Pharmacophore-based design and discovery of (-)-meptazinol carbamates as dual modulators of cholinesterase and amyloidogenesis.

Multifunctional carbamate-type acetylcholinesterase (AChE) inhibitors with anti-amyloidogenic properties like phenserine are potential therapeutic agents for Alzheimer's disease (AD). We reported here the design of new carbamates using pharmacophore model strategy to modulate both cholinesterase and amyloidogenesis. A five-feature pharmacophore model was generated based on 25 carbamate-type training set compounds. (-)-Meptazinol carbamates that superimposed well upon the model were designed and synthesized, which exhibited nanomolar AChE inhibitory potency and good anti-amyloidogenic properties in in vitro test. The phenylcarbamate 43 was highly potent (IC50 31.6 nM) and slightly selective for AChE, and showed low acute toxicity. In enzyme kinetics assay, 43 exhibited uncompetitive inhibition and reacted by pseudo-irreversible mechanism. 43 also showed amyloid-ß (Aß) lowering effects (51.9% decrease of Aß42) superior to phenserine (31% decrease of total Aß) in SH-SY5Y-APP695 cells at 50 µM. The dual actions of 43 on cholinergic and amyloidogenic pathways indicated potential uses as symptomatic and disease-modifying agents.

A novel assay to determine acetylcholinesterase activity: The application potential for screening of drugs against Alzheimer's disease.

Acetycholinesterase (AChE) that regulates hydrolysis of acetylcholine (ACh) in the brain, is an important target for treatment of Alzheimer's disease (AD), a feature of which is ACh deficiency. However, the methods to precisely determine AChE activity are still under development. We developed a new method to exploit acetylcholine-d4 as a surrogate substrate of ACh and measure product choline-d4 via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay detected activity of AChE present in the normal mouse brain, which is consistent with the standard Ellman assay that determines products spectrophotometrically. In AD mouse models, the result of LC-MS/MS assay showed significant higher AChE activity than that seen in control normal mice, while treatment of AD mice with an AChE inhibitor, huperzine A, led to partial decreases in AChE activity. Our results suggest that this surrogate-based LC-MS/MS method is a new, sensitive and convenient assay for the determination of AChE activity, providing a useful means for screening active compounds that target AChE.

Determination of a novel carbamate AChE inhibitor meserine in mouse plasma, brain and rat plasma by LC-MS/MS: application to pharmacokinetic study after intravenous and subcutaneous administration.

In this paper a simple and sensitive method for determination of a novel phenylcarbamate AChE inhibitor, meserine, in mouse plasma, brain and rat plasma was evaluated using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Separation was achieved on an Alltech Alltima-C18 column (150mm×2.1mm, 3µm, Deerfield, IL, USA) with isocratic elution at a flow rate of 0.35ml/min. Detection was performed under the multiple reaction monitoring (MRM) mode using an electrospray ionization (ESI) in the positive ion mode. The protein precipitation and liquid-liquid extraction methods were used for the pretreatment of plasma and brain homogenates, respectively. The calibration curves of meserine showed good linearity over the concentration range of 0.5-1000ng/ml for mouse and rat plasma and 0.5-500ng/ml for mouse brain. The intra- and inter-day precision were less than 9.34% and the accuracy was from 95.34% to 107.78% for QC samples. The validated method was successfully applied to a preclinical pharmacokinetic study of meserine in mice and rats after intravenous and subcutaneous administration. The results showed that this novel drug could easily cross the blood-brain barrier to reach the site of drug action. Meserine was rapidly absorbed with a high subcutaneous absolute bioavailability (>90%).

Meserine, a novel carbamate AChE inhibitor, ameliorates scopolamine-induced dementia and alleviates amyloidogenesis of APP/PS1 transgenic mice.

AIMS: To investigate whether Meserine, a novel phenylcarbamate derivative of (-)-meptazinol, possesses beneficial activities against cholinergic deficiency and amyloidogenesis, the two major pathological characteristics of Alzheimer's disease (AD). METHODS: Ellman's assay and Morris water maze were used to detect acetylcholinesterase (AChE) activity and evaluate spatial learning and memory ability, respectively. Both high content screening and Western blotting were carried out to detect ß-amyloid precursor protein (APP), while RT-PCR and ELISA were conducted to detect APP-mRNA and ß-amyloid peptide (Aß). RESULTS: In scopolamine-induced dementia mice, Meserine (1 mg/kg, i.p.) significantly ameliorated spatial learning and memory deficits, which was consistent with its in vitro inhibitory ability against AChE (recombinant human AChE, IC50 = 274 ± 49 nM). Furthermore, Meserine (7.5 mg/kg) injected intraperitoneally once daily for 3 weeks lowered APP level by 28% and Aß42 level by 42% in APP/PS1 transgenic mouse cerebrum. This APP modulation action might be posttranscriptional, as Meserine reduced APP by about 30% in SH-SY5Y-APP695 cells but did not alter APP-mRNA level. And both APP and Aß42 lowering action of Meserine maintained longer than that of rivastigmine. CONCLUSION: Meserine executes dual actions against cholinergic deficiency and amyloidogenesis and provides a promising lead compound for symptomatic and modifying therapy of AD.

Novel bis-(-)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property.

The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(-)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC(50) values of 9.63µM (for ZLA) and 8.64µM (for ZLB), and prevent AChE-induced amyloid-ß (Aß) aggregation with IC(50) values of 49.1µM (for ZLA) and 55.3µM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aß aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD.

Surrogate based accurate quantification of endogenous acetylcholine in murine brain by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Cholinergic dysfunction is known as a hallmark feature of Alzheimer's disease (AD). Measurement of endogenous acetylcholine (ACh) in specific brain regions is important in understanding the pathology of AD and in designing and evaluating novel cholinomimetic agents for the treatment of AD. Since ACh is an endogenous neurotransmitter, there is no real blank matrix available to construct standard curves. It has been a challenging task to determine ACh in complex brain matrices. To overcome these difficulties, we employed a surrogate analyte strategy using ACh-d(4) instead of ACh to generate calibration curves and Ch-d(9) as internal standard (IS). The brain samples were deproteinized by acetonitrile with IS. Analytes and IS were separated by a HILIC column with the mobile phase composed of 20 mM ammonium formate in water-acetonitrile (30:70, v/v, adjusted to pH 3.0 with formic acid) and monitored in multiple reaction monitoring (MRM) mode using a positive electrospray source. The concentrations of endogenous ACh were calculated based on the peak area ratio of the analyte to the IS using a regression equation for the corresponding surrogate standard (ACh-d(4)). The lower limit of detection was 0.2 ng/mL and linearity was maintained over the range of 10-1000 ng/mL. Compared to other currently available methods, this approach offers improved accuracy and precision for efficient analysis of ACh. The proposed method was proved successfully by evaluating the action of typical acetylcholinesterase inhibitor huperzine A in senescence accelerated mouse prone 8 (SAMP8).

Pilocarpine protects cobalt chloride-induced apoptosis of RGC-5 cells: involvement of muscarinic receptors and HIF-1 alpha pathway.

The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl(2))-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1 alpha (HIF-1 alpha), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 microM CoCl(2) for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 microM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 microM pilocarpine could significantly prevent CoCl(2)-induced HIF-1 alpha translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1 alpha, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1 alpha pathway. The findings suggest that HIF-1 alpha pathway as a "master switch" may be used as a therapeutic target in the cholinergic treatment of glaucoma.

Peripheral cholinoceptor antagonist anisodamine counteracts cholinergic adverse effects and facilitates cognitive amelioration of rivastigmine.

Rivastigmine is a potent acetyl- and butyrylcholinesterase inhibitor widely used for cognitive improvement in Alzheimer's disease (AD) therapy. However, dose-limiting adverse effects restrict its tolerability and clinical outcomes. This study explored new combined therapy, in which peripheral cholinergic adverse effects and central cognitive amelioration of rivastigmine were differentiated by a peripheral cholinoceptor antagonist anisodamine. The results demonstrated that rivastigmine (0.75 and 2.0 mg/kg) could significantly reverse the scopolamine-induced cognitive deficit in mice through passive avoidance test. Nevertheless, a high dose of rivastigmine (3.25 mg/kg) would compromise cognitive amelioration and produce obvious adverse effects, including hypersalivation, intestinal hyperperistalsis and muscle cramp. Interestingly, concomitant administration of anisodamine (10 mg/kg) effectively counteracted both the muscarinergic and nicotinergic adverse effects, while facilitating cognitive amelioration of rivastigmine (3.25 mg/kg). These findings provide an insight into the feasibility of combined therapy with cholinesterase inhibitors and peripheral cholinoceptor antagonists for the treatment of AD.

Novel piperazine derivative PMS1339 exhibits tri-functional properties and cognitive improvement in mice.

Amyloid-beta-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimer's disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Abeta aggregation inhibition and cognitive improvement. PMS1339 could significantly inhibit both mice brain AChE (IC50=4.41+/-0.63 microM) and sera butyrylcholinesterase (BuChE, IC50=1.09+/-0.20 microM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95+/-2.31 microM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Abeta aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimer's disease.

Muscarinic activation attenuates abnormal processing of beta-amyloid precursor protein induced by cobalt chloride-mimetic hypoxia in retinal ganglion cells.

beta-Amyloid peptide (Abeta), the major pathological factor in Alzheimer's disease, has recently been reported to be implicated in the development of glaucoma. In this study, we explored the effect of muscarinic activation on abnormal processing of beta-amyloid precursor protein (APP) induced by a risk factor hypoxia in retinal ganglion cells. Hypoxia mimetic compound cobalt chloride could increase the generation of Abeta via up-regulating the expression of APP as well as the expression of beta-secretase and gamma-secretase, whereas muscarinic receptor agonist pilocarpine could significantly attenuate this abnormal pathway, thereby resulting in a decreased amyloidogenic cleavage of APP. This finding may provide an insight into better understanding of pathophysiology for the retinal neurodegenerative disease and searching for its new modifying approach.

Protection of PMS777, a new AChE inhibitor with PAF antagonism, against amyloid-beta-induced neuronal apoptosis and neuroinflammation.

Amyloid-beta (Abeta) plays a central role in the neuroinflammation and cholinergic neuronal apoptosis in Alzheimer's disease, and thus has been considered as a main determinant of this disease. In the previous study, we reported that PMS777, a novel bis-interacting ligand for acetylcholinesterase (AChE) inhibition and platelet-activating factor (PAF) receptor antagonism, could significantly attenuate PAF-induced neurotoxicity. Continuing our efforts, we further investigated the protective effect of PMS777 on Abeta-induced neuronal apoptosis in vitro and neuroinflammation in vivo. PMS777 (1-100 microM) was found to inhibit Abeta-induced human neuroblastoma SH-SY5Y cell apoptosis in a concentration-dependent manner. Concurrently, PMS777 increased ratio of bcl-2 to bax mRNA, and inhibited both mRNA expression and activity of caspase-3 in SH-SY5Y cells after the exposure with Abeta. In vivo experimental study demonstrated that PMS777 could attenuate Abeta-induced microglial and astrocytic activation in the rat hippocampus after systemic administration. These results suggest that PMS777 potently protects against Abeta-induced neuronal apoptosis and neuroinflammation, and warrants further investigations in connection with its potential value in the treatment of Alzheimer's disease.

PMS777, a bis-interacting ligand for PAF receptor antagonism and AChE inhibition, attenuates PAF-induced neurocytotoxicity in SH-SY5Y cells.

(1) HIV-1 and viral proteins-evoked chronic brain inflammation, which is characterized by microglial activation, is the pivotal neuropathogenesis of HIV-1-associated dementia (HAD). Platelet-activating factor (PAF), mainly released from activated microglia and acts as a high potent inflammatory mediator and a neurotoxin, is indicated to be a principle initiator of neuroinflammation, neuronal dysfunction, and apoptosis related to HAD. Thus, bis-interacting ligands of acetylcholinesterase (AChE) inhibition and PAF receptor antagonism would be of great interest in the therapeutic potential of HAD not only for improvement of cognitive performance, but also for disease-modifying. (2). We have previously reported that a novel tetrahydrofuran-derived bis-interacting ligand PMS777 had satisfying potencies for PAF receptor blockade and AChE inhibition, and markedly improved cholinergic dysfunction-induced cognitive impairment in mice. Continuing with our research, we further investigated the neuroprotective activities of PMS777 on PAF-triggered neuronal injury in human neuroblastoma SH-SY5Y cells. (3) The bis-interacting ligand PMS777 (10 muM) obviously alleviated PAF-induced cell apoptosis in SH-SY5Y cells. Pretreatment with PMS777 also markedly inhibited intracellular Ca(2+) overload, down-regulation of anti-apoptotic bcl-2 mRNA, stimulation of pro-apoptotic bax mRNA expression and activation of caspase-3 pathway. Also, PMS777 could fine-tune pro-inflammatory cyclooxygenase-2 (cox-2) mRNA expression in PAF-treated cells. (4) These results suggest that PMS777 possesses a neuroprotective profile via anti-apoptotic/inflammatory signaling and warrant further investigations in connection with the potential value of this compound in HAD treatment.