Selective inhibition of human acetylcholinesterase by xanthine derivatives: In vitro inhibition and molecular modeling investigations

The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC₅₀ = 6.40 μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC₅₀ = 7.25 μM) < pentoxifylline (hAChE IC₅₀ = 6.60 μM) ? propentofylline (hAChE IC₅₀ = 6.40 μM). These compounds were less potent relative to the reference agent donepezil (hAChE IC₅₀ = 0.04 μM). Moreover, they all exhibited selective inhibition of hAChE with no inhibition of hBuChE (IC₅₀ > 50 μM) relative to the reference agent donepezil (hBuChE IC₅₀ = 13.60 μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia.     

Detection and quantification of fatty acid ethyl esters in meconium by headspace-solid-phase microextraction and gas chromatography–mass spectrometry

Meconium fatty acid ethyl esters (FAEEs) are currently used as biomarkers to detect heavy prenatal alcohol exposure. We introduce a novel technique to quantify FAEEs in meconium using headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS). This method improves on previous approaches by decreasing sample preparation time, eliminating the need for organic solvents, and reducing the required sample size. Using 50 mg of meconium, the detection limits of FAEEs ranged from 0.05 to 0.16 nmol/g and had good reproducibility making it ideal for routine analysis of clinical samples.     

Discovery of isoalloxazine derivatives as a new class of potential anti-Alzheimer agents and their synthesis

This article describes discovery of a novel and new class of cholinesterase inhibitors as potential therapeutics for Alzheimer’s disease. A series of novel isoalloxazine derivatives were synthesized and biologically evaluated for their potential inhibitory outcome for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds exhibited high activity against both the enzymes AChE as well as BuChE. Of the synthesized compounds, the most potent isoalloxazine derivatives (7m and 7q) showed IC₅₀ values of 4.72 μM and 5.22 μM respectively against AChE; and, 6.98 μM and 5.29 μM respectively against BuChE. These two compounds were further evaluated for their anti-aggregatory activity for β-amyloid (Aβ) in presence and absence of AChE by performing Thioflavin-T (ThT) assay and Congo red (CR) binding assay. In order to evaluate cytotoxic profile of these two potential compounds, cell viability assay of SH-SY5Y human neuroblastoma cells was performed. Further, to understand the binding behavior of these two compounds with AChE and BuChE enzymes, docking studies have been reported.     

Microwave assisted synthesis of novel hybrid tacrine-sulfonamide derivatives and investigation of their antioxidant and anticholinesterase activities

A novel series of tacrine derivatives containing sulfonamide group were synthesized and their inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. The result showed that all the synthesized tacrine-sulfonamides (VIIIa–o) exhibited inhibitory activity on both cholinesterases. VIIIg showed the highest inhibitory activity on AChE IC₅₀ = 0.009 μM. This value is 220-fold greater than that of galantamine (IC₅₀ = 2.054 μM) and 6-fold greater than tacrine (IC₅₀ = 0.055 μM). VIIIf displayed the strongest inhibition of BuChE (IC₅₀ = 2.250 μM), which is close to donepezil (IC₅₀ = 2.680 μM) and 8-fold greater than that of galantamine (IC₅₀ = 18.130 μM) Furthermore, all of the synthesized tacrine derivatives showed higher inhibition of BuChE than that of galantamine. In addition, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical scavenging abilities of the synthesized compounds were investigated for the antioxidant activity. Among them, VIIIb (IC₅₀ = 94.390 ± 2.310 μM) showed significantly better ABTS cation radical scavenging ability than all of the new synthesized compounds.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

Synthesis and evaluation of multi-target-directed ligands for the treatment of Alzheimer’s disease based on the fusion of donepezil and melatonin

A novel series of compounds obtained by fusing the acetylcholinesterase (AChE) inhibitor donepezil and the antioxidant melatonin were designed as multi-target-directed ligands for the treatment of Alzheimer’s disease (AD). In vitro assay indicated that most of the target compounds exhibited a significant ability to inhibit acetylcholinesterase (eeAChE and hAChE), butyrylcholinesterase (eqBuChE and hBuChE), and β-amyloid (Aβ) aggregation, and to act as potential antioxidants and biometal chelators. Especially, 4u displayed a good inhibition of AChE (IC₅₀ value of 193 nM for eeAChE and 273 nM for hAChE), strong inhibition of BuChE (IC₅₀ value of 73 nM for eqBuChE and 56 nM for hBuChE), moderate inhibition of Aβ aggregation (56.3% at 20 μM) and good antioxidant activity (3.28 trolox equivalent by ORAC assay). Molecular modeling studies in combination with kinetic analysis revealed that 4u was a mixed-type inhibitor, binding simultaneously to catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4u could chelate metal ions, reduce PC12 cells death induced by oxidative stress and penetrate the blood–brain barrier (BBB). Taken together, these results strongly indicated the hybridization approach is an efficient strategy to identify novel scaffolds with desired bioactivities, and further optimization of 4u may be helpful to develop more potent lead compound for AD treatment.     

New cholinesterase inhibitors for Alzheimer’s disease: Structure Activity Studies (SARs) and molecular docking of isoquinolone and azepanone derivatives

A library of isoquinolinone and azepanone derivatives were screened for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. The strategy adopted included (a) in vitro biological assays, against eel AChE (EeAChE) and equine serum BuChE (EqBuChE) in order to determine the compounds IC₅₀ and their dose-response activity, consolidated by (b) molecular docking studies to evaluate the docking poses and interatomic interactions in the case of the hit compounds, validated by STD-NMR studies. Compound (1f) was identified as one of these hits with an IC₅₀ of 89.5 μM for EeAChE and 153.8 μM for EqBuChE, (2a) was identified as a second hit with an IC₅₀ of 108.4 μM (EeAChE) and 277.8 μM (EqBuChE). In order to gain insights into the binding mode and principle active site interactions of these molecules, (R)-(1f) along with 3 other analogues (also as the R-enantiomer) were docked into both RhAChE and hBuChE models. Galantamine was used as the benchmark. The docking study was validated by performing an STD-NMR study of (1f) with EeAChE using galantamine as the benchmark.     

Culture temperature affects gene expression and metabolic pathways in the 2-methylisoborneol-producing cyanobacterium Pseudanabaena galeata

A volatile metabolite, 2-methylisoborneol (2-MIB), causes an unpleasant taste and odor in tap water. Some filamentous cyanobacteria produce 2-MIB via a two-step biosynthetic pathway: methylation of geranyl diphosphate (GPP) by methyl transferase (GPPMT), followed by the cyclization of methyl-GPP by monoterpene cyclase (MIBS). We isolated the genes encoding GPPMT and MIBS from Pseudanabaena galeata, a filamentous cyanobacterium known to be a major causal organism of 2-MIB production in Japanese lakes. The predicted amino acid sequence showed high similarity with that of Pseudanabaena limnetica (96% identity in GPPMT and 97% identity in MIBS). P. galeata was cultured at different temperatures to examine the effect of growth conditions on the production of 2-MIB and major metabolites. Gas chromatograph–mass spectrometry (GC–MS) measurements showed higher accumulation of 2-MIB at 30 °C than at 4 °C or 20 °C after 24 h of culture. Real-time-RT PCR analysis showed that the expression levels of the genes encoding GPPMT and MIBS decreased at 4 °C and increased at 30 °C, compared with at 20 °C. Furthermore, metabolite analysis showed dramatic changes in primary metabolite concentrations in cyanobacteria grown at different temperatures. The data indicate that changes in carbon flow in the TCA cycle affect 2-MIB biosynthesis at higher temperatures.     

Omega-3 index determined by gas chromatography with electron impact mass spectrometry

Omega-3 index is a relatively new concept, defined as the sum of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) expressed as a percentage of the total fatty acids in red blood cell membranes. This index reflects medium to long-term intake of omega-3 polyunsaturated fatty acids and could be a useful tool in epidemiological studies. The standard technique used for fatty acid analysis and quantification has been gas chromatography (GC) with flame ionization detection. This method is robust and has good precision and sensitivity. However, a major disadvantage is inability to confirm spectrometrically the identity of fatty acids detected, which is important especially in complex biological samples. The current study measures omega-3 index in 12 healthy human volunteers using GC-mass spectrometry (MS). Both the intra-assay and day-to-day variations were well within 5% with linearity of response extending to a concentration of 250 μg/ml (830 μmol/L) of EPA. The limit of detection of EPA was 0.36 μg/ml (1.2 μmol/L). About 25 fatty acids were consistently detected in red blood cells from healthy volunteers including cis and trans isomers. The omega-3 index ranged from 2.4% to 6.2% among the 12 volunteers examined and there was no difference between samples taken in the fasting and postprandial states. EPA and DHA concentrations ranged from 3.53 to 105.89 μg/ml (11.7–350 μmol/L) and 12.19 to 214.42 μg/ml (37.1–652.7 μmol/L), respectively. Thus a GC–MS method has been developed for measuring the omega-3 index. Further studies are required to determine the role of this index as a predictor of disease.     

Green synthesis of silver nanoparticles by Conocarpus Lancifolius plant extract and their antimicrobial and anticancer activities

Due to drug addiction and the emergence of antibiotic resistance in pathogens, the disease load and medication intake have risen worldwide. The alternative treatment for drug-resistant infections is Nano formulation-based antimicrobial agents. The plant extract of Conocarpus Lancifolius fruits was used to synthesize silver nanoparticles in the current study, and it was further employed as an antimicrobial and anticancer agent. Nanoparticles have been characterized by UV–visible spectrometer revealed the notable peak of λ_max = 410–442 nm, which confirms the reduction of silver ion to elemental silver nanoparticles, and the biological moieties in the synthesis were further confirmed by FTIR analysis. The stability and crystalline nature of materials were approved by XRD analysis and expected the size of the nanomaterials of 21 to 173 nm analyzed by a nanophox particle-size analyzer. In vitro, synthesized materials act as an antibacterial agent against Streptococcus pneumonia and Staphylococcus aureus. The inhibition zones of 18 and 24 mm have been estimated to be antibacterial activity against both bacteria. The potency of up to 100% of AgNPs for bacterial strains was incubated overnight at 60 μg/ml. Based on our results, biogenic AgNPs reveal significant activity against fungal pathogen Rhizopusus stolonifera and Aspergillus flavus that cause leading infectious diseases. Additionally, nanomaterials were biocompatible and demonstrated the potential anticancer activities against MDA MB-231 cells after 24-hour exposure.     

Synthesis and biological evaluation of a new series of berberine derivatives as dual inhibitors of acetylcholinesterase and butyrylcholinesterase

A series of novel berberine derivatives were designed, synthesized, and biologically evaluated as inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Among these derivatives, compound 48a, berberine linked with 3-methylpyridinium by a 2-carbon spacer, was found to be a potent inhibitor of AChE, with an IC₅₀ value of 0.048 μM and compound 40c, berberine linked with 2-thionaphthol by a 4-carbon spacer, acted as the most potent inhibitor for BuChE with an IC₅₀ value of 0.078 μM. Kinetic studies and molecular modeling simulations of the AChE-inhibitor complex indicated that a mixed-competitive binding mode existed for these berberine derivatives.     

Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase inhibitors: Synthesis,biological evaluation and molecular modeling studies

A series of new biphenyl bis-sulfonamide derivatives 2a–3p were synthesized in good to excellent yield (76–98%). The inhibitory potential of the synthesized compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was investigated. Most of the screened compounds showed modest in vitro inhibition for both AChE and BChE. Compared to the reference compound eserine (IC₅₀ 0.04 ± 0.0001 μM for AChE) and (IC₅₀ 0.85 ± 0.0001 μM for BChE), the IC₅₀ values of these compounds were ranged from 2.27 ± 0.01 to 123.11 ± 0.04 μM for AChE and 7.74 ± 0.07 to <400 μM for BuChE. Among the tested compounds, 3p was found to be the most potent against AChE (IC₅₀ 2.27 ± 0.01 μM), whereas 3g exhibited the highest inhibition for BChE (IC₅₀ 7.74 ± 0.07 μM). Structure–activity relationship (SAR) of these compounds was developed and elaborated with the help of molecular docking studies.     

Variance-based signal conditioning technique: Comparison to a wavelet-based technique to improve spike detection in multiunit intrafascicular recordings

Detection of single unit action potentials (APs) from peripheral nerve recordings is complicated by low signal-to-noise ratio (SNR) due to the activity of nearby muscles, interference from more distant nerve fibers, and thermal noise from the neural interface. In this study, we propose a novel signal conditioning technique for multiunit signals (i.e. a signal comprised of multiple units coming from different nerve fibers), based on the variance to be applied prior to detection of APs. The proposed technique was tested on experimental and simulated intrafascicular recordings; and was compared to a wavelet-based conditioning (also applied before AP detection). The outputs of both conditioning schemes were sent to an AP detection algorithm that used a simple threshold (equal to the standard deviation of the signal). The overall performance of the detection phase was superior when using the wavelet-based conditioned signal especially for SNR ≤ 2 dB. However, when using the variance-based conditioned signal, the AP detection phase resulted in lower number of false positives for SNR > 2 dB. The novel variance-based method improves the SNR by attenuating the background noise between APs and can be applied as pre-conditioning processing for AP detection.     

Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput

We report on the development and characterization of automated metal-free multiple-column nanoLC instrumentation for sensitive and high-throughput analysis of phosphopeptides with mass spectrometry. The system employs a multiple-column capillary LC fluidic design developed for high-throughput analysis of peptides (Anal. Chem. 2001, 73, 3011–3021), incorporating modifications to achieve broad and sensitive analysis of phosphopeptides. The integrated nanoLC columns (50 μm i.d. × 30 cm containing 5 μm C18 particles) and the on-line solid phase extraction columns (150 μm i.d. × 4 cm containing 5 μm C18 particles) were connected to automatic switching valves with non-metal chromatographic accessories, and other modifications to avoid the exposure of the analyte to any metal surfaces during handling, separation, and electrospray ionization. The nanoLC developed provided a separation peak capacity of ～250 for phosphopeptides (and ～400 for normal peptides). A detection limit of 0.4 fmol was obtained when a linear ion trap tandem mass spectrometer (Finnegan LTQ) was coupled to a 50-μm i.d. column of the nanoLC. The separation power and sensitivity provided by the nanoLC–LTQ enabled identification of ～4600 phosphopeptide candidates from ～60 μg COS-7 cell tryptic digest followed by IMAC enrichment and ～520 tyrosine phosphopeptides from ～2 mg of human T cells digests followed by phosphotyrosine peptide immunoprecipitation.     

Antibiofilm activity and post antifungal effect of lemongrass oil on clinical Candida dubliniensis isolate

Candidal infections are often difficult to eradicate due to the resistance of biofilms to antifungal agents. This study aimed at determining the effects of lemongrass (Cymbopogon citratus DC) oil against Candida dubliniensis in both planktonic and biofilms form. The results from broth microdilution method revealed that the minimum inhibitory and minimum fungicidal concentration of lemongrass oil on C. dubliniensis were 0.43 and 0.86 mg/ml, respectively. Employing a formazan salt (XTT tetrazolium) reduction assay for biofilm study, the results showed that the average percentage (mean ± SD) inhibition of lemongrass oil (0.43 mg/ml) on biofilm formation was 91.57 ± 1.31%, while it exhibited more than 80% killing activity against C. dubliniensis in biofilm at concentrations of 1.7 mg/ml. In addition, a significant reduction (P = 0.03) of candidal adhesion to acrylic occurred after a 15 min exposure to 1.7 mg/ml of lemongrass oil. Moreover, limited exposure of yeasts to lemongrass oil at subcidal concentration can suppress growth for more than 24 h. Altogether, the results obtained indicate that lemongrass oil possessed antifungal and antibiofilm activities and could modulate candidal colonization. Therefore, the efficacy of lemongrass oil merits further development of this agent for the therapy of oral candidiasis.     

Design,synthesis and evaluation of novel tacrine-(β-carboline) hybrids as multifunctional agents for the treatment of Alzheimer’s disease

A series of tacrine-(β-carboline) hybrids (11a–q) were designed, synthesized and evaluated as multifunctional cholinesterase inhibitors against Alzheimer’s disease (AD). In vitro studies showed that most of them exhibited significant potency to inhibit acetylcholinesterase (eeAChE and hAChE), butyrylcholinesterase (BuChE) and self-induced β-amyloid (Aβ) aggregation, Cu²⁺-induced Aβ (1–42) aggregation, and to chelate metal ions. Especially, 11l presented the greatest ability to inhibit cholinesterase (IC₅₀, 21.6 nM for eeAChE, 63.2 nM for hAChE and 39.8 nM for BuChE), good inhibition of Aβ aggregation (65.8% at 20 μM) and good antioxidant activity (1.57 trolox equivalents). Kinetic and molecular modeling studies indicated that 11l was a mixed-type inhibitor, binding simultaneously to the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 11l could chelate metal ions, reduce PC12 cells death induced by oxidative stress and penetrate the blood–brain barrier (BBB). These results suggested that 11l might be an excellent multifunctional agent for AD treatment.     

Synthesis,biological evaluation and molecular modeling of substituted 2-aminobenzimidazoles as novel inhibitors of acetylcholinesterase and butyrylcholinesterase

A series of novel 2-aminobenzimidazole derivatives were synthesized under microwave irradiation. Their biological activities were evaluated on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). A number of the 2-aminobenzimidazole derivatives showed good inhibitory activities to AChE and BuChE. Among them, compounds 9, 12 and 13 were found to be >25-fold more selective for BuChE than AChE. No evidence of cytotoxicity was observed by MTT assay in PC12 cells or HepG2 cells exposed to 100 μM of the compounds. Molecular modeling studies indicate that the benzimidazole moiety of compounds 9, 12 and 13 forms a face-to-face π–π stacking interaction in a ‘sandwich’ form with the indole ring of Trp82 (4.09 Å) in the active gorge, and compounds 12 and 13 form a hydrogen bond with His438 at the catalytic site of BuChE. In addition, compounds 12 and 13 fit well into the hydrophobic pocket formed by Ala328, Trp430 and Tyr332 of BuChE. Our data suggest the 2-aminobenzimidazole drugs as promising new selective inhibitors for AChE and BuChE, potentially useful to treat neurodegenerative diseases.     

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography–mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85 μm PA exposed at 100 °C for 40 min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2 ng/g, serum: 0.2 ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.     

Partial regeneration of β-cells in the islets of Langerhans by Nymphayol a sterol isolated from Nymphaea stellata (Willd.) flowers

Reduction of the β-cell mass is critical in the pathogenesis of diabetes mellitus. The discovery of agents which induce regeneration of pancreatic β-cells would be useful to develop new therapeutic approaches to treat diabetes. The present study was aimed at identifying a new agent for the control of diabetes through regeneration of pancreatic β cells and insulin secretory potential. Nymphaea stellata flower chloroform extract (NSFCExt) showed significant plasma glucose lowering effect. Further NSFCExt was utilized to isolate and identify the lead compound based on bioassay guided fractionation; we found Nymphayol (25,26-dinorcholest-5-en-3β-ol) a new crystal [space group P2₁ (No. 4), a = 9.618(5), b = 7.518(5), c = 37.491(5)]. It was purified by repeat column. The structure was determined on the basis of X-ray crystallography and spectral data. Oral administration of Nymphayol for 45 days significantly (p < 0.05) lowered the blood glucose level and more importantly it effectively increased the insulin content in diabetic rats. In addition, Nymphayol increased the number of β cell mass enormously. Islet-like cell clusters in the islets of Langerhans were clearly observed based on histochemical and immunohistochemical study.