Discovery of new acetylcholinesterase inhibitors for Alzheimer's disease: virtual screening and in vitro characterisation.

For more than two decades, the development of potent acetylcholinesterase (AChE) inhibitors has been an ongoing task to treat dementia associated with Alzheimer's disease and improve the pharmacokinetic properties of existing drugs. In the present study, we used three docking-based virtual screening approaches to screen both ZINC15 and MolPort databases for synthetic analogs of physostigmine and donepezil, two highly potent AChE inhibitors. We characterised the in vitro inhibitory concentration of 11 compounds, ranging from 14 to 985 µM. The most potent of these compounds, S-I 26, showed a fivefold improved inhibitory concentration in comparison to rivastigmine. Moderate inhibitors carrying novel scaffolds were identified and could be improved for the development of new classes of AChE inhibitors.

Multitarget drugs of plants origin acting on Alzheimer's disease.

The etiopathology of Alzheimer's disease (AD) is extremely complex and heterogeneous, often associated with comorbidities. As a result it may be unlikely that AD may be mitigated by drug acting on a single specific target. The current tendency in drug design and discovery in AD is the rational design or "serendipitous" discovery of new drug entities challenging multiple targets. Since two of the presently approved drugs for AD are based on natural products (galantamine and the physostigmine-derivative rivastigmine), many plants are now under investigation as a potential source of new drugs. Multifunctional drugs often have their origin in natural sources. This review is limited to plant chemicals having different targets with actual (galantamine) or promising (drugs from Crocus sativus, Ginkgo biloba, Salvia species, and Huperzia serrata) clinical evidence in people with dementia or AD.

Improved thin-layer chromatography bioautographic assay for the detection of actylcholinesterase inhibitors in plants.

INTRODUCTION: Thin-layer chromatography (TLC) bioautographic method is a simple and rapid method to screen acetylcholinesterase inhibitors from plant extracts. However, the high consumption of enzyme (6 U/mL) in current methods makes the procedure expensive, which is an obstacle to scientific research centers lacking funding. OBJECTIVE: To develop a new low-cost TLC bioautographic method. METHODOLOGY: A series of compounds, as substrates, were synthesised and their ability to be hydrolysed by acetylcholinesterase was evaluated by the HPLC method. RESULTS: 4-Methoxyphenyl acetate (14) was proved to be an appropriate substrate for TLC bioautographic assay. Therefore a new and cheap TLC bioautographic assay was set up. The mechanism of this new method is that the enzyme converts 4-methoxylphenyl acetate into 4-methoxyphenol, which reacts with a solution of potassium ferricyanide ([K3(FeCN)6]) and iron chloride hexahydrate (FeCl3·6H2O) to make an aquamarine blue coloured background on the TLC plates. Regions of the TLC plate which contain acetylcholinesterase inhibitors show up as light yellow spots against the background. The consumption of enzyme (1 U/mL) in the new method is low and the detection limit of two known acetylcholinesterase inhibitors, huperzine A (0.0001 µg) and physostigmine (0.001 µg), for this assay are close to published values. CONCLUSION: A low-cost TLC bioautographic method was developed, which will benefit research groups pursuing natural acetylcholinesterase inhibitors.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species.

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.

Molybdenum-catalyzed asymmetric allylic alkylation of 3-alkyloxindoles: reaction development and applications.

We report a full account of our work towards the development of Mo-catalyzed asymmetric allylic alkylation reactions with 3-alkyloxindoles as nucleophiles. The reaction is complementary to the Pd-catalyzed reaction with regard to the scope of oxindole nucleophiles. A number of 3-alkyloxindoles were alkylated successfully under mild conditions to give products with excellent yields and good-to-excellent enantioselectivities. Applications of this method to the preparation of indoline alkaloids such as (-)-physostigmine, ent-(-)-debromoflustramine B, and the indolinoquinoline rings of communesin B are reported.

Tetrahydrofurobenzofuran cymserine, a potent butyrylcholinesterase inhibitor and experimental Alzheimer drug candidate, enzyme kinetic analysis.

Synaptic loss, particularly related to the forebrain cholinergic system, is considered to be an early event that leads to Alzheimer's disease (AD) and has led to the development of acetylcholinesterase inhibitors (AChE-Is) as the mainstay of treatment for several degenerative disorders that culminate in dementia. The primary dose-limiting toxicities of all clinically available AChE-Is are, similar to useful actions on cognition, cholinergically mediated and they ultimately limit the value of this drug class in achieving anything but symptomatic improvements. In addition, AChE levels in brain areas associated with AD decline with disease progression, which likely ultimately limits the therapeutic utility of this drug class. New research indicates that selective inhibition of butyrylcholinesterase (BuChE), a closely related enzyme that is markedly elevated in AD brain, increases acetylcholine (ACh) and augments cognition in rodents free of the characteristic undesirable actions of AChE-Is. BuChE inhibition hence represents an innovative treatment approach for AD, and agents are currently being synthesized to optimally achieve this. The novel compound, tetrahydrofurobenzofuran cymserine (THFBFC), is derived from our effort to produce a potent and BuChE-selective inhibitor as a candidate to test the hypothesis that BuChE-Is would be efficacious and better tolerated than AChE-Is in AD. Herein, we applied innovative enzyme kinetic analyses to characterize the quantitative interaction of THFBFC with human BuChE. These provided values for the agent's IC(50), together with specific new kinetic constants, such as K (T50), K (T1/2), R (I), (o)K (RT), (o)P(max), K(PT) and PT(1/2), to aid define target concentrations for clinical translation. Additional classical kinetic parameters, including K(i), K(m)or K(s), k(cat) or V(max) and V (mi) were also determined. THFBFC proved to be a potent competitive inhibitor of human BuChE and, like its isomer dihydrobenzodioxepine cymserine, is a potentially interesting AD drug candidate.

Cardio-selective inhibitory effect of the betel nut extract: possible explanation.

Chewing of betel nut, the seed of Areca catechu, is associated with a host of physical and psychological effects while it is also traditionally used in constipation and hypertension. In this study, we report the cardio-selective cholinomimetic activity of the betel nut crude extract (Ac.Cr). Ac.Cr, that tested positive for saponins, tannins, phenols, alkaloids and terpenes, exhibited dose-dependent atropine-sensitive inhibition of isolated guinea-pig atrial contractility with an EC50 value of 0.93 microg/ml (0.57-1.51, 95% CI). In rabbit jejunum, Ac.Cr showed atropine-sensitive spasmogenicity with an EC50 of 7.31 microg/ml (5.41-9.88, 95% CI) showing that it is around 8 times more potent in the cardiac than the intestinal preparation. Both carbachol and physostigmine exhibited acetylcholine-like stimulant activity in jejunum with the latter being more potent in jejunum than in atrial tissues. Activity-directed fractionation of Ac.Cr yielded fractions with similar cholinergic activity in atria and jejunum except the aqueous fraction being 6 times more potent in the atria. Arecoline, the known betel nut compound with cholinergic activity showed similar potency in both tissues while catechin and tannic acid exhibited intestinal spasmolytic effect but were inactive in atria. The results show the cardio-selective inhibitory effect of Ac.Cr which might possibly be due to selective gut-spasmolytic behaviour of catechin and tannic acid thus reducing the cholinomimetic activity of Ac.Cr in the gut though the preferential binding of the constituents of betel nut extract at muscarinic receptor subtypes in heart cannot be ignored.

Identification of novel small molecule inhibitors of amyloid precursor protein synthesis as a route to lower Alzheimer's disease amyloid-beta peptide.

A wealth of independent research with transgenic mice, antibodies, and vaccines has pointed to a causative role of the amyloid-beta peptide (A beta) in Alzheimer's disease (AD). Based on these and earlier associative studies, A beta represents a promising target for development of therapeutics focused on AD disease progression. Interestingly, a cholinesterase inhibitor currently in clinical trials, phenserine, has been shown to inhibit production of both amyloid precursor protein (APP) and A beta. We have shown that this inhibition occurs at the post-transcriptional level with a specific blocking of the synthesis of APP relative to total protein synthesis (Shaw et al., 2001). However, the dose of phenserine necessary to block APP production is far higher than that needed to elicit its anticholinesterase activity, and it is these latter actions that are dose limiting in vivo. The focus of this study was to screen 144 analogs of phenserine to identify additional small molecules that inhibit APP protein synthesis, and thereby A beta production, without possessing potent acetylcholinesterase (AChE) inhibitory activity. An enzyme-linked immunosorbent assay was used to identify analogs capable of suppressing APP production following treatment of human neuroblastoma cells with 20 muM of compound. Eight analogs were capable of dose dependently reducing APP and A beta production without causing cell toxicity in further studies. Several of these analogs had little to no AChE activities. Translation of APP and A beta actions to mice was demonstrated with one agent. They thus represent interesting lead molecules for assessment in animal models, to define their tolerance and utility as potential AD therapeutics.

Skin permeation of physostigmine from fatty acids-based formulations: evaluating the choice of solvent.

This study was conducted to gain an understanding of the enhancement mechanism of fatty acids in skin permeation of physostigmine (PHY) by using a series of fatty acids and two solvents of opposing lipophilicity (propylene glycol (PG) and mineral oil (MO)). Interaction between fatty acid and drug was proven using NMR and conductivity measurements that showed a dependence on type of solvent used. Permeation flux of physostigmine from mineral oil-based formulations to skin was increased as solubility of physostigmine in mineral oil was enhanced in the presence of fatty acids having a longer chain. Thus, the dominant role of fatty acids in mineral oil was to increase solubility of physostigmine in the formulations that increased the driving force for physostigmine permeation through skin. As for propylene glycol, enhancement caused by fatty acids was attributed to their ability to increase the lipophilicity of formulation and to disrupt the lipid bilayers within the stratum corneum (SC). In conclusion, fatty acids enhancement for drug permeation across the skin was found to be dependent on the solvent used. Among various formulations here, oleic acid in mineral oil yielded fast permeation of PHY with a short lag time, which may be a good vehicle for transdermal delivery of PHY.