Computational and experimental studies on the interaction between butyrylcholinesterase and fluoxetine: implications in health and disease

1. Butyrylcholinesterase (BChE) is a serine esterase that plays a role in the detoxification of natural as well as synthetic ester-bond-containing compounds. Alterations in BChE activity are associated with a number of diseases. Cholinergic system abnormalities in particular are correlated with the formation of senile plaques in Alzheimer’s disease (AD), and administration of cholinesterase inhibitors is a common therapeutic approach used to treat AD.

2. Here, our aim was to study the interaction between BChE and fluoxetine.

3. Molecular docking simulations revealed that fluoxetine penetrated deep into the active-site gorge of BChE and that it was engaged in stabilizing noncovalent interactions with multiple subsites. In substrate kinetic studies, the V_m, K_m, k_cat and k_cat/K_m values were found to be 20.59?±?0.36?U mg^?1 protein, 194?±?14?µM, 1.3?×?10⁸?s^?1 and 6.7?×?10⁵?µM^?1s^?1, respectively. Based on inhibitory studies, fluoxetine appeared to inhibit BChE competitively, with an IC₅₀ value of 104?µM and a K_i value of 36.3?±?4.7?µM.

4. Overall, both the low K_i value and the high number of BChE–fluoxetine interactions suggest that fluoxetine is a potent inhibitor of BChE, although in vivo mechanisms for the direct effects of BChE inhibition on various pathologies remain to be further investigated.

     

Dipeptidyl carboxypeptidase 1 (DCP1) and butyrylcholinesterase (BCHE) gene interactions with the apolipoprotein E epsilon4 allele as risk factors in Alzheimer's disease and in Parkinson's disease with coexisting Alzheimer pathology

Alzheimer's disease (AD) and Parkinson's disease (PD) are genetically heterogeneous. Dipeptidyl carboxypeptidase 1 (DCP1) and butyrylcholinesterase (BCHE) genes may modify the risk of these disorders. We investigated whether common polymorphisms present in these genes operate as risk factors for AD and PD in Finnish subjects, independently or in concert with the apolipoprotein E ε4 allele (APOE ε4). Eighty late onset sporadic AD patients, 53 PD patients (34 of whom had concomitant AD pathology), and 67 control subjects were genotyped for the insertion (I)/deletion (D) polymorphism of DCP1 and the K variant of BCHE. In logistic regression analysis, the DCP1 *I allele in combination with APOE ε4 significantly increased the risk of AD (OR 30.0, 95% CI 7.3-123.7), compared to subjects carrying neither of the alleles. Similar analysis showed that the risk of AD was significantly increased in subjects carrying both the BCHE wild type (*WT/*WT) genotype and ε4 (OR 9.9, 95% CI 2.9-33.8), compared to those without this BCHE genotype and ε4. Further, the risk of PD with AD pathology was significantly increased for carriers of DCP1 *I and ε4 (OR 8.0, 95% CI 2.1-31.1). We thus conclude that, in Finns, interaction between DCP1 *I and ε4 increases the risk of AD as well as of PD with coexisting Alzheimer pathology, which underlines the importance of the DCP1 I/D polymorphism in the development of Alzheimer neuropathology, whereas the wild type BCHE genotype in combination with ε4 had a combined effect with regard to the risk of AD.


Keywords: Alzheimer's disease; Parkinson's disease; dipeptidyl carboxypeptidase 1; butyrylcholinesterase     

Interacting with α7 nAChR is a new mechanism for AChE to enhance the inflammatory response in macrophages

Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses.     

Ovariectomy Enhances Acetylcholinesterase Activity But Does Not Alter Ganglioside Content in Cerebral Cortex of Female Adult Rats

In the present work we investigated the effect of ovariectomy on acetylcholinesterase (AChE) activity and ganglioside content in cerebral cortex of female rats. We also studied the activity of butyrylcholinesterase (BuChE) in serum of these animals. Adult Wistar rats were divided into three groups: (1) naive females (control), (2) sham-operated females and (3) castrated females (ovariectomy). Thirty days after ovariectomy, rats were sacrificed by decapitation without anaesthesia. Blood was collected and the serum used for BuChE determination. Cerebral cortex was homogenized to determine AChE activity and extracted with chlorophorm:methanol for ganglioside evaluation. Results showed that rats subjected to ovariectomy presented a significant increase of AChE activity, but did not change the content and the profile of gangliosides in cerebral cortex when compared to sham or naive rats. BuChE activity was decreased in serum of rats ovariectomized. Our findings suggest that the alteration in the activity of brain AChE, as well as serum BuChE activity caused by ovariectomy may contribute to the impaired cognition and/or other neurological dysfunction found in post-menopausal women.     

Identification of embelin,a 3-undecyl-1,4-benzoquinone from Embelia ribes as a multitargeted anti-Alzheimer agent

Beta-secreatse (BACE-1) and cholinesterases are clinically validated targets of Alzheimer's disease (AD), for which natural products have provided immense contribution. The multifaceted nature of AD signifies the need of multitargeted agents to tackle this disease. In the search of new natural products as dual BACE-1/cholinesterase inhibitors, a library of pure natural products was screened for inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE-1. The screening efforts have identified 1,4-benzoquinone “embelin,” a natural product derived from Embelia ribes displaying inhibition of all three enzymes, with IC₅₀ values of 2.5, 5.4, and 2.1 μM, respectively. This screen has also identified isoquinoline alkaloids papaverine and L-tetrahydropalmatine as AChE inhibitors. Kinetic study has shown that embelin inhibits EeAChE and EqBChE with ki values of 4.59 and 0.57 μM, in an uncompetitive and noncompetitive manner, respectively. The interactions of embelin with allosteric peripheral anionic site of cholinesterases, has further supported the results of kinetic study. Embelin has also enhanced the activity of P-gp in LS-180 cells, the efflux pump which is involved in the clearance of amyloid-β from AD brain. Further, the cell viability study in neuronal cell line has indicated the excellent therapeutic window of embelin. These results are indicative of the fact that embelin is a multitargeted agent playing role in stopping the formation of amyloid-β oligomers (via inhibition of BACE-1), improves cholinergic-transmission (via inhibition of AChE/BChE) and increases amyloid-β clearance (via P-gp induction).     

Synthesis and biological evaluation of phloroglucinol derivatives possessing α‐glycosidase,acetylcholinesterase, butyrylcholinesterase,carbonic anhydrase inhibitory activity

A series of novel phloroglucinol derivatives were designed, synthesized, characterized spectroscopically and tested for their inhibitory activity against selected metabolic enzymes, including α‐glycosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase I and II (hCA I and II). These compounds displayed nanomolar inhibition levels and showed K_i values of 1.14–3.92 nM against AChE, 0.24–1.64 nM against BChE, 6.73–51.10 nM against α‐glycosidase, 1.80–5.10 nM against hCA I, and 1.14–5.45 nM against hCA II.

     

Cholinesterase Inhibitors for the Treatment of Alzheimer's Disease:: Getting On and Staying On

Background: Cholinesterase (ChE) inhibitors currently used in the treatment of Alzheimer's disease (AD) are the acetylcholinesterase (AChE)-selective inhibitors, donepezil and galantamine, and the dual AChE and butyrylcholinesterase (BuChE) inhibitor, rivastigmine. In addition to differences in selectivity for AChE and BuChE, ChE inhibitors also differ in pharmacokinetic and pharmacodynamic properties, and these differences could significantly impact on safety, tolerability, and efficacy.Objective: The aim of this article was to provide an overview of the ChE inhibitors widely used in AD, focusing on key pharmacologic differences among agents and how these may translate into important differences in safety, tolerability, and efficacy in clinical practice.Methods: Using published literature collected over time by the author, a review was conducted, focusing on the pharmacology and clinical data of donepezil, galantamine, and rivastigmine.Results: All ChE inhibitors have the potential to induce centrally mediated cholinergic adverse events (AEs), such as nausea and vomiting, if the dose is increased too rapidly or in increments that are too large. These AEs, which are most likely to occur during the “getting on,” or dose-escalation, phase of treatment, may result in patients discontinuing treatment early without achieving optimum therapeutic benefit. To reduce the incidence of these AEs, a slow dose-escalation schedule has been established in clinical practice, consisting of a “start low, go slow” procedure with a minimum of 4 weeks between dose increases. After “getting on” treatment, maintaining treatment in the long term, or “staying on,” may be achieved with good safety, tolerability, and sustained symptomatic efficacy across the key symptom domains (activities of daily living, behavior, and cognition).Conclusions: ChE inhibitors provide symptomatic benefit in AD across key symptom domains. Factors influencing the safety, tolerability, and efficacy of these agents in clinical practice include ChE enzymes inhibited, brain and brain-region ChE selectivity, and metabolism route. Class-specific cholinergic AEs can be minimized using slow, flexible dose escalation.     

Syntheses,Cholinesterases Inhibition,and Molecular Docking Studies of Pyrido[2,3‐b]pyrazine Derivatives

Cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), have a role in cholinergic deficit which evidently leads to Alzheimer's disease (AD). Inhibition of cholinesterases with small molecules is an attractive strategy in AD therapy. This study demonstrates synthesis of pyrido[2,3‐b]pyrazines ( 6a ‐ 6q ) series, their inhibitory activities against both cholinesterases, AChE and BChE, and molecular docking studies. The bioactivities data of pyrido[2,3‐b]pyrazines showed 3‐(3′‐nitrophenyl)pyrido[2,3‐b]pyrazine 6n a potent dual inhibitor among the series against both AChE and BChE with IC₅₀ values of 0.466 ± 0.121 and 1.89 ± 0.05 μm , respectively. The analogues 3‐(3′‐methylphenyl)pyrido[2,3‐b]pyrazine 6c and 3‐(3′‐fluorophenyl)pyrido[2,3‐b]pyrazine 6f were found to be selective inhibition for BChE with IC₅₀ values of 0.583 ± 0.052 μm and AChE with IC₅₀ value of 0.899 ± 0.10 μm , respectively. Molecular docking studies of the active compounds suggested the putative binding modes with cholinesterases. The potent compounds among the series could potentially serves as good leads for the development of new cholinesterase inhibitors.