Can blood morphology, oxidative stress, and cholinesterase activity determine health status of pigeon Columba livia f. urbana?

Environmental studies in Northern Poland are example of the functioning of ecophysiological relationships under anthropogenic impact. The aim of our studies was to investigate sex-dependent effects on the alterations in the concentration of chemical elements in soil samples collected from habitats of feral pigeon Columba livia f. urbana from Northern Poland, as well as feathers, biomarkers of oxidative stress, antioxidant defense, and total cholinesterase activity in tissues (liver, kidney, brain). Concentration of Si, Zn, and Pb in feathers of pigeons was significant. The levels of Si and Zn were higher in feathers of females from non-polluted, while higher Pb levels were found only in females from polluted areas (p = 0.000). This was confirmed by MANOVA of biomarkers of antioxidant defense, elements concentration, and revealing the order of effects: tissue type > environment > sex. Erythrocytes of males living in polluted areas were more fragile to hemolytic agents resulting in a higher percentage of hemolyzed erythrocytes. The effects of polluted environment on the level of carbonyl derivatives of oxidatively modified proteins compared to the effects of sex were more pronounced in the case of kidney (p = 0.000) and hepatic tissues (p = 0.000). Polluted areas were associated with significant increase in SOD activity in the brain and hepatic tissues of pigeons (p = 0.000). Health status of feral pigeons is significantly different in conditions of environmental destabilization.

Design, synthesis, and pharmacological evaluation of indole-piperidine amides as Blood-brain barrier permeable dual cholinesterase and ß-secretase inhibitors.

Heterocyclic compounds play a crucial role in the discovery of therapeutics. Alzheimer's disease (AD) is an unfathomable sporadic neurodegenerative disorder that involves multiple pathological pathways. The failure of current single-target small molecules to address AD's underlying causes has prompted interest in discovering multi-target directed ligands (MTDLs) to slow down the disease's progression. Herein we report the synthesis and biological evaluation of indole-piperidine amides as MTDLs for AD. The 5,6-dimethoxy-indole N-(2-(1-benzylpiperidine) carboxamide (23a) inhibits hAChE and hBACE-1 with IC50 values of 0.32 and 0.39 µM, respectively. The MTDL 23a is a mixed-type inhibitor of both hAChE and hBACE-1 with Ki values of 0.26 µM and 0.46 µM, respectively. The MD simulation studies revealed that both AChE and BACE-1 experience minor conformational changes on binding with 23a. In the PAMPA-BBB assay, analog 23a demonstrated CNS permeability, indicating the possibility for future investigation in preclinical models of AD.

In vitro and in vivo Biological Evaluation of Newly Tacrine-Selegiline Hybrids as Multi-Target Inhibitors of Cholinesterases and Monoamine Oxidases for Alzheimer's Disease.

Purpose: Alzheimer's disease (AD) is the most common neurodegenerative disease, and its multifactorial nature increases the difficulty of medical research. To explore an effective treatment for AD, a series of novel tacrine-selegiline hybrids with ChEs and MAOs inhibitory activities were designed and synthesized as multifunctional drugs. Methods: All designed compounds were evaluated in vitro for their inhibition of cholinesterases (AChE/BuChE) and monoamine oxidases (MAO-A/B) along with their blood-brain barrier permeability. Then, further biological activities of the optimizing compound 7d were determined, including molecular model analysis, in vitro cytotoxicity, acute toxicity studies in vivo, and pharmacokinetic and pharmacodynamic property studies in vivo. Results: Most synthesized compounds demonstrated potent inhibitory activity against ChEs/MAOs. Particularly, compound 7d exhibited good and well-balanced activity against ChEs (hAChE: IC50 = 1.57 µM, hBuChE: IC50 = 0.43 µM) and MAOs (hMAO-A: IC50 = 2.30 µM, hMAO-B: IC50 = 4.75 µM). Molecular modeling analysis demonstrated that 7d could interact simultaneously with both the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE in a mixed-type manner and also exhibits binding affinity towards BuChE and MAO-B. Additionally, 7d displayed excellent permeability of the blood-brain barrier, and under the experimental conditions, it elicited low or no toxicity toward PC12 and BV-2 cells. Furthermore, 7d was not acutely toxic in mice at doses up to 2500 mg/kg and could improve the cognitive function of mice with scopolamine-induced memory impairment. Lastly, 7d possessed well pharmacokinetic characteristics. Conclusion: In light of these results, it is clear that 7d could potentially serve as a promising multi-functional drug for the treatment of AD.

A method for the efficient evaluation of substrate-based cholinesterase imaging probes for Alzheimer's disease.

Cholinesterase (ChE) enzymes have been identified as diagnostic markers for Alzheimer disease (AD). Substrate-based probes have been synthesised to detect ChEs but they have not detected changes in ChE distribution associated with AD pathology. Probes are typically screened using spectrophotometric methods with pure enzyme for specificity and kinetics. However, the biochemical properties of ChEs associated with AD pathology are altered. The present work was undertaken to determine whether the Karnovsky-Roots (KR) histochemical method could be used to evaluate probes at the site of pathology. Thirty thioesters and esters were synthesised and evaluated using enzyme kinetic and KR methods. Spectrophotometric methods demonstrated all thioesters were ChE substrates, yet only a few provided staining in the brain with the KR method. Esters were ChE substrates with interactions with brain ChEs. These results suggest that the KR method may provide an efficient means to screen compounds as probes for imaging AD-associated ChEs.

Chemo- and bio-informatics insight into anti-cholinesterase potentials of berries and leaves of Myrtus communis L., Myrtaceae: an in vitro/in silico study.

BACKGROUND: Myrtus communis L. (MC) has been used in Mesopotamian medicine. Here, the cholinesterase (ChE) inhibitory potential of its methyl alcohol extracts has been investigated and computationally dissected. METHOD: The ChE inhibition has been measured based on usual Ellman's colorimetric method compared to a canonical ChE inhibitor, eserine. Through a deep text mining, the structures of phytocompounds (= ligands) of MC were curated from ChemSpider, PubChem, and ZINC databases and docked into protein targets, AChE (PDB 1EVE) and BChE (PDB 1P0I) after initial in silico preparedness and binding affinity (BA; kcal/mol) reported as an endpoint. The calculation of ADMET (absorption, distribution, metabolism, excretion, and toxicity) features of phytocompounds were retrieved from SwissADME ( http://www.swissadme.ch/ ) and admetSAR software to predict the drug-likeness or lead-likeness fitness. The Toxtree v2.5.1, software platforms ( http://toxtree.sourceforge.net/ ) have been used to predict the class of toxicity of phytocompounds. The STITCH platform ( http://stitch.embl.de ) has been employed to predict ChE-chemicals interactions. RESULTS: The possible inhibitory activities of AChE of extracts of leaves and berries were 37.33 and 70.00%, respectively as compared to that of eserine while inhibitory BChE activities of extracts of leaves and berries of MC were 19.00 and 50.67%, respectively as compared to that of eserine. Phytochemicals of MC had BA towards AChE ranging from -7.1 (carvacrol) to -9.9 (ellagic acid) kcal/mol. In this regard, alpha-bulnesene, (Z)-gamma-Bisabolene, and beta-bourbonene were top-listed low toxic binders of AChE, and (Z)-gamma-bisabolene was a more specific AChE binder. Alpha-cadinol, estragole, humulene epoxide II, (a)esculin, ellagic acid, patuletin, juniper camphor, linalyl anthranilate, and spathulenol were high class (Class III) toxic substances which among others, patuletin and alpha-cadinol were more specific AChE binders. Among intermediate class (Class II) toxic substances, beta-chamigrene was a more specific AChE binder while semimyrtucommulone and myrtucommulone A were more specific BChE binders. CONCLUSION: In sum, the AChE binders derived from MC were categorized mostly as antiinsectants (e.g., patuletin and alpha-cardinal) due to their predicted toxic classes. It seems that structural amendment and stereoselective synthesis like adding sulphonate or sulphamate groups to these phytocompounds may make them more suitable candidates for considering in preclinical investigations of Alzheimer's disease.

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.

The ESTHER database on alpha/beta hydrolase fold proteins - An overview of recent developments.

The ESTHER database, dedicated to ESTerases and alpha/beta-Hydrolase Enzymes and Relatives (https://bioweb.supagro.inra.fr/ESTHER/general?what=index), offers online access to a continuously updated, sequence-based classification of proteins harboring the alpha/beta hydrolase fold into families and subfamilies. In particular, the database proposes links to the sequences, structures, ligands and huge diversity of functions of these proteins, and to the related literature and other databases. Taking advantage of the promiscuity of enzymatic function, many engineered esterases, lipases, epoxide-hydrolases, haloalkane dehalogenases are used for biotechnological applications. Finding means for detoxifying those protein members that are targeted by insecticides, herbicides, antibiotics, or for reactivating human cholinesterases when inhibited by nerve gas, are still active areas of research. Using or improving the capacity of some enzymes to breakdown plastics with the aim to recycle valuable material and reduce waste is an emerging challenge. Most hydrolases in the superfamily are water-soluble and act on or are inhibited by small organic compounds, yet in a few subfamilies some members interact with other, unrelated proteins to modulate activity or trigger functional partnerships. Recent development in 3D structure prediction brought by AI-based programs now permits analysis of enzymatic mechanisms for a variety of hydrolases with no experimental 3D structure available. Finally, mutations in as many as 34 of the 120 human genes compiled in the database are now linked to genetic diseases, a feature fueling research on early detection, metabolic pathways, pharmacological treatment or enzyme replacement therapy. Here we review those developments in the database that took place over the latest decade and discuss potential new applications and recent and future expected research in the field.

Two residues determine nicotinic acetylcholine receptor requirement for RIC-3.

Nicotinic acetylcholine receptors (N-AChRs) mediate fast synaptic signaling and are members of the pentameric ligand-gated ion channel (pLGIC) family. They rely on a network of accessory proteins in vivo for correct formation and transport to the cell surface. Resistance to cholinesterase 3 (RIC-3) is an endoplasmic reticulum protein that physically interacts with nascent pLGIC subunits and promotes their oligomerization. It is not known why some N-AChRs require RIC-3 in heterologous expression systems, whereas others do not. Previously we reported that the ACR-16 N-AChR from the parasitic nematode Dracunculus medinensis does not require RIC-3 in Xenopus laevis oocytes. This is unusual because all other nematode ACR-16, like the closely related Ascaris suum ACR-16, require RIC-3. Their high sequence similarity limits the number of amino acids that may be responsible, and the goal of this study was to identify them. A series of chimeras and point mutations between A. suum and D. medinensis ACR-16, followed by functional characterization with electrophysiology, identified two residues that account for a majority of the receptor requirement for RIC-3. ACR-16 with R/K159 in the cys-loop and I504 in the C-terminal tail did not require RIC-3 for functional expression. Mutating either of these to R/K159E or I504T, residues found in other nematode ACR-16, conferred a RIC-3 requirement. Our results agree with previous studies showing that these regions interact and are involved in receptor synthesis. Although it is currently unclear what precise mechanism they regulate, these residues may be critical during specific subunit folding and/or assembly cascades that RIC-3 may promote.

Exploring the Potential of Sulfonamide-Dihydropyridine Hybrids as Multitargeted Ligands for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease that has a heavy social and economic impact on all societies and for which there is still no cure. Multitarget-directed ligands (MTDLs) seem to be a promising therapeutic strategy for finding an effective treatment for this disease. For this purpose, new MTDLs were designed and synthesized in three steps by simple and cost-efficient procedures targeting calcium channel blockade, cholinesterase inhibition, and antioxidant activity. The biological and physicochemical results collected in this study allowed us the identification two sulfonamide-dihydropyridine hybrids showing simultaneous cholinesterase inhibition, calcium channel blockade, antioxidant capacity and Nrf2-ARE activating effect, that deserve to be further investigated for AD therapy.

Selected herbicides screened for toxicity and analysed as inhibitors of both cholinesterases.

Sets of 346 herbicides in use and 163 no longer in use were collected from open access online sources and compared in silico with cholinesterases inhibitors (ChI) and drugs in terms of physicochemical profile and estimated toxic effects on human health. The screening revealed at least one potential adverse consequence for each herbicide class assigned according to their mode of action on weeds. The classes with most toxic warnings were K1, K3/N, F1 and E. The selection of 11 commercial herbicides for in vitro biological tests on human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the enzymes involved in neurotoxicity and detoxification of various xenobiotics, respectively, was based mainly on the structural similarity with inhibitors of cholinesterases. Organophosphate anilofos and oxyacetanilide flufenacet were the most potent inhibitors of AChE (25 µM) and BChE (6.4 µM), respectively. Glyphosate, oxadiazon, tembotrione and terbuthylazine were poor inhibitors with an estimated IC50 above 100 µM, while for glyphosate the IC50 was above 1 mM. Generally, all of the selected herbicides inhibited with a slight preference towards BChE. Cytotoxicity assays showed that anilofos, bensulide, butamifos, piperophos and oxadiazon were cytotoxic for hepatocytes (HepG2) and neuroblastoma cell line (SH-SY5Y). Time-independent cytotoxicity accompanied with induction of reactive oxygen species indicated rapid cell death in few hours. Our results based on in silico and in vitro analyses give insight into the potential toxic outcome of herbicides in use and can be applied in the design of new molecules with a less impact on humans and the environment.

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (Acari: Ixodidae).

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens.

Small Molecular Fluorescent Probes for Alzheimer's Disease Associated Active Species.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the main cause of dementia worldwide. As the pathogenesis of AD is quite complicated, there is continuous attention to AD-associated active species, such as amyloid-ß plaques, neurofibrillary tangles, metal ions, reactive oxygen/nitrogen/sulphur species, cholinesterase, viscosity, formaldehyde and so on. To this end, a series of small molecular fluorescent probes for these active species have been explored for early diagnosis and even remedy of AD. Herein, we systematacially summarize the versatile fluorescent probes mainly in recent three years, including the relationship between the structure and properties as well as the targeted diagnosis and imaging application of all these fluorescent probes. Moreover, the challenges and perspectives of the AD-related fluorescent probes are briefly explicated. We firmly expect this review may provide guidance for constructing new AD-relevant fluorescent probes and promote the clinical study of AD.

A meta-analysis of normal human blood cholinesterase activities determined by a modified electrometric method.

Measurements of blood cholinesterase (ChE) activities, including those of erythrocytes (EChE), plasma or serum (PChE), and whole blood (WBChE), are used to assess exposure to ChE-inhibiting pesticides. The purpose of this review was to report normal reference ChE activities in the blood of healthy adult humans, as determined by a modified electrometric method. We performed a systematic review following PRISMA guidelines. Single-group meta-analysis of means of PChE, EChE, and WBChE activities of adult healthy subjects was conducted using the random effects model. The programs used were Open-Meta Analyst and Meta-Essentials Version 1.5. Studies selected for analysis comprised 21, 19, and 4 reports of normal reference/baseline PChE, EChE, and WBChE activities in 690, 635, and 121 healthy adult males and/or females, respectively. The meta-analysis showed normal reference values of the means (effect sizes) with 95% CI of PChE, EChE, and WBChE activities of healthy adult subjects, which were 1.078 (1.015, 1.142), 1.075 (1.024, 1.125) and 1.331 (1.226, 1.436), respectively. By the subgroup analysis, heterogeneity (I2>89%) was considerably reduced in females to 4.4% and 30.1% for PChE and EChE, respectively. Funnel plots indicated no publication bias. However, Egger's regression confirmed the symmetry of the data points for PChE and WBChE activities with a significant effect on EChE. This meta-analysis showed normal reference values of PChE, EChE, and WBChE activities, measured by a modified electrometric method, in healthy adult humans.

Ultrasound-Assisted Synthesis of Piperidinyl-Quinoline Acylhydrazones as New Anti-Alzheimer's Agents: Assessment of Cholinesterase Inhibitory Profile, Molecular Docking Analysis, and Drug-like Properties.

Alzheimer's disease (AD) is one of the progressive neurological disorders and the main cause of dementia all over the world. The multifactorial nature of Alzheimer's disease is a reason for the lack of effective drugs as well as a basis for the development of new structural leads. In addition, the appalling side effects such as nausea, vomiting, loss of appetite, muscle cramps, and headaches associated with the marketed treatment modalities and many failed clinical trials significantly limit the use of drugs and alarm for a detailed understanding of disease heterogeneity and the development of preventive and multifaceted remedial approach desperately. With this motivation, we herein report a diverse series of piperidinyl-quinoline acylhydrazone therapeutics as selective as well as potent inhibitors of cholinesterase enzymes. Ultrasound-assisted conjugation of 6/8-methyl-2-(piperidin-1-yl)quinoline-3-carbaldehydes (4a,b) and (un)substituted aromatic acid hydrazides (7a-m) provided facile access to target compounds (8a-m and 9a-j) in 4-6 min in excellent yields. The structures were fully established using spectroscopic techniques such as FTIR, 1H- and 13C NMR, and purity was estimated using elemental analysis. The synthesized compounds were investigated for their cholinesterase inhibitory potential. In vitro enzymatic studies revealed potent and selective inhibitors of AChE and BuChE. Compound 8c showed remarkable results and emerged as a lead candidate for the inhibition of AChE with an IC50 value of 5.3 ± 0.51 µM. The inhibitory strength of the optimal compound was 3-fold higher compared to neostigmine (IC50 = 16.3 ± 1.12 µM). Compound 8g exhibited the highest potency and inhibited the BuChE selectively with an IC50 value of 1.31 ± 0.05 µM. Several compounds, such as 8a-c, also displayed dual inhibitory strength, and acquired data were superior to the standard drugs. In vitro results were further supported by molecular docking analysis, where potent compounds revealed various important interactions with the key amino acid residues in the active site of both enzymes. Molecular dynamics simulation data, as well as physicochemical properties of the lead compounds, supported the identified class of hybrid compounds as a promising avenue for the discovery and development of new molecules for multifactorial diseases, such as Alzheimer's disease (AD).

(S)-N-Benzyl-1-phenyl-3,4-dihydroisoqunoline-2(1H)-carboxamide Derivatives, Multi-Target Inhibitors of Monoamine Oxidase and Cholinesterase: Design, Synthesis, and Biological Activity.

A series of (S)-1-phenyl-3,4-dihydroisoquinoline-2(1H)-carboxamide derivatives was synthesized and evaluated for inhibitory activity against monoamine oxidase (MAO)-A and-B, acetylcholine esterase (AChE), and butyrylcholine esterase (BChE). Four compounds (2i, 2p, 2t, and 2v) showed good inhibitory activity against both MAO-A and MAO-B, and two compounds (2d and 2j) showed selective inhibitory activity against MAO-A, with IC50 values of 1.38 and 2.48 µM, respectively. None of the compounds showed inhibitory activity against AChE; however, 12 compounds showed inhibitory activity against BChE. None of the active compounds showed cytotoxicity against L929cells. Molecular docking revealed several important interactions between the active analogs and amino acid residues of the protein receptors. This research paves the way for further study aimed at designing MAO and ChE inhibitors for the treatment of depression and neurodegenerative disorders.

The kinetic and molecular docking analysis of interactions between three V-type nerve agents and four human cholinesterases.

G and V-type nerve agents represent the most toxic chemical warfare agents. Their primary toxicity was the consequence of the covalent inhibition of the pivotal acetylcholinesterase (AChE), which induces overstimulation of cholinergic receptors and overaccumulation of cholines, eventually leading to death by respiratory arrest. The inhibitory and reactivation kinetics of cholinesterase (ChE) are essential for the toxicology and countermeasures of nerve agents. Medical defensive research on V-type nerve agents (V agents) has been mainly reported on VX and VR. Here we demonstrated the first systematical kinetic analysis between the type of ChE [native or recombinant human AChE and butyrylcholinesterase (BChE)] and three V agents, including VX, VR, and Vs, another isomer of VX, and highlighted the effects of native and recombinant ChE differences. The spontaneous reactivation and aging kinetics data of Vs-inhibited BChEs were firstly reported here. The results showed that AChE was more easily inhibited by three V agent compared to BChE, regardless of whether it is native or recombinant. The increased inhibitory potency order on AChE was VX, Vs, then VR, and on BChE was VX, then Vs and VR. The difference between native and recombinant ChE could influence the inhibition, aging, and spontaneous reactivation kinetics of three V agents, whether AChE or BChE, which was systematically revealed for the first time. For inhibition kinetics, the ki of three V agents for recombinant AChE was significantly higher than native AChE, and the stronger the inhibitory potency of V agents, the more pronounced difference in ki. In terms of aging and spontaneous reactivation kinetics, recombinant ChE was found to be more prone to spontaneous reactivation, but more resistant to aging compared to native ChE, particularly for AChE. The performed covalent molecular docking results partially explained the effects of differences between native and recombinant ChE on enzyme kinetics from the perspective of binding energy and conformation.

Deletion induced splicing in RIC3 drives nicotinic acetylcholine receptor regulation with implications for endoplasmic reticulum stress in human astrocytes.

Nicotinic acetylcholine receptor (nAChR) dysregulation in astrocytes is reported in neurodegenerative disorders. Modulation of nAChRs through agonists confers protection to astrocytes from stress but regulation of chaperones involved in proteostasis with pathological implications is unclear. Resistance to inhibitors of cholinesterase 3 (RIC3), a potential chaperone of nAChRs is poorly studied in humans. We characterized RIC3 in astrocytes derived from an isogenic wild-type and Cas9 edited "del" human iPSC line harboring a 25 bp homozygous deletion in exon2. Altered RIC3 transcript ratio due to deletion induced splicing and an unexpected gain of α7nAChR expression were observed in "del" astrocytes. Transcriptome analysis showed higher expression of neurotransmitter/G-protein coupled receptors mediated by cAMP and calcium/calmodulin-dependent kinase signaling with increased cytokines/glutamate secretion. Functional implications examined using tunicamycin induced ER stress in wild-type astrocyte stress model showed cell cycle arrest, RIC3 upregulation, reduction in α7nAChR membrane levels but increased α4nAChR membrane expression. Conversely, tunicamycin-treated "del" astrocytes showed a comparatively higher α4nAChR membrane expression and upsurged cAMP signaling. Furthermore, reduced expression of stress markers CHOP, phospho-PERK and lowered XBP1 splicing in western blot and qPCR, validated by proteome-based pathway analysis indicated lowered disease severity. Findings indicate (i) a complex RNA regulatory mechanism via exonic deletion induced splicing; (ii) RIC-3 as a disordered protein having contrasting effects on co-expressed nAChR subtypes under basal/stress conditions; and (iii) RIC3 as a potential drug target against ER stress in astrocytes for neurodegenerative/nicotine-related brain disorders. Cellular rescue mechanism through deletion induced exon skipping may encourage ASO-based therapies for tauopathies.

Computational design of new tacrine analogs: an in silico prediction of their cholinesterase inhibitory, antioxidant, and hepatotoxic activities.

Tacrine, the first drug approved for the treatment of Alzheimer's disease (AD), is a non-competitive cholinesterase inhibitor withdrawn due to its acute hepatotoxicity. However, new non-hepatotoxic forms of tacrine have been actively researched. Moreover, several recent reports have shown that oxidative stress is the cause of damage and plays a role in the pathogenesis of several neurodegenerative diseases including AD. The aim of the present study is the design of new easily synthesized tacrine analogs with less hepatotoxicity and potent antioxidant activity. In this context, a library of 34 novel tacrine analogs bearing an antioxidant fragment was designed and evaluated for its hepatotoxicity as well as anticholinesterase and antioxidant activities using computational methods. As a result, six new tacrine analogs have been proposed as potential inhibitors of cholinesterase with antioxidant activity and low or no hepatotoxicity. Furthermore, ADME calculations suggest that these compounds are promising oral drug candidates. Communicated by Ramaswamy H. Sarma.

Exploration of a library of piperonylic acid-derived hydrazones possessing variable aryl functionalities as potent dual cholinesterase and monoamine oxidase inhibitors.

A library of piperonylic acid-derived hydrazones possessing variable aryl moiety was synthesized and investigated for their multifunctional properties against cholinesterases (ChEs) and monoamine oxidases (MAOs). The in vitro enzymatic assay results revealed that the tested hydrazones have exhibited excellent cholinesterase inhibition profile. Compound 4i, (E)-N'-(2,3-dichlorobenzylidene)benzo[d][1,3]dioxole-5-carbohydrazide showed promising dual inhibitory profile against AChE (0.048 ± 0.007 µM), BChE (0.89 ± 0.018 µM), and MAO-B (0.95 ± 0.12 µM) enzymes. SAR exploration revealed that the truncation of the linker connecting both the aryl binding sites of the semicarbazone scaffold, by one atom, has relatively suppressed the AChE inhibitory potential. Kinetic studies disclosed that the compound 4i reversibly inhibited AChE enzyme in a competitive manner (Ki = 8.0 ± 0.076 nM), while it displayed a non-competitive and reversible inhibition profile against MAO-B (Ki = 9.6 ± 0.021 µM). Moreover, molecular docking studies of synthesized compounds against ChEs and MAOs provided the crucial molecular features that enable their close association and interaction with the target enzymes. All atomistic simulation studies confirmed the stable association of compound 4i within the active sites of AChE and MAO-B. In addition, theoretical ADMET prediction studies demonstrated the acceptable pharmacokinetic profile of the dual inhibitors. In summary, the attempted lead simplification study afforded a potent dual ChE-MAO-B inhibitor compound that merits further investigation.

Novel AP2238-clorgiline hybrids as multi-target agents for the treatment of Alzheimer's disease: Design, synthesis, and biological evaluation.

Cholinesterase and monoamine oxidase are potential targets for the therapy of Alzheimer's disease. A series of novel AP2238-clorgiline hybrids as multi-target agents were designed, synthesized and investigated in vitro for their inhibition of cholinesterases and monoamine oxidases. Many compounds displayed balanced and good inhibitory activity against AChE, BuChE and MAO-B with an obvious selective inhibitory effect on MAO-B. Among them, Compound 5l showed the most balanced potency to inhibit ChEs (eeAChE: IC50 = 4.03 ± 0.03 µM, eqBuChE: IC50 = 5.64 ± 0.53 µM; hAChE: IC50 = 8.30 ± 0.04 µM, hBuChE: IC50 = 1.91 ± 0.06 µM) and hMAO-B (IC50 = 3.29 ± 0.09 µM). Molecular modeling and kinetic studies showed that 5l was a mixed inhibitor for both AChE and BuChE, and a competitive MAO-B inhibitor. Compound 5l exhibited no toxicity to PC12 and BV-2 cells at 12.5 µM and no acute toxicity at a dosage of 2500 mg/kg. Moreover, 5l can improve the memory function of mice with scopolamine-induced memory impairment and have an excellent ability to cross the blood-brain barrier. Overall, these findings suggested that compound 5l could be deemed as a promising, balanced multi-target drug candidate against Alzheimer's disease.