Potent Butyrylcholinesterase Inhibitors Research Articles

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.

Dialkyl phenyl phosphates as novel selective inhibitors of butyrylcholinesterase

A series of dialkyl phenyl phosphates (DAPPs) were synthesized and evaluated in silico and in vitro for inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Among the compounds examined, several DAPPs were shown to be potent inhibitors of butyrylcholinesterase, while having little activity against acetylcholinesterase. The most potent and selective inhibitors were di- n-butyl phenyl phosphate ( K i = 43 μM), di- n-pentyl phenyl phosphate ( K i = 6 μM), and di-cyclohexyl phenyl phosphate ( K i = 7 μM), the first which was shown to be a competitive inhibitor while the latter two being partial competitive inhibitors. Flexible docking simulations suggested that relative binding affinities generally increased as a function of alkyl chain length, while the strength and nature of inhibitory activity depended on whether the compound bound deeply or midway in the active site gorge, or in the proposed peripheral site.

Rivastigmine is a potent inhibitor of acetyl- and butyrylcholinesterase in Alzheimer's plaques and tangles

Acetylcholinesterase and butyrylcholinesterase activities emerge in association with plaques and tangles in Alzheimer's disease. These pathological cholinesterases, with altered properties, are suggested to participate in formation of plaques. The present experiment assessed the ability of rivastigmine, a clinically utilized agent that inhibits acetylcholinesterase and butyrylcholinesterase activities, to inhibit cholinesterases in plaques and tangles. Cortical sections from cases of Alzheimer's disease were processed using cholinesterase histochemistry in the presence or absence of rivastigmine. Optical densities of stained sections were utilized as a measure of inhibition. The potency of rivastigmine was compared with those of other specific inhibitors. Optimum staining for cholinesterases in neurons and axons was obtained at pH 8.0. Cholinesterases in plaques, tangles and glia were stained best at pH 6.8. Butyrylcholinesterase-positive plaques were more numerous than acetylcholinesterase-positive plaques. Rivastigmine inhibited acetylcholinesterase in all positive structures in a dose-dependent manner (10 −6–10 −4 M). However, even at the highest concentration, faint activity remained. In contrast, rivastigmine resulted in complete inhibition of butyrylcholinesterase in all structures at 10 −5 M. Rivastigmine was equipotent to the specific acetylcholinesterase inhibitor BW284C51 and more potent than the butyrylcholinesterase inhibitors iso-OMPA and ethopropazine. In conclusion, rivastigmine is a potent inhibitor of acetylcholinesterase and a more potent inhibitor of butyrylcholinesterase in plaques and tangles. Unlike other cholinesterase inhibitors tested, rivastigmine inhibited cholinesterases in normal and pathological structures with the same potency. Thus, at the therapeutic concentrations used, rivastigmine is likely to result in inhibition of pathological cholinesterases, with the potential of interfering with the disease process.

A practical conversion of natural physostigmine into the potent butyrylcholinesterase inhibitor N1, N8-bisnorcymserine

A rapid novel synthetic route to the potent reversible butyrylcholinesterase inhibitor (−)- N 1, N 8-bisnorcymserine ( 1) is reported from physostigmine ( 2) in a 20% total yield. Details on the formation of the imino-quinone 6 obtained in the oxidation of N 1-benzylnoresermethole ( 4) and its conversion into N 1-bisnoreseroline ( 7) are given. As expected, the product of this synthesis, ( 1), had identical biological activity to the same agent produced by total synthesis.

A comparative study in rats of the in vitro and in vivo pharmacology of the acetylcholinesterase inhibitors tacrine, donepezil and NXX-066

The in vitro and in vivo effects of the novel acetylcholinesterase inhibitors donepezil and NXX-066 have been compared to tacrine. Using purified acetylcholinesterase from electric eel both tacrine and donepezil were shown to be reversible mixed type inhibitors, binding to a similar site on the enzyme. In contrast, NXX-066 was an irreversible non-competitive inhibitor. All three compounds were potent inhibitors of rat brain acetylcholinesterase (IC 50 [nM]; tacrine: 125±23; NXX-066: 148±15; donepezil: 33±12). Tacrine was also a potent butyrylcholinesterase inhibitor. Donepezil and tacrine displaced [ 3H]pirenzepine binding in rat brain homogenates (IC 50 values [μM]; tacrine: 0.7; donepezil: 0.5) but NXX-066 was around 80 times less potent at this M 1-muscarinic site. Studies of carbachol stimulated increases in [Ca 2+]i in neuroblastoma cells demonstrated that both donepezil and tacrine were M 1 antagonists. Ligand binding suggested little activity of likely pharmacological significance with any of the drugs at other neurotransmitter sites. Intraperitoneal administration of the compounds to rats produced dose dependent increases in salivation and tremor (ED 50 [μmol/kg]; tacrine: 15, NXX-066: 35, donepezil: 6) with NXX-066 having the most sustained effect on tremor. Following oral administration, NXX-066 had the slowest onset but the greatest duration of action. The relative potency also changed, tacrine having low potency (ED 50 [μmol/kg]; tacrine: 200, NXX-066: 30, donepezil: 50). Salivation was severe only in tacrine treated animals. Using in vivo microdialysis in cerebral cortex, both NXX-066 and tacrine were found to produce a marked (at least 30-fold) increase in extracellular acetylcholine which remained elevated for more than 2 h after tacrine and 4 h after NXX-066.

Amitriptyline: a potent inhibitor of butyrylcholinesterase from human serum

1. 1. The effect of amitriptyline on human serum butyrylcholinesterase (acylcholine acylhydrolase E.C.3.1.1.8) has been investigated. From the Lineweaver-Burk plot and the plot of v versus amitriptyline concentration, it was concluded that amitriptyline inhibition is partially competitive, and the kinetic parameters have been calculated as Ks=0.11 mM, α=1425 and Ki=0.01 mM 2. 2. Because amitriptyline is a partial competitive inhibitor of butyrylcholinesterase, acquired deficiency may be seen in patients treated with amitriptyline and may cause complications in operations.

Tetrahydroaminoacridine affects the cholinergic function of blood-brain barrier

In rat brain microvessels, tetrahydroaminoacridine (THA) caused a significant inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities, in a dose-dependent manner. THA resulted to be a more potent inhibitor of BuChE than AChE. Lineweaver-Burk plots showed that Km (app) and V were altered by THA, indicating mixed competitive/non-competitite inhivition. The results of the present study also established that the three molecular forms of BuChE (G 1, G 2 and G 4), recently described to be present in brain microvessels, are inhibited after THA treatment.

Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide : A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase ( IC 50 = 2 × 10 −7 M) but not of acetylcholinesterase ( IC 50 = 4 × 10 −4 M) . For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K d of 4.5 × 10 −7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.

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A series of novel ferulic acid-O-alkylamines derivatives were designed, synthesized, and evaluated as multitarget-directed ligands against Alzheimer's disease. In vitro studies displayed that all the synthesized target compounds showed impressive inhibitory activity against butyrylcholinesterase (BuChE), significant inhibition/disaggregation of self-induced β-amyloid (Aβ) aggregation and acted as potential antioxidants. Particularly, compound 7f, one of the most potent BuChE inhibitor (IC50 value of 0.021 μM for equine serum BuChE, 8.63 μM for ratBuChE and 0.07 μM for human serum BuChE), was found to be a good acetylcholinesterase (AChE) inhibitor (IC50 = 2.13 μM for electric eel AChE, 1.8 μM for ratAChE and 3.82 μM for human erythrocytes AChE), and the result of molecular docking provided an explanation for its selective BuChE inhibitory activity. Compound 7f also had noteworthy inhibitory effects on self-induced Aβ1-42 aggregation (50.8 ± 0.82%) and was found to disaggregate self-induced Aβ1-42 aggregation (38.7 ± 0.65%), which was further elucidated by the transmission electron microscopy. Meanwhile, compound 7f showed the modest antioxidant activity (0.55 eq of Trolox), good protective effect against H2O2-induced PC12 cell injury, with low toxicity. Moreover, compound 7f could cross the blood-brain barrier (BBB) in vitro. Significantly, compound 7f did not exhibit any acute toxicity in mice at doses up to 1000 mg/kg, and the step-down passive avoidance test showed this compound significantly reversed scopolamine-induced memory deficit in mice. Taken together, the results indicated that compound 7f is a very promising multifunctional agent in the treatment of Alzheimer's disease, particularly the advanced stages of AD.