarget design approaches. Hybrid compounds designed by linking tacrine or flavonoid motifs to H3 R antagonists were obtained with high affinity for both targets, and compounds designed by merging the H3 R antagonist pharmacophore with known anticholinesterase molecules were also reported. These reports strongly suggest that key modifications in the lipophilic region (including a second basic group) seem to be a strategy to reach novel compounds, allied with longer linker groups to a basic region. Some compounds have already demonstrated efficacy in memory models, although the pharmacokinetic and toxicity profile should be considered when designing further compounds. In conclusion, the key features to be considered when designing novel H3 R/ChEs inhibitors with improved pharmacological profile were herein summarized.

A Comprehensive Review of Cholinesterase Modeling and Simulation.

The present article reviews published efforts to study acetylcholinesterase and butyrylcholinesterase structure and function using computer-based modeling and simulation techniques. Structures and models of both enzymes from various organisms, including rays, mice, and humans, are discussed to highlight key structural similarities in the active site gorges of the two enzymes, such as flexibility, binding site location, and function, as well as differences, such as gorge volume and binding site residue composition. Catalytic studies are also described, with an emphasis on the mechanism of acetylcholine hydrolysis by each enzyme and novel mutants that increase catalytic efficiency. The inhibitory activities of myriad compounds have been computationally assessed, primarily through Monte Carlo-based docking calculations and molecular dynamics simulations. Pharmaceutical compounds examined herein include FDA-approved therapeutics and their derivatives, as well as several other prescription drug derivatives. Cholinesterase interactions with both narcotics and organophosphate compounds are discussed, with the latter focusing primarily on molecular recognition studies of potential therapeutic value and on improving our understanding of the reactivation of cholinesterases that are bound to toxins. This review also explores the inhibitory properties of several other organic and biological moieties, as well as advancements in virtual screening methodologies with respect to these enzymes.

Structure Elucidation and Cholinesterase Inhibition Activity of Two New Minor Amaryllidaceae Alkaloids.

Two new minor Amaryllidaceae alkaloids were isolated from Hippeastrum × hybridum cv. Ferrari and Narcissus pseudonarcissus cv. Carlton. The chemical structures were identified by various spectroscopic (one- and two-dimensional (1D and 2D) NMR, circular dichroism (CD), high-resolution mass spectrometry (HRMS) and by comparison with literature data of similar compounds. Both isolated alkaloids were screened for their human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE) inhibition activity. One of the new compounds, a heterodimer alkaloid of narcikachnine-type, named narciabduliine (2), showed balanced inhibition potency for both studied enzymes, with IC50 values of 3.29 ± 0.73 µM for hAChE and 3.44 ± 0.02 µM for hBuChE. The accommodation of 2 into the active sites of respective enzymes was predicted using molecular modeling simulation.

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions.

Organophosphorus compounds (OPCs) are able to interact with various biological targets in living organisms, including enzymes. The binding of OPCs to enzymes does not always lead to negative consequences for the body itself, since there are a lot of natural biocatalysts that can catalyze the chemical transformations of the OPCs via hydrolysis or oxidation/reduction and thereby provide their detoxification. Some of these enzymes, their structural differences and identity, mechanisms, and specificity of catalytic action are discussed in this work, including results of computational modeling. Phylogenetic analysis of these diverse enzymes was specially realized for this review to emphasize a great area for future development(s) and applications.

Identification, characterization and expression analyses of cholinesterases genes in Yesso scallop (Patinopecten yessoensis) reveal molecular function allocation in responses to ocean acidification.

Cholinesterases are key enzymes in central and peripheral cholinergic nerve system functioning on nerve impulse transmission in animals. Though cholinesterases have been identified in most vertebrates, the knowledge about the variable numbers and multiple functions of the genes is still quite meagre in invertebrates, especially in scallops. In this study, the complete cholinesterase (ChE) family members have been systematically characterized in Yesso scallop (Patinopecten yessoensis) via whole-genome scanning through in silico analysis. Ten ChE family members in the genome of Yesso scallop (designated PyChEs) were identified and potentially acted to be the largest number of ChE in the reported species to date. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of these genes. The expression profiles of PyChEs were determined in all developmental stages, in healthy adult tissues, and in mantles under low pH stress (pH 6.5 and 7.5). Spatiotemporal expression suggested the ubiquitous functional roles of PyChEs in all stages of development, as well as general and tissue-specific functions in scallop tissues. Regulation expressions revealed diverse up- and down-regulated expression patterns at most time points, suggesting different functional specialization of gene superfamily members in response to ocean acidification (OA). Evidences in gene number, phylogenetic relationships and expression patterns of PyChEs revealed that functional innovations and differentiations after gene duplication may result in altered functional constraints among PyChEs gene clusters. Collectively, our results provide the potential clues that the selection pressures coming from the environment were the potential inducement leading to function allocation of ChE family members in scallop.

The synthesis and cholinesterase inhibitory activities of solasodine analogues with seven-membered F ring.

Solasodine analogues containing a seven-membered F ring with a nitrogen atom placed at position 22a were prepared from diosgenin or tigogenin in a four-step synthesis comprising of the simultaneous opening of the F-ring and introduction of cyanide in position 22α, activation of the 26-hydroxyl group as mesylate, nitrile reduction, and N-cyclization. Solasodine, six obtained 22a(N)-homo analogues, as well as four 26a-homosolasodine derivatives and their open-chain precursors (13 in total) were tested as potential inhibitors of acetyl- and butyryl-cholinesterases and showed activity at micromolar concentrations. The structure-activity relationship study revealed that activities against studied esterases are affected by the structure of E/F rings and the substitution pattern of ring A. The most potent compound 8 acted as non-competitive inhibitors and exerted IC50 = 8.51 µM and 7.05 µM for eeAChE and eqBChE, respectively. Molecular docking studies revealed the hydrogen bond interaction of 8 with S293 of AChE; further rings are stabilized via hydrophobic interaction (ring A) or interaction with Y341 and W286 (rings B and C). Biological experiments showed no neurotoxicity of differentiated SH-SY5Y cells. More importantly, results from neuroprotective assay based on glutamate-induced cytotoxicity revealed that most derivatives had the ability to increase the viability of differentiated SH-SY5Y cells in comparison to galantamine and lipoic acid assayed as standards. The newly synthesized solasodine analogues are able to inhibit and to bind cholinesterases in noncompetitive mode of inhibition and exhibited neuroprotection potential of differentiated neuroblastoma cells after Glu-induced toxicity.

Comparative mapping of selected structural determinants on the extracellular domains of cholinesterase-like cell-adhesion molecules.

Cell adhesion generally involves formation of homophilic or heterophilic protein complexes between two cells to form transcellular junctions. Neural cell-adhesion members of the α/ß-hydrolase fold superfamily of proteins use their extracellular or soluble cholinesterase-like domain to bind cognate partners across cell membranes, as illustrated by the neuroligins. These cell-adhesion molecules currently comprise the synaptic organizers neuroligins found in all animal phyla, along with three proteins found only in invertebrates: the guidance molecule neurotactin, the glia-specific gliotactin, and the basement membrane protein glutactin. Although these proteins share a cholinesterase-like fold, they lack one or more residues composing the catalytic triad responsible for the enzymatic activity of the cholinesterases. Conversely, they are found in various subcellular localisations and display specific disulfide bonding and N-glycosylation patterns, along with individual surface determinants possibly associated with recognition and binding of protein partners. Formation of non-covalent dimers typical of the cholinesterases is documented for mammalian neuroligins, yet whether invertebrate neuroligins and their neurotactin, gliotactin and glutactin relatives also form dimers in physiological conditions is unknown. Here we provide a brief overview of the localization, function, evolution, and conserved versus individual structural determinants of these cholinesterase-like cell-adhesion proteins. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Cryo-electron microscopy of cholinesterases, present and future.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) exist in a variety of oligomeric forms, each with defined cellular and subcellular distributions. Although crystal structures of AChE and BChE have been available for many years, structures of the physiologically relevant ChE tetramer were only recently solved by cryo-electron microscopy (cryo-EM) single-particle analysis. Here, we briefly review how these structures contribute to our understanding of cholinesterase oligomerization, highlighting the advantages of using cryo-EM to resolve structures of protein assemblies that cannot be expressed recombinantly. We argue that the next frontier in cholinesterase structural biology is to image membrane-anchored ChE oligomers directly in their native environment-the cell.

Synthesis of Hantzsch Adducts as Cholinesterases and Calcium Flux inhibitors, Antioxidants and Neuroprotectives.

We report herein the design, synthesis, biological evaluation, and molecular modelling of new inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), able to block Ca+2 channels also showing antioxidant and neuroprotective activities. The new MTDL, dialkyl 2,6-dimethyl-4-(4-((5-aminoalkyl)oxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate 3a-p, have been obtained via Hantzsch reaction from appropriate and commercially available precursors. Pertinent biological analysis has prompted us to identify MTDL 3h [dimethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate] as an attractive inhibitor of AChE (1.8 µM) and BuChE (2 µM), Ca+2 channel antagonist (47.72% at 10 µM), and antioxidant (2.54 TE) agent, showing significant neuroprotection 28.68% and 38.29% against H2O2, and O/R, respectively, at 0.3 µM, thus being considered a hit-compound for further investigation in our search for anti-Alzheimer's disease agents.

Computational studies on cholinesterases: Strengthening our understanding of the integration of structure, dynamics and function.

Computational approaches have proved valuable in elucidating structure/function relationships in the cholinesterases in the context of their unusual three-dimensional structure. In this review we survey several recent studies that have enhanced our understanding of how these enzymes function, and have utilized computational approaches both to modulate their activity and to improve the design of lead compounds for their inhibition. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Neuropharmacology:2.

New Tetrahydroacridine Hybrids with Dichlorobenzoic Acid Moiety Demonstrating Multifunctional Potential for the Treatment of Alzheimer's Disease.

A series of new tetrahydroacridine and 3,5-dichlorobenzoic acid hybrids with different spacers were designed, synthesized, and evaluated for their ability to inhibit both cholinesterase enzymes. Compounds 3a, 3b, 3f, and 3g exhibited selective butyrylcholinesterase (EqBuChE) inhibition with IC50 values ranging from 24 to 607 nM. Among them, compound 3b was the most active (IC50 = 24 nM). Additionally, 3c (IC50 for EeAChE = 25 nM and IC50 for EqBuChE = 123 nM) displayed dual cholinesterase inhibitory activity and was the most active compound against acetylcholinesterase (AChE). Active compound 3c was also tested for the ability to inhibit Aß aggregation. Theoretical physicochemical properties of the compounds were calculated using ACD Labs Percepta and Chemaxon. A Lineweaver-Burk plot and docking study showed that 3c targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Moreover, 3c appears to possess neuroprotective activity and could be considered a free-radical scavenger. In addition, 3c did not cause DNA damage and was found to be less toxic than tacrine after oral administration; it also demonstrated little inhibitory activity towards hyaluronidase (HYAL), which may indicate that it possesses anti-inflammatory properties. The screening for new in vivo interactions between 3c and known receptors was realized by yeast three-hybrid technology (Y3H).

Effect of food contamination and collection material in the measurement of biomarkers in saliva of horses.

This study aims to evaluate the effect of the presence of food and the material used in a panel of biomarkers in saliva of horses. For the food effect study, clean saliva was incubated with a known amount of food consisting of oats, hay or grass. Significant changes were observed when saliva was incubated with oats for total protein (P = .050) and phosphorus (P = .008), with grass for total protein (P = .037), salivary alpha-amylase (sAA, P = .018), total esterase (TEA, P = .018), butyrilcholinesterase (BChE, P = .037), adenosine deaminase (ADA, P = .037), and total bilirubin (P = .018), and with hay for sAA (P = .018), phosphorus (P = .037), γ-glutamyl transferase (gGT, P = .004), and creatine kinase (CK, P = .016). For the material-based collection study, saliva using a sponge and a cotton role at the same time were collected and compared. Lower values were obtained in clean saliva collected with cotton role compared to sponge for sAA (P = .030), TEA (P = .034), BChE (P = .003), gGT (P = .002) and cortisol (P < .001) In conclusion, the presence of food and the material used for its collection, can influence the results obtained when analytes are measured in saliva of horses.

Inhibitors of Cholinesterases in Pharmacology: the Current Trends.

Inhibitors of cholinesterases are a wide group of low molecular weight compounds with a significant role in the current pharmacology. Besides the pharmacological importance, they are also known as toxic compounds like military nerve agents. In the pharmacology, drugs for Alzheimer disease, myasthenia gravis and prophylaxis of poisoning by nerve agents can be mentioned as the relevant applications. Besides this, anti-inflammation and antiphrastic drugs are other pharmacological applications of these inhibitors. This review is focused on a survey of cholinesterase inhibitors with known or expected pharmacological impact and indications of their use. Recent literature with comments is provided here as well.

Propargylamine-derived multi-target directed ligands for Alzheimer's disease therapy.

Current options for the treatment of Alzheimers disease have been restricted to prescription of acetylcholinesterase inhibitors or N-methyl-d-aspartate receptor antagonist, memantine. Propargylamine-derived multi-target directed ligands, such as ladostigil, M30, ASS234 and contilisant, involve different pathways. Apart from acting as inhibitors of both cholinesterases and monoamine oxidases, they show improvement of cognitive impairment, antioxidant activities, enhancement of iron-chelating activities, protect against tau hyperphosphorylation, block metal-associated oxidative stress, regulate APP and Aß expression processing by the non-amyloidogenic α-secretase pathway, suppress mitochondrial permeability transition pore opening, and coordinate protein kinase C signaling and Bcl-2 family proteins. Other hybrid propargylamine derivatives are also reported.

Exploring Biological Activity of 4-Oxo-4H-furo[2,3-h]chromene Derivatives as Potential Multi-Target-Directed Ligands Inhibiting Cholinesterases, ß-Secretase, Cyclooxygenase-2, and Lipoxygenase-5/15.

A series of 5-oxo-5H-furo[3,2-g]chromene-6-carbaldehydes and their hydrazone derivatives were evaluated as potential multi-target-directed ligands in vitro against cholinesterases, ß-secretase, cyclooxygenase-2, and lipoxygenase-15 (LOX-15), as well as for free radical-scavenging activities. The most active compounds against LOX-15 were also evaluated for activity against the human lipoxygenase-5 (LOX-5). Kinetic studies against AChE, BChE, and ß-secretase (BACE-1) were performed on 2-(3-fluorophenyl)- (3b) and 2-(4-chlorophenyl)-6-[(4-trifluoromethylphenyl)hydrazonomethyl]furo[3,2-h]chromen-5-one (3e) complemented with molecular docking (in silico) to determine plausible protein-ligand interactions on a molecular level. The docking studies revealed hydrogen and/or halogen bonding interactions between the strong electron-withdrawing fluorine atoms of the trifluoromethyl group with several residues of the enzyme targets, which are probably responsible for the observed increased biological activity of these hydrazone derivatives. The two compounds were found to moderately inhibit COX-2 and lipoxygenases (LOX-5 and LOX-15). Compounds 3b and 3e were also evaluated for cytotoxicity against the breast cancer MCF-7 cell line and Hek293-T cells.

Natural Monoterpenes: Much More than Only a Scent.

Terpenes are a widespread group of secondary metabolites that can be found in various family plants such as the Lamiaceae. In view of their numerous valuable biological activities, the industrial production of concrete terpenes and essential oils rich in the substances is intensively studied. Monoterpenes constitute a significant part of the whole group of the aforementioned secondary metabolites. This is due to their numerous biological activities and their ability to permeate the skin. Despite the fact that these substances have gain popularity, they are not comprehensively characterized. The presented review is based on studies of the biological activities of the most important monoterpenes and the essential oils rich in these compounds. The authors focused attention on antioxidant activity, inhibition towards acetyl- and butyrylcholinesterase, and α-amylase and α-glucosidase, antifungal, hepatoprotective, sedative properties, and their skin permeation enhancement.

Critical evaluation of current Alzheimer's drug discovery (2018-19) & futuristic Alzheimer drug model approach.

Alzheimer's disease (AD), a neurodegenerative disease responsible for death of millions of people worldwide is a progressive clinical disorder which causes neurons to degenerate and ultimately die. It is one of the common causes of dementia wherein a person's incapability to independently think, behave and decline in social skills can be quoted as major symptoms. However the early signs include the simple non-clinical symptoms such as forgetting recent events and conversations. Onset of these symptoms leads to worsened conditions wherein the AD patient suffers severe memory impairment and eventually becomes unable to work out everyday tasks. Even though there is no complete cure for AD, rigorous research has been going on to reduce the progress of AD. Currently, a very few clinical drugs are prevailing for AD treatment. So this is the need of hour to design, develop and discovery of novel anti-AD drugs. The main factors for the cause of AD according to scientific research reveals structural changes in brain proteins such as beta amyloid, tau proteins into plaques and tangles respectively. The abnormal proteins distort the neurons. Despite the high potencies of the synthesized molecules; they could not get on the clinical tests up to human usage. In this review article, the recent research carried out with respect to inhibition of AChE, BuChE, NO, BACE1, MAOs, Aß, H3R, DAPK, CSF1R, 5-HT4R, PDE, σ1R and GSK-3ß is compiled and organized. The summary is focused mainly on cholinesterases, Aß, BACE1 and MAOs classes of potential inhibitors. The review also covers structure activity relationship of most potent compounds of each class of inhibitors alongside redesign and remodeling of the most significant inhibitors in order to expect cutting edge inhibitory properties towards AD. Alongside the molecular docking studies of the some final compounds are discussed.