Mutasynthesis of Physostigmines in Myxococcus xanthus.

The alkaloid physostigmine is an approved anticholinergic drug and an important lead structure for the development of novel therapeutics. Using a complementary approach that merged chemical synthesis with pathway refactoring, we produced a series of physostigmine analogues with altered specificity and toxicity profiles in the heterologous host Myxococcus xanthus. The compounds that were generated by applying a simple feeding strategy include the promising drug candidate phenserine, which was previously accessible only by total synthesis.

Computational insight into the anticholinesterase activities and electronic properties of physostigmine analogs.

Aim: Alzheimer's disease (AD) is known to be themajor cause of dementia among the elderly. The structural properties and binding interactions of the AD drug physostigmine (-)-phy, and its analogues (-)-hex and (-)-phe and (+)-phe, were examined, as well as their impact on the conformational changes of two different AD target enzymes AChE and BChE. Materials & methods: The conformational changes were studied using molecular dynamics and structural properties using Quantum mechanics. Results & conclusions: The binding free energy (ΔGbind) and the change in the free energy surface (FES) computed from the funnel metadynamics (FMD) simulation, both support the idea that inhibitors (-)-phe and (-)-hex have better binding activities toward enzyme AChE, and that (-)-phe is stronger in binding than the present AD drug (-)-phy.

Acetylcholine suppresses shoot formation and callusing in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby.

We present experimental evidence to show that acetylcholine (ACh) causes decrease in shoot formation in leaf explants of tomato (Lycopersicon esculentum Miller var Pusa Ruby) when cultured on shoot regeneration medium. The optimum response was obtained at 10(-4) M ACh-enriched medium. ACh also causes decrease in percentage of cultures forming callus and reduces the callus mass. Inhibitors of enzymatic hydrolysis of ACh, neostigmine and physostigmine, also suppresses callogenesis and caulogenesis. On the other hand, the breakdown products of Ach, choline and acetate, do not alter the morphogenic response induced on the shoot regeneration medium. Neostigmine showed optimal reduction in shoot formation at 10(-5) M. The explants cultured on neostigmine augmented medium showed decline in the activity of ACh hydrolyzing enzyme acetylcholinesterase. ACh and neostigmine added together showed marked reduction in callus mass. These results strongly support the role of ACh as a natural regulator of morphogenesis in tomato plants.

Revival of physostigmine - a novel HPLC assay for simultaneous determination of physostigmine and its metabolite eseroline designed for a pharmacokinetic study of septic patients.

BACKGROUND: Physostigmine, commonly used as an antidote in anticholinergic poisoning, is reported to have additional pharmacological effects, such as activation of the cholinergic anti-inflammatory pathway in sepsis models. Due to the narrow therapeutic range of physostigmine and its metabolite eseroline, however, the plasma concentrations of these substances need to be determined so as to understand their effect and ensure safety in the treatment of septic patients. METHODS: To determine physostigmine and its metabolite eseroline, a rapid and sensitive high performance liquid chromatography (HPLC) method has been developed and validated. Spiked plasma samples were cleaned up and concentrated using a simple liquid-liquid extraction (LLE) procedure with N-methylphysostigmine as internal standard. Separation was achieved using reversed-phase HPLC on a Kinetex C18 column with gradient elution and fluorescence detection (254 nm excitation/355 nm emission). RESULTS: LLE produced clean extracts and a mean recovery of 80.3% for eseroline and 84.9% for physostigmine. The HPLC assay revealed a limit of detection (LOD) of 0.025 ng/mL and a lower limit of quantification (LLOQ) of 0.05 ng/mL for both analytes. Linearity was observed at 0.05-10.0 ng/mL (r²>0.999). Intra- and inter-day precision ranged from 0.7% to 6.6%, and intra- and inter-day accuracy 97.5%-110.0%. CONCLUSIONS: The presented method is useful for human drug level monitoring of physostigmine and eseroline in accordance with current guidelines. Remarkably low plasma concentrations can be quantified after LLE with gradient elution and fluorescence detection, making this a suitable method for pharmacokinetic studies in a clinical setting.

Acetylcholinesterase biosensor for carbamate drugs based on tetrathiafulvalene-tetracyanoquinodimethane/ionic liquid conductive gels.

A highly sensitive acetylcholinesterase biosensor was developed for detection of carbamate drugs based on TTF-TCNQ-ionic liquid gel thiocholine sensor. The TTF-TCNQ-ionic/ionic liquid gel was characterized by FT-IR and scanning electron microscopy. The electrocatalytic behavior of TTF-TCNQ-ionic liquid gels toward oxidation of thiocholine was thoroughly investigated. 1-Ethyl-3-methylimidazolium tetracyanoborate gel based sensor allowed amperometric detection of thiocholine at +400 mV vs. Ag/AgCl with a high sensitivity of 55.9±1.2 µA mM(-1)cm(-2) and a low detection limit equal to 7.6 µM. The catalytic rate constant and diffusion constant of thiocholine were estimated from chronoamperometric data. The proposed biosensor based on AChE immobilized in sol-gel matrix was used for the detection of two carbamate therapeutic drugs. Very low detection limits of 26 pM eserine and 0.3 nM neostigmine were achieved. The analysis of spiked tap water proved the biosensor capability to be used as a screening method for detection of carbamate drugs in wastewaters.

Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials.

Physostigmine and galanthamine bind in the presence of agonist at the canonical and noncanonical subunit interfaces of a nicotinic acetylcholine receptor.

Galanthamine and physostigmine are clinically used cholinomimetics that both inhibit acetylcholinesterase and also interact directly with and potentiate nAChRs. As with most nAChR-positive allosteric modulators, the location and number of their binding site(s) within nAChRs are unknown. In this study, we use the intrinsic photoreactivities of [(3)H]physostigmine and [(3)H]galanthamine upon irradiation at 312 nm to directly identify amino acids contributing to their binding sites in the Torpedo californica nAChR. Protein sequencing of fragments isolated from proteolytic digests of [(3)H]physostigmine- or [(3)H]galanthamine-photolabeled nAChR establish that, in the presence of agonist (carbamylcholine), both drugs photolabeled amino acids on the complementary (non-α) surface of the transmitter binding sites (γTyr-111/γTyr-117/δTyr172). They also photolabeled δTyr-212 at the δ-ß subunit interface and γTyr-105 in the vestibule of the ion channel, with photolabeling of both residues enhanced in the presence of agonist. Furthermore, [(3)H]physostigmine photolabeling of γTyr-111, γTyr-117, δTyr-212, and γTyr-105 was inhibited in the presence of nonradioactive galanthamine. The locations of the photolabeled amino acids in the nAChR structure and the results of computational docking studies provide evidence that, in the presence of agonist, physostigmine and galanthamine bind to at least three distinct sites in the nAChR extracellular domain: at the α-γ interface (1) in the entry to the transmitter binding site and (2) in the vestibule of the ion channel near the level of the transmitter binding site, and at the δ-ß interface (3) in a location equivalent to the benzodiazepine binding site in GABA(A) receptors.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is an attractive analytical tool for high-throughput screening because of its rapid scan time and ability to detect compounds without need for labels. Impediments to the use of ESI-MS for screening have been the relatively large sample consumed and slow sample introduction rates associated with commonly used flow injection analysis. We have previously shown that by segmenting nanoliter plugs of sample with air, an array of discrete samples can be delivered to a platinum-coated emitter tip for ESI-MS analysis with throughput as high as 0.8 Hz and carry-over between samples less than 0.1%. This method was applied to screening for inhibitors of acetylcholinesterase as a demonstration of the potential of segmented flow ESI-MS for such applications. Each enzyme assay consumed 10 nL of sample. At 1 microL/min infusion rate, 102 samples were analyzed, corresponding to a 0.65 Hz sample analysis rate. Linear quantification of choline was achieved from 200 microM to 10 mM using this method and Z' values were over 0.8 for the assay. Detailed pharmacologic dose-response curves of selected inhibitors were also measured in high-throughput fashion to validate the method.

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine.

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.

Differential effects of two hexahydropyrroloindole carbamate-based anticholinesterase drugs on the amyloid beta protein pathway involved in Alzheimer's disease.

One of the main hallmarks of Alzheimer's disease (AD) is the brain deposition of senile plaques made up of toxic amyloid beta-peptide (Abeta), which is derived from a larger protein called the beta-amyloid precursor protein (APP). Both APP processing and cholinesterase activity are affected in the AD brain, but, yet, cholinesterase inhibitors (ChEI) remain the primary Food and Drug Administration approved drugs for AD within the United States. Herein, we evaluated the effects of two clinically relevant drugs on the APP pathway, which is presumably involved in AD pathogenesis. Specifically, we compared the actions of the classical ChEI physostigmine (PHY) and its analog phenserine (PHE) on neuronal cell viability, on IC50 and on levels of different amyloid proteins. Interestingly, these drugs share the same chemical backbone, inhibit acetylcholinesterase with similar potency, but differentially affect APP processing. PHE treatment decreased levels of APP in the human neuroblastoma cells (p=0.009) whereas PHY showed a similar but less-pronounced trend, which did not attain statistical significance. PHE treatment significantly decreased levels of Abeta in human neuroblastoma cells (p=0.02) whereas PHY showed no significant change under the same conditions. The divergent actions of these two structurally related drugs on the amyloid pathway indicate that the mechanisms underpinning the cholinergic and the amyloid-lowering properties for this class of drugs are independent of each other.

In vivo butyrylcholinesterase activity is not increased in Alzheimer's disease synapses.

OBJECTIVE: We tested the premise that cholinesterase inhibitor therapy should target butyrylcholinesterase (BuChE) in Alzheimer's disease (AD), not acetylcholinesterase (AChE) alone, because both enzymes hydrolyze acetylcholine, and BuChE is increased in AD cerebral cortex. METHODS: To examine this issue in vivo, we quantified human cerebral cortical BuChE activity using tracer kinetic estimates (k(3)) of 1-[(11)C]methyl-4-piperidinyl n-butyrate ([(11)C]BMP) hydrolysis determined by positron emission tomography. Validation of the putative positron emission tomography method included regional distribution, positive correlation with age, and attenuation by the nonselective cholinesterase inhibitor physostigmine, but no attenuation by the AChE-selective inhibitor donepezil. Positron emission tomography scans in AD patients (n = 15) and control subjects (n = 12) measured both BuChE (using [(11)C]BMP) and AChE activity (using N-[(11)C] methylpiperidin-4-yl propionate, an established method). RESULTS: As expected, AChE activity in AD cerebral cortex was decreased to 75 +/- 13% of normal (p = 0.00001). Contrary to prediction, accompanying BuChE activity also was decreased to 82 +/- 14% of normal (p = 0.001). INTERPRETATION: Failure to observe increased [(11)C]BMP hydrolysis in vivo makes it less likely that incremental BuChE contributes importantly to acetylcholine hydrolysis in AD. The findings do not support the premise that inhibitor therapy should target BuChE so as to prevent increased levels of BuChE from hydrolyzing acetylcholine in AD cerebral cortex.

Characterization of cholinesterase activity from different tissues of Nile tilapia (Oreochromis niloticus).

Cholinesterases (ChE) from brain, muscle and liver in Nile tilapia (Oreochromis niloticus) were characterized using three substrates: acetylthiocholine iodide, propionylthiocholine iodide, and butyrylthiocholine iodide. Eserine was used as a total ChE inhibitor; BW284c51 and iso-OMPA were used as selective inhibitors for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), respectively. The results indicate that AChE is the enzyme present in brain, whereas in both liver and muscle, the presence of atypical ChEs are suggested. These findings indicate that characterization of ChE is necessary prior to use in monitoring programs.

Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay.

In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H(2)O(2) system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-serum-HRP-H(2)O(2) system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at < or =4 degrees C. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O(2) uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta-methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme.

Involvement of cholinergic mechanisms in the central control of respiration in the cane toad, Bufo marinus.

Chemical substrates, central sites and central mechanisms underlying the regulation of breathing in lower vertebrates have not been well characterized. The present study was undertaken to determine the effect of pH changes and cholinergic agents on the central control of respiration in the cane toad, Bufo marinus. Adult toads were anesthetized, catheterized and unidirectionally ventilated before exposing the brainstem. An airtight buccal cannula was also inserted through the tympanum to record buccal pressure. The animal was decerebrated, anesthetic removed and the responses to pH changes of solutions bathing the ventral surface of the medulla (VSM) were tested by superfusing the VSM with mock cerebrospinal fluid (mCSF) of pH 7.8-normal, 7.2-acidic and 8.4-basic. The acidic solution increased respiratory activity, the basic solution decreased activity and the normal solution had no effect. In addition, cholinergeric agents (acetylcholine-ACh, physostigmine-Phy, nicotine-Nic, and atropine-Atr) dissolved in mCSF were applied bilaterally onto the VSM using filter paper pledgets. ACh, Phy and Nic increased episodic breathing frequency by 14.3+/-9.7, 9.4+/-5.4 and 29.1+/-11.8 %, respectively, whereas, Atr caused a decrease (-26.6+/-16.6%). These agents had no effect on blood pressure. It is therefore, concluded that the VSM is pH sensitive and a cholinergic mechanism is involved in the central modulation of respiration in Bufo.

Quantitative measurement of cerebral acetylcholinesterase using.

[11C]physostigmine, an acetylcholinesterase inhibitor, has been shown to be a promising positron emission tomography ligand to quantify the cerebral concentration of the enzyme in animals and humans in vivo. Here, a quantitative and noninvasive method to measure the regional acetylcholinesterase concentration in the brain is presented. The method is based on the observation that the ratio between regions rich in acetylcholinesterase and white matter, a region almost entirely deprived of this enzyme, was found to become approximately constant after 20 to 30 minutes, suggesting that at late time points the uptake mainly contains information about the distribution volume. Taking the white matter as the reference region, a simplified reference tissue model, with effectively one reversible tissue compartment and three parameters, was found to give a good description of the data in baboons. One of these parameters, the ratio between the total distribution volumes in the target and reference regions, showed a satisfactory correlation with the acetylcholinesterase concentration measured postmortem in two baboon brains. Eight healthy male subjects were also analyzed and the regional enzyme concentrations obtained again showed a good correlation with the known acetylcholinesterase concentrations measured in postmortem studies of human brain.

Limited donepezil inhibition of acetylcholinesterase measured with positron emission tomography in living Alzheimer cerebral cortex.

Based on surrogate assays of peripheral red blood cells, reports state that widely prescribed doses of donepezil hydrochloride provide nearly complete inhibition of cerebral cortical acetylcholinesterase activity in the treatment of Alzheimer's disease (AD). To test this, direct positron emission tomography measures of cerebral acetylcholinesterase activity were made in AD patients before and after treatment with donepezil (5 and 10 mg/day) for at least 5 weeks and compared with similar measures in normal controls who were untreated or after acute administration of another AChE inhibitor, physostigmine salicylate (1.5 mg/hr). After physostigmine, acetylcholinesterase inhibition averaged 52% in normal cerebral cortex. After donepezil, cerebral cortical inhibition in AD brain averaged only 27%. Clinical trials of this donepezil dose schedule are not testing the effect of nearly complete cerebral cortical inhibition.

Influence of muscarinic ligands on the amplitudes of the evoked surface potential's late components in the optic tectum of the urodele Plethodon jordani.

Recordings of field potentials from the tectal surface of an urodele amphibian were obtained in an in vitro preparation under influence of various muscarinic drugs. Bath applied acetylcholine (ACh) led to no change in the amplitudes or the shape of the evoked potentials. If the ACh-esterase blocker (-)-physostigmine was applied synchronously, the late components of the surface potential increased in amplitude. The non-selective cholinergic agonist carbachol showed a similar effect which was partially diminished by the nicotinic antagonist d-tubocurarine chloride (d-TC) and the muscarinic antagonist atropine sulfate. The application of the non-selective muscarinic agonist (+)-pilocarpine hydrochloride led to an increase of the late oligo- and polysynaptic events. This effect was reduced by the M(1)-antagonist pirenzepine dihydrochloride. The presented findings suggest that muscarinic receptors play a more important role in tectal processing than assumed in previous studies which emphasized the role of nicotinic receptors.

Using single photon emission tomography (SPECT) and positron emission tomography (PET) to trace the distribution of muscarinic acetylcholine receptor (MACHR) binding radioligands.

Two [18F] labeled ligands for the mAChR were prepared and evaluated in rodents and nonhuman primates. The properties of both compounds, one an agonist and the other an antagonist, were consistent with M2 subtype specificity.

Development and in vivo assessment of a transdermal system for physostigmine.

A copolymer was developed as a transdermal (TD) system for physostigmine. The loading was carried out with a solution of physostigmine (PHY) base (20 mg/ml) in water/ethanol: 80/20 (v/v) at 40 degrees C for 3 h. The PHY load was 5.3 mg/cm2 (n = 3). Desorption carried out in vitro showed that 70% was desorbed during the first 6 h. More than 50% of the PHY was degraded within 45 min in skin homogenate. The TD was tested in vivo in rabbits during a 24 h experiment. PHY was quantified using a validated HPLC method. AUC0-24 h was 245.2 +/- 337.2 h.ng/ml. The mean pad flux reached 4.6 +/- 6.3 micrograms/cm2 from 0 to 24 h and, 24 h after the application of the pad, 110 micrograms/cm2 of PHY had been passed through the skin. After removed of the patch, plasma concentrations first increased from 15.8 +/- 28.6 ng/ml (at 24 h) to 21.4 +/- 36.7 ng/ml, then decreased with an elimination half-life of 0.7 +/- 0.2 h. AChE inhibition percentages increased from 6.5 +/- 2.3% to 16.0 +/- 27.7%. In vitro and in vivo studies in rabbit have shown that this system is suitable for further investigations in order to obtain a possible carrier for PHY therapy.