Protein-mimicking nanowire-inspired electro-catalytic biosensor for probing acetylcholinesterase activity and its inhibitors.

A highly sensitive electrochemical biosensor based on the synthetized L-Cysteine-Ag(I) coordination polymer (L-Cys-Ag(I) CP), which looks like a protein-mimicking nanowire, was constructed to detect acetylcholinesterase (AChE) activity and screen its inhibitors. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce hydrogen peroxide (H2O2), thus L-Cys-Ag(I) CP possesses the electro-catalytic property to H2O2 reduction. Herein, the protein-mimicking nanowire-based platform was capable of investigating successive of H2O2 effectively by amperometric i-t (current-time) response, and was further applied for the turn-on electrochemical detection of AChE activity. The proposed sensor is highly sensitive (limit of detection is 0.0006 U/L) and is feasible for screening inhibitors of AChE. The model for AChE inhibition was further established and two traditional AChE inhibitors (donepezil and tacrine) were employed to verify the feasibility of the system. The IC50 of donepezil and tacrine were estimated to be 1.4 nM and 3.5 nM, respectively. The developed protocol provides a new and promising platform for probing AChE activity and screening its inhibitors with low cost, high sensitivity and selectivity.

Design, synthesis, in-silico and biological evaluation of novel donepezil derivatives as multi-target-directed ligands for the treatment of Alzheimer's disease.

A novel series of donepezil based multi-functional agents "(E)-5,6-dimethoxy-2-(4-(4-substituted piperazin-1-yl)benzylidene)-2,3-dihydro-1H-inden-1-ones" have been designed and synthesized as potential anti-Alzheimer's agents. In-vitro studies revealed that these compounds demonstrated moderate to good AChE and Aß aggregation inhibitory activity. These derivatives are also endowed with admirable antioxidant activity. Among the entire series compounds IP-9, IP-13 and IP-15 appeared as most active multi-functional agents and displayed marked AChE inhibitory, Aß disaggregation and antioxidant activity. Studies indicate that IP-13 and IP-15 showed better AChE inhibitory activity than the standard drug donepezil and IP-9, IP-13 as well as IP-15 exhibited better Aß aggregation inhibitory activity than curcumin. These compounds (IP-9, IP-13 and IP-15) successfully diminished H2O2 induced oxidative stress in SH-SY5Y cells and displayed excellent neuroprotective activity against H2O2 as well as Aß induced toxicity in SH-SY5Y cells in a concentration dependent manner. Moreover, these derivatives did not exert any significant toxicity in neuronal SH-SY5Y cells in cytotoxicity assay. To elucidate the plausible binding mode of the compounds IP-9, IP-13 and IP-15, molecular docking studies and molecular dynamics (MD) simulation studies were also performed and the results indicate their significant interactions with the active sites of AChE as well as Aß1-42 peptide. Thus, the present study evidently showed that IP-9, IP-13 and IP-15 are potent multi-functional agents against Alzheimer's disease and might serve as promising lead candidates for anti-Alzheimer drug development.

"Heart-cut" bidimensional achiral-chiral liquid chromatography applied to the evaluation of stereoselective metabolism, in vivo biological activity and brain response to chiral drug candidates targeting the central nervous system.

A "heart-cut" two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC-LC-MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide ((±)-1), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)-1 and its two hepatic metabolites. In the "heart-cut" two-dimension achiral-chiral configuration, (±)-1 and (±)-1-d4 eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)-1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs. Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal.

Investigation of functional selenium nanoparticles as potent antimicrobial agents against superbugs.

Developing highly effective antibacterial agents is important for a wide range of applications. However, the emergence of multiple antibiotic-resistant bacteria poses a public health threat. Many developed agents have limited practical application due to chemical instability, low biocompatibility, and poor long-term antibacterial efficiency. In the following study, we synthesize a synergistic nanocomposite by conjugating quercetin (Qu) and acetylcholine (Ach) to the surface of Se nanoparticles (Qu-Ach@SeNPs). Quercetin has been reported to exhibit a wide range of biological activities related to their antibacterial activity and acetylcholine as a neurotransmitter, which can combine with the receptor on the bacterial cell. Arrows indicate NPs and arrowheads indicate compromised cell walls. The study demonstrated how Qu-Ach@SeNPs exhibit a synergistically enhanced antibacterial performance against the multidrug-resistant superbugs (MDRs) compared to Qu@SeNPs and Ach@SeNPs alone. Qu-Ach@SeNPs are effective against MDRs, such as Methicillin-resistant Staphylococcus aureus (MRSA), at a low dose. The mechanistic studies showed that Qu-Ach@SeNPs attach to the bacterial cell wall, causing irreversible damage to the membrane, and thereby achieving a remarkable synergistic antibacterial effect to inhibit MRSA. The findings suggested that the synergistic properties of quercetin and acetylcholine enhance the antibacterial activity of SeNPs. In this way, Qu-Ach@SeNPs comprise a new class of inorganic nano-antibacterial agents that can be used as useful applications in biomedical devices. STATEMENT OF SIGNIFICANCE: The Qu-Ach@SeNPs have low cytotoxicity when tested on normal human cells in vitro. Qu-Ach@SeNPs are effective against MDRs, such as Methicillin-resistant S. aureus (MRSA), at a low dose. Importantly, Qu-Ach@SeNPs showed no emergence of resistance. These results suggest that Qu-Ach@SeNPs have excellent antibacterial activities. These agents can serve as good antibacterial agents against superbugs. Our data suggest that these antibacterial agents may have widespread application in the field of medicine for combating infectious diseases caused by MDRs, as well as other infectious diseases.

Quantitative Determination of Betaine, Choline, Acetylcholine, and 20-Hydroxyecdysone Simultaneously from Atriplex Species by UHPLC-UV-MS.

A simple, rapid, and sensitive UHPLC-UV-MS method was developed for the quantitative determination of betaine (1), choline (2), acetylcholine (3), and 20-hydroxyecdysone (4) from various species of Atriplex. The baseline separation of the four analytes was achieved on a reversed phase C 18 column within nine minutes. The mobile phase was composed of 50 mM ammonium formate in 2% methanol-water containing 5 mM sodium dodecyl sulfate (pH = 8.2) and methanol with 0.01% ammonium hydroxide. The analytical method was validated for recovery, precision, limits of detection (LOD), and limits of quantification (LOQ). The developed method was applied for the characterization and quantitation of analytes from plant parts of different Atriplex species, including A. canescens, A. fruticulosa, A. fasciculata, A. semibaccata, and A. lentiformis. Compounds 1-4 were found in a range of 0.53-1.61%, detection under limit of quantification (DUL)-0.74, DUL-0.0038, and 0-0.10% (w/w, mg in 100 mg plant material), respectively, in test samples. In leaf and fruit of A. canescens, a high content of 1, 2, and 4 were identified. The content of 1, 2, and 4 in A. canescens explains the potential implications of this native US plant for human health and nutrition. The result of this study provides a new method to analyze these phytoconstituents simultaneously in a mixture.

Direct, rapid, and label-free detection of enzyme-substrate interactions in physiological buffers using CMOS-compatible nanoribbon sensors.

We demonstrate the versatility of Al2O3-passivated Si nanowire devices ("nanoribbons") in the analysis of enzyme-substrate interactions via the monitoring of pH change. Our approach is shown to be effective through the detection of urea in phosphate buffered saline (PBS), and penicillinase in PBS and urine, at limits of detection of <200 µM and 0.02 units/mL, respectively. The ability to extract accurate enzyme kinetics and the Michaelis-Menten constant (Km) from the acetylcholine-acetylcholinesterase reaction is also demonstrated.

Insights into the binding of GABA to the insect RDL receptor from atomistic simulations: a comparison of models.

The resistance to dieldrin (RDL) receptor is an insect pentameric ligand-gated ion channel (pLGIC). It is activated by the neurotransmitter γ-aminobutyric acid (GABA) binding to its extracellular domain; hence elucidating the atomistic details of this interaction is important for understanding how the RDL receptor functions. As no high resolution structures are currently available, we built homology models of the extracellular domain of the RDL receptor using different templates, including the widely used acetylcholine binding protein and two pLGICs, the Erwinia Chrysanthemi ligand-gated ion channel (ELIC) and the more recently resolved GluCl. We then docked GABA into the selected three dimensional structures, which we used as starting points for classical molecular dynamics simulations. This allowed us to analyze in detail the behavior of GABA in the binding sites, including the hydrogen bond and cation-π interaction networks it formed, the conformers it visited and the possible role of water molecules in mediating the interactions; we also estimated the binding free energies. The models were all stable and showed common features, including interactions consistent with experimental data and similar to other pLGICs; differences could be attributed to the quality of the models, which increases with increasing sequence identity, and the use of a pLGIC template. We supplemented the molecular dynamics information with metadynamics, a rare event method, by exploring the free energy landscape of GABA binding to the RDL receptor. Overall, we show that the GluCl template provided the best models. GABA forming direct salt-bridges with Arg211 and Glu204, and cation-π interactions with an aromatic cage including Tyr109, Phe206 and Tyr254, represents a favorable binding arrangement, and the interaction with Glu204 can also be mediated by a water molecule.

Cellular membrane phospholipids act as a depository for quaternary amine containing drugs thus competing with the acetylcholine/nicotinic receptor.

We previously demonstrated that ammonium- or guanidinium-phosphate interactions are key to forming noncovalent complexes (NCXs) through salt bridge formation with G-protein coupled receptors (GPCR), which are immersed in the cell membrane's lipids. The present work highlights MALDI ion mobility coupled to orthogonal time-of-flight mass spectrometry (MALDI IM oTOF MS) as a method to determine qualitative and relative quantitative affinity of drugs to form NCXs with targeted GPCRs' epitopes in a model system using, bis-quaternary amine based drugs, α- and ß- subunit epitopes of the nicotinic acetylcholine receptor' (nAChR) and phospholipids. Bis-quaternary amines proved to have a strong affinity for all nAChR epitopes and negatively charged phospholipids, even in the presence of the physiological neurotransmitter acetylcholine. Ion mobility baseline separated isobaric phosphatidyl ethanolamine and a matrix cluster, providing an accurate estimate for phospholipid counts. Overall this technique is a powerful method for screening drugs' interactions with targeted lipids and protein respectively containing quaternary amines and guanidinium moieties.

Synthesis and characterization of a novel potato starch derivative with cationic acetylcholine groups.

A novel substance, cationic acetylcholine potato starch (CAPS), was developed for the first time. The synthesis process had three steps: first, carboxymethyl potato starch (CMPS) was synthesized under sodium hydroxide alkaline condition and in isopropyl alcohol organic media; second, bromocholine chloride (BCC) was synthesized with sulphuric acid as a catalytic agent; finally, CAPS was synthesized by the reaction of CMPS with BCC in N,N'-dimethylformamide (DMF). The degree of substitution (DS) of CAPS was determined by ammonia gas-sensing electrode and elemental analysis. CAPS was characterized by Fourier transformed infrared (FTIR) and near infrared (FTNIR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD) and differential scanning calorimetry (DSC).

Uncaria rhynchophylla ameliorates cognitive deficits induced by D-galactose in mice.

The stem with hooks of Uncaria rhynchophylla is a component herb of many traditional formulae for the treatment of neurodegenerative diseases. However, scientific evidence of the efficacy of Uncaria rhynchophylla in the treatment of Alzheimer's disease (AD) in animal models is lacking. Thus, in the present study, we investigated whether the 70 % aqueous ethanol extract of Uncaria rhynchophylla (EUR) could protect against D-galactose (D-gal)-induced cognitive deficits in mice. Mice were given a subcutaneous injection of D-gal (50 mg/kg) and orally administered EUR (100, 200, or 400 mg/kg) daily for 8 weeks. The effect of EUR on D-gal-induced cognitive deficits was evaluated by measuring behavioral and neurochemical parameters of AD and the antioxidant status of brain tissue. The results showed that EUR (200 or 400 mg/kg) significantly increased exploratory behavior (assessed by an open-field test) and improved spatial learning and memory function (assessed by the Morris water maze test) in D-gal-treated mice. In addition, EUR (200 or 400 mg/kg) significantly increased the levels of acetylcholine and glutathione and decreased the activity of acetylcholinesterase and the level of malondialdehyde in the brains of D-gal-treated mice. These results indicate that EUR ameliorates cognitive deficits induced by D-gal in mice, and that this action may be mediated, at least in part, by the inhibition of acetylcholinesterase activity and the enhancement of the antioxidant status of brain tissue.

In vitro compatibility study of cephalosporin with intraocular irrigating solutions and intracameral medications.

BACKGROUND: To study the compatibility of cephalosporins with intraocular irrigating solutions and intracameral medications commonly used in cataract surgery. DESIGN: The was an in vitro experiment conducted in the Research Laboratory of the Department of Microbiology, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. SAMPLES: Three cephalosporins--cefazolin, cefuroxime and ceftazidime--were separately diluted and mixed with irrigating solutions and intracameral medications to form 192 samples and 12 control solutions. METHODS: The cephalosporins were dissolved in normal saline and further diluted to the concentration of 1 mg in 0.1 mL with normal saline, Ringer's solution, balanced salt solution and fortified balanced salt solutions. These were mixed with balanced salt solutions or fortified balanced salt solutions, with adrenaline, acetylcholine or carbachol and kept at 37°C for 2 h. The concentrations of free cephalosporins were measured with rapid high-performance liquid chromatography at baseline (0 h) and at 2 h. MAIN OUTCOME MEASURES: Free concentrations of cephalosporins at 2 h were compared with mean baseline (0 h) value. A difference of 3 standard deviations or more was considered statistically significant. RESULTS: At 2 h there was a significant drop in the cefuroxime concentration in preparations in which cefuroxime was diluted with normal saline (P < 0.01). In all preparations, the final concentrations of cephalosporins were higher than the minimal inhibitory concentrations (MIC(90)) for microbials commonly isolated from the external eye. CONCLUSION: Cefazolin, cefuroxime and ceftazidime were compatible with irrigating solutions and intracameral medications commonly used in cataract surgery.

Perinatal L-arginine and antioxidant supplements reduce adult blood pressure but not ameliorate the altered vascular function in spontaneously hypertensive rats.

Spontaneously hypertensive rat (SHR) offspring from L-arginine- and antioxidant-supplemented SHR dams had persistent lower blood pressure in adulthood. We investigated the influence of vascular mechanism in this effect. We analyzed response to acetylcholine and phenylephrine in aorta and superior mesenteric arteries from Wistar-Kyoto (WKY), SHR, and SHR perinatally supplemented with L-arginine and 4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl (TEMPOL; SHR-suppl). Supplements reduced blood pressure persistently in SHR. Relaxation to acetylcholine was greater in WKY than SHR and remained unmodified in SHR-suppl compared with SHR. Acute TEMPOL did not alter relaxation to acetylcholine in WKY but increased it similarly in SHR and SHR-suppl. Phenylephrine contraction was increased in SHR compared to WKY. In SHR-suppl, this response was similar to SHR. Endothelium removal or N-nitro-L-arginine methyl ester (L-NAME) increased contraction to phenylephrine more in WKY than SHR. In SHR-suppl, this was similar to SHR. In both SHR and SHR-suppl, TEMPOL similarly reduced phenylephrine response. This effect was prevented by L-NAME. Results exposed reinforce the concept that oxidative stress during perinatal period is a contributing factor to the development of hypertension in SHR. Results also reveal that the beneficial effect of this supplementation does not appear to be related to improved endothelial function, suggesting that other regulatory mechanisms of blood pressure may be involved.

Nicotinic pharmacophore: the pyridine N of nicotine and carbonyl of acetylcholine hydrogen bond across a subunit interface to a backbone NH.

Pharmacophore models for nicotinic agonists have been proposed for four decades. Central to these models is the presence of a cationic nitrogen and a hydrogen bond acceptor. It is now well-established that the cationic center makes an important cation-pi interaction to a conserved tryptophan, but the donor to the proposed hydrogen bond acceptor has been more challenging to identify. A structure of nicotine bound to the acetylcholine binding protein predicted that the binding partner of the pharmacophore's second component was a water molecule, which also hydrogen bonds to the backbone of the complementary subunit of the receptors. Here we use unnatural amino acid mutagenesis coupled with agonist analogs to examine whether such a hydrogen bond is functionally significant in the alpha4beta2 neuronal nAChR, the receptor most associated with nicotine addiction. We find evidence for the hydrogen bond with the agonists nicotine, acetylcholine, carbamylcholine, and epibatidine. These data represent a completed nicotinic pharmacophore and offer insight into the design of new therapeutic agents that selectively target these receptors.

Molecular modeling of the alpha9alpha10 nicotinic acetylcholine receptor subtype.

This study reports the comparative molecular modeling, docking and dynamic simulations of human alpha9alpha10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and alpha-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary alpha10(-) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal alpha9(+), and Asp114 in the complementary alpha10(-) subunit, might confer the potency and selectivity to alpha-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.

Novel acetylcholine and carbamoylcholine analogues: development of a functionally selective alpha4beta2 nicotinic acetylcholine receptor agonist.

A series of carbamoylcholine and acetylcholine analogues were synthesized and characterized pharmacologically at neuronal nicotinic acetylcholine receptors (nAChRs). Several of the compounds displayed low nanomolar binding affinities to the alpha4beta2 nAChR and pronounced selectivity for this subtype over alpha3beta4, alpha4beta4, and alpha7 nAChRs. The high nAChR activity of carbamoylcholine analogue 5d was found to reside in its R-enantiomer, a characteristic most likely true for all other compounds in the series. Interestingly, the pronounced alpha4beta2 selectivities exhibited by some of the compounds in the binding assays translated into functional selectivity. Compound 5a was a fairly potent partial alpha4beta2 nAChR agonist with negligible activities at the alpha3beta4 and alpha7 subtypes, thus being one of the few truly functionally selective alpha4beta2 nAChR agonists published to date. Ligand-protein docking experiments using homology models of the amino-terminal domains of alpha4beta2 and alpha3beta4 nAChRs identified residues Val111(beta2)/Ile113(beta4), Phe119(beta2)/Gln121(beta4), and Thr155(alpha4)/Ser150(alpha3) as possible key determinants of the alpha4beta2/alpha3beta4-selectivity displayed by the analogues.

Shoot-and-Trap: use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography.

Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the "off-switch" at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a "backdoor." The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.

Comparative interaction of few antihypertensive drugs with cyclosporine-A in rats.

The maximal endothelial dependent relaxation of isolated aortic rings to cumulative doses of acetylcholine was significantly decreased in the Cyclosporine-A (CSA, 20 mg kg(-1) day(-1)) treated animals compared to olive oil (CSA vehicle) treated control. Administration of antihypertensive drugs like diltiazem, enalapril or propranolol to CSA treated animals augmented the endothelial damage induced by CSA. These drugs also increased the bioavailability of CSA. However, administration of losartan to CSA treated animals produced a significant increase in endothelial dependent relaxation as compared to CSA treated control but did not affect the bioavailability of CSA significantly. The results suggest that losartan is safer compared to other antihypertensives for the treatment of CSA induced hypertension.

The influence of pomegranate fruit extract in comparison to regular pomegranate juice and seed oil on nitric oxide and arterial function in obese Zucker rats.

Metabolic syndrome includes most widely distributed clinical conditions such as obesity, hypertension, dislipidemia, and diabetes. Pomegranate fruit extract (PFE), rich in polyphenolic antioxidants, reduces the expression of oxidation-sensitive genes at the sites of perturbed shear-stress. The aim of this study was to evaluate the effect of PFE in comparison to regular pomegranate juice (PJ) and seed oil on the biological actions of nitric oxide (NO) and the arterial function in obese Zucker rats, a model of metabolic syndrome. Our results indicated that supplementation with PFE or PJ significantly decreased the expression of vascular inflammation markers, thrombospondin (TSP), and cytokine TGFbeta1 (P<0.05), whereas seed oil supplementation had a significant effect only on TSP-1 expression (P <0.05). Plasma nitrate and nitrite (NO(x)) levels were significantly increased by PFE and PJ (P<0.05). Furthermore, the effect of PFE in increasing endothelial NO synthase (eNOS) expression was comparable to that of PJ. These data highlight possible clinical applications of PFE in metabolic syndrome.

Neuro-bioenergetic concepts in cancer prevention and treatment.

Cancer remains one of the most difficult and elusive disorders to prevent and treat, despite great efforts in research and treatment over the last 30 years. Researchers have tried to understand the pathogenesis of cancer by discovering the single cellular mechanism or pathway derived from a genetic mutation. There are limited efforts made toward discovering a unified concept of cancer. We propose a neuro-bioenergetic concept of cancer pathogenesis based on the central mechanism of cellular hyperexcitability via inducible overexpression of voltage-gated ion channels, ligand-gated channels and neurotransmitters. Exploration of this concept could lead to a better understanding of the cause of cancer as well as developing more effective and specific strategies toward cancer prevention and treatment.