Fungal biotransformations of anticholinesterase norbelladine derivatives to obtain new products and mimic mammalian metabolism

Norbelladine is the natural precursor of all alkaloids of the Amaryllidaceae family. These compounds have been reported to exert interesting biological activities. Particularly, galantamine is currently used in the palliative treatment of Alzheimer’s disease for its cholinergic effect. The biotransformation of two norbelladine analogues exhibiting anticholinesterase activity was studied using 11 fungi belonging to Aspergillus, Rhizopus, Cunninghamella and Fusarium genera. The substrates were refractory to biotransformation by all fungi screened, except by A. clavatus. Based on GC-MS analyses, we demonstrated that its enzymes were able to catalyse the oxidative cleavage of the C-N bond of the secondary amine of 4’-O-methylnorbelladine. On the other hand, the hindered phenolic hydroxy group at the 3’ position of the brominated derivative was regioselectively methylated. The latter compound was chemically synthesised for better characterisation and the biological assays showed that this metabolite exerted a lower inhibition of BChE and lost the anti-AChE activity. The metabolic pathways involved here were useful to mimic phase I and II xenobiotic metabolism in mammals and thus predict the products that could be formed. A new source of biocatalytic tools to obtain new protoalkaloid derivatives was also discovered.     

Amelioration of intracerebroventricular streptozotocin induced cognitive impairment by Evolvulus alsinoides in rats: In vitro and in vivo evidence

Evolvulus alsinoides, also known as Shankpushapi, is a commonly used traditional medicine for enhancing memory. We evaluated the in vitro free radical scavenging and enzymes [acetylcholinesterase, butyrylcholinestrase, glycogen synthase kinase-3-β (GSK-3-β), rho kinase (ROCK II), prolyl endopeptidase (PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)] inhibitory activities of aqueous and hydro-alcoholic extracts of E. alsinoides. Hydro-alcoholic extract of E. alsinoides demonstrated more free radical scavenging activity as compared to aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-β, ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as compared to aqueous extract but no significant difference in phenolic content of the two extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and 500 mg/kg, p.o.) was selected for in vivo study in intracerebroventricularly injected streptozotocin (STZ) induced cognitive impairment in male Wistar rats. Elevated plus maze, passive avoidance and Morris water maze were used for assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rat brain at the end of the study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced cognitive impairment by reducing the oxidative stress, improving cholinergic function and preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides.     

PMS777, a New Cholinesterase Inhibitor with Anti-Platelet Activated Factor Activity,Regulates Amyloid Precursor Protein Processing In Vitro

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by progressive impairment of memory and cognition. Previous data have shown that beta-amyloid (Aβ) cascade plays a central role in AD pathophysiology and thus drugs regulate amyloid precursor protein (APP) metabolism may have therapeutic potential. Here the effects of PMS777, a new cholinesterase inhibitor with anti-platelet activated factor activity, on APP processing were investigated. Using SH-SY5Y^APP695 cells, it showed that PMS777 treatment caused significant decreased secretion of sAPPα into the conditioned media without affecting cellular holoAPP synthesis. When PC12 cells were incubated with PMS777, the same effect was observed. The data also indicated that 10 μM PMS777 incubation decreased the release of Aβ42 into the cell media as compared with vehicle group in SH-SY5Y^APP695 cells. Pretreatment of cells with M-receptor scopolamine antagonized the decreased secretion of sAPPα induced by PMS777, but N-receptor α-bungarotoxin pretreatment did not have such an effect. These results indicated that PMS777 could modulate APP processing in vitro and that decreasing Aβ generation might demonstrate its therapeutic potential in AD.     

A water extract of Curcuma longa L. (Zingiberaceae) rescues PC12 cell death caused by pyrogallol or hypoxia/reoxygenation and attenuates hydrogen peroxide induced injury in PC12 cells

A number of studies indicate that free radicals are involved in the neurodegeneration in Alzheimer's disease (AD). The role of superoxide anion (O2*-) in neuronal cell injury induced by reactive oxygen species (ROS) was examined in PC12 cells using pyrogallol (1,2,3-benzenetrior), a donor to release O2*-. Pyrogallol induced PC12 cell death at concentrations, which evidently increased intracellular O2*-, as assessed by O2*- sensitive fluorescent precursor hydroethidine (HEt). A water extract of Curcuma longa L. (Zingiberaceae) (CLE), having O2*- scavenging activity rescued PC12 cells from pyrogallol-induced cell death. Hypoxia/reoxygenation injury of PC12 cells was also blocked by CLE. The present study was also conducted to examine the effect of CLE on H2O2 -induced toxicity in rat pheochromocytoma line PC12 by measuring cell lesion, level of lipid peroxidation and antioxidant enzyme activities. Following a 30 min exposure of the cells to H2O2 (150 microM), a marked decrease in cell survival, activities of glutathione peroxidase and catalase as well as increased production of malondialdehyde (MDA) were found. Pretreatment of the cells with CLE (0.5-10 microg/ml) prior to H2O2 exposure significantly elevated the cell survival, antioxidant enzyme activities and decreased the level of MDA. The above-mentioned neuroprotective effects are also observed with tacrine (THA, 1 microM), suggesting that the neuroprotective effects of cholinesterase inhibitor might partly contribute to the clinical efficacy in AD treatment. Further understanding of the underlying mechanism of the protective effects of these radical scavengers reducing intracellular O2*- on neuronal cell death may lead to development of new therapeutic treatments for hypoxic/ischemic brain injury.     

Amino acids defining the acyl pocket of an invertebrate cholinesterase

Amphioxus (Branchiostoma floridae) cholinesterase 2 (ChE2) hydrolyzes acetylthiocholine (AsCh) almost exclusively. We constructed a homology model of ChE2 on the basis of Torpedo californica acetylcholinesterase (AChE) and found that the acyl pocket of the enzyme resembles that of Drosophila melanogaster AChE, which is proposed to be comprised of Phe330 (Phe290 in T. californica AChE) and Phe440 (Val400), rather than Leu328 (Phe288) and Phe330 (Phe290), as in vertebrate AChE. In ChE2, the homologous amino acids are Phe312 (Phe290) and Phe422 (Val400). To determine if these amino acids define the acyl pocket of ChE2 and its substrate specificity, and to obtain information about the hydrophobic subsite, partially comprised of Tyr352 (Phe330) and Phe353 (Phe331), we performed site-directed mutagenesis and in vitro expression. The aliphatic substitution mutant F312I ChE2 hydrolyzes AsCh preferentially but also butyrylthiocholine (BsCh), and the change in substrate specificity is due primarily to an increase in k_cat for BsCh; K_m and K_ss are also altered. F422L and F422V produce enzymes that hydrolyze BsCh and AsCh equally due to an increase in k_cat for BsCh and a decrease in k_cat for AsCh. Our data suggest that Phe312 and Phe422 define the acyl pocket. We also screened mutants for changes in sensitivity to various inhibitors. Y352A increases the sensitivity of ChE2 to the bulky inhibitor ethopropazine. Y352A decreases inhibition by BW284c51, consistent with its role as part of the choline-binding site. Aliphatic replacement mutations produce enzymes that are more sensitive to inhibition by iso-OMPA, presumably by increasing access to the active site serine. Y352A, F353A and F353V make ChE2 considerably more resistant to inhibition by eserine and neostigmine, suggesting that binding of these aromatic inhibitors is mediated by π–π or cation–π interactions at the hydrophobic site. Our results also provide information about the aromatic trapping of the active site histidine and the inactivation of ChE2 by sulfhydryl reagents.     

石首鱼科鱼肌肉胆碱酯酶对有机磷化合物反应灵敏度的研究

胆碱酯酶对有机磷化合物的反应灵敏度高,特异性强,但不同来源的生物材料的胆碱酯酶在灵敏度上差异甚大。鲈形目石首鱼科的小黄鱼、大黄鱼、白姑鱼、鳘和黄姑鱼肌肉胆碱酯酶对沙林和ＶＸ均相当敏感,两种化合物对它们的胆碱酯酶抑制的ＰＩ５０值几乎均在７．０以上。进一步研究,这些胆碱酯酶可能成为分析有磷化合物和农药的有效试剂,对筛选生物活性物质有一定的指导意义。     

疏水载体固定化胆碱酯酶的研究

首次摸索了带有疏水基团琼脂珠固定化鸭血清胆碱酯酶的最佳条件,固定化酶活力回收为７０一７６％。探讨了不同疏水基团对固定化酶偶联率、稳定性及对有机磷化合物灵敏度的影响。α－萘氨基、Ｐ－甲苯氨基和苄氨基载体固定化酶连续操作半哀期分别为２２．３ｈ,２１．７ｈ,１２．８ｈ,对有机磷化合物Ｖｘ和ＧＢ的灵敏度分别提高了２．２～１２．６倍和２．３～４．９倍,这对于在环境保护、食品检验、军事侦检等领域检测微量有机磷化合物具有重要意义。     

Amine substitution of quinazolinones leads to selective nanomolar AChE inhibitors with ‘inverted’ binding mode

Selective and nanomolar acetylcholinesterase inhibitors were obtained by connecting tri- and tetracyclic quinazolinones—previously described as moderately active and unselective cholinesterase (ChE) inhibitors—via a hydroxyl group in para position to an anilinic nitrogen with different amines linked via a three carbon atom spacer. These tri- and tetracyclic quinazolinones containing different alicyclic ring sizes and connected to tertiary amines were docked to a high-resolution hAChE crystal structure to investigate the preferred binding mode in relation to results obtained by experimental structure–activity relationships. While the ‘classical orientation’ locating the heterocycle in the active site was rarely found, an alternative binding mode with the basic aliphatic amine in the active center (‘inverted’ orientation) was obtained for most compounds. Analyses of extended SARs based on this inverted binding mode are able to explain the compounds’ binding affinities at AChE.     

Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.     

The effect of environmental parameters on growth, cholinesterase activity and glutathione S-transferase activity in the earthworm (Apporectodea caliginosa)

The effect of age and environmental parameters on growth, cholinesterase (ChE), and glutathione S-transferase (GST) activities were assessed in juvenile Apporectodea caliginosa earthworms. Earthworms were maintained in three types of soil- loam, sandy, and clay- at a range of moisture contents (15-30%), and temperatures (5-20 C). Earthworm age (1-3 months) had no significant affect on ChE activity. Growth rates were influenced by all environmental parameters tested and these effects were interrelated. Optimum conditions for growth appeared to be in loam or sandy soil with 25-30% moisture at 10-15 C. The GST activity was influenced by soil temperature and activity was significantly higher at 15 C than at 5 C and 10 C. Soil type also influenced GST activity and this influence was dependent on moisture content. In sandy soil GST activity was significantly lower at 30% moisture than at lower moisture contents, in loam soil GST activity was significantly higher at 15% moisture than at higher moisture contents, while in clay soil GST activity was not affected by moisture content. These results indicate that in field experiments when evaluating GST activity soil temperature and soil type need to be consistent between control and 'contaminated sites'. ChE activity was only affected by temperature, so this should be considered when comparing control and treatedareas.