Mechanism of thioflavin T binding to amyloid fibrils

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.     

Peripheral site ligand-oxime conjugates: A novel concept towards reactivation of nerve agent-inhibited human acetylcholinesterase

A conceptually novel approach to the design of reactivators of nerve agent-inhibited acetylcholinesterase (AChE) is presented. The concept comprises the linkage of a peripheral site ligand via a spacer to a reactivating moiety with the eventual goal to develop non-ionic reactivators with sufficient affinity for AChE to induce reactivation and potentially improved blood-brain barrier penetration. Herein, the first step towards that goal—the synthesis and biological evaluation of a peripheral site ligand conjugated to a charged pyridinium oxime is discussed. It was found, that the introduction of the peripheral site ligand not only increased affinity of the construct for AChE but also enhanced reactivation of nerve agent-inhibited AChE.     

Mechanism of thioflavin T accumulation inside cells overexpressing P-glycoprotein or multidrug resistance-associated protein: role of lipophilicity and positive charge

Alzheimer's disease is characterized by the presence of amyloid deposition. Thioflavin T (ThT) has been one of the molecules of choice to attempt the detection of these amyloid deposits. However, it has been reported that ThT was unable to cross blood-brain barrier (BBB). Our aim was to understand the mechanism according to which it has been said that ThT is not able to cross the BBB. For this purpose we have used cellular models overexpressing P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP1), two proteins overexpressed in BBB. Our results show that: (i) ThT is able to cross membranes and to penetrate inside the cells; (ii) ThT is a P-gp substrate; (iii) ThT is poor MRP1 substrate. In conclusion, our results suggest that two factors could be involved in the low accumulation of ThT in the brain: ThT is a P-gp substrate and its lipophilicity is low.     

Identification of a structural site on acetylcholinesterase that promotes neurite outgrowth and binds laminin-1 and collagen IV

The cell adhesion and neurite outgrowth-promoting function of acetylcholinesterase has been localised to the area of the peripheral anionic site. In order to precisely determine the site involved, we used synthetic peptides representing sequences of the peripheral anionic site and its surrounds, and investigated their binding to a panel of monoclonal antibodies that inhibit cell adhesion/neurite outgrowth and/or to recognise the peripheral anionic site. Binding to laminin-1 and collagen IV was also investigated. A relationship between recognition of the sequence 37-50, representing a surface loop adjacent to the peripheral anionic site, and the degree of inhibition of cell adhesion was observed; both laminin-1 and collagen IV also bound this loop with high affinity. Neurite outgrowth on coverslips coated with this peptide was similar to those coated with acetylcholinesterase itself. Adhesion-inhibiting antibodies also recognised the omega loop 69-96, as did laminin-1 and collagen IV. Laminin also bound the sequences 55-66 and 340-353, recognised by the antibodies to varying degrees, but collagen did not. All these peptides were able to promote neurite outgrowth to some degree. No binding to the amyloid-binding omega loop 275-304 by the ligands was observed, nor did the antibodies recognise this consistently. No relationship was observed between the degree of inhibition of acetylcholinesterase and inhibition of neurite outgrowth by the antibodies from which we conclude that the neurite outgrowth function is non-cholinergic. In conclusion, we have identified a specific conformational structure on acetylcholinesterase, comprising adjacent surface loops between residues 37-50 and 69-96, with additional involvement of the sequences 55-66 and 340-353, that mediates cell adhesion and neurite outgrowth.     

Thioflavin T fluorescence assay for β-lactoglobulin fibrils hindered by DAPH

The molecule 4,5-dianilinophthalimide was recently found to be an efficient compound in disaggregating amyloid fibrils involved in the Alzheimer’s disease. In this study we have investigated whether the compound 4,5-dianilinophthalimide was able to disaggregate fibrils derived from β-lactoglobulin. In addition to a Thioflavin T fluorescence assay, flow-induced birefringence was used as an independent technique to measure the total length concentration of the fibrils. An additional advantage of the latter technique is that not only the total length concentration, but also the length distribution of the fibrils can be measured. The results from flow-induced birefringence showed that the total amount of fibrils and also the length distribution of the fibrils was not influenced by the addition of 4,5-dianilinophthalimide, even though this was suggested by the results of the Thioflavin T assay. The results of flow-induced birefringence were confirmed by rheological measurements and transmission electron microscopy. Our findings show that the use of a Thioflavin T assay in order to probe the possible disaggregating effect of certain compounds can give misleading results.     

Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure-activity relationships for inhibitions by aryl carbamates

Substituted phenyl-N-butyl carbamates (1-9) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1-9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dissociation constant of the enzyme inhibitor adduct (Ki), the inactivation constant of the enzyme-inhibitor adduct (k2), and the bimolecular inhibition constant (k(i)) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1-9 and the Hammett substituent constant (sigma), are observed, and the reaction constants (ps) are -1.36, 0.35 and -1.01, respectively. Therefore, the above reaction may form a positive charged enzyme-inhibitor intermediate at the peripheral anionic site of the enzyme and may follow the irreversible inactivation by a conformational change of the enzyme.     

Probing the peripheral anionic site of acetylcholinesterase with quantitative structure activity relationships for inhibition by biphenyl-4-acyoxylate-4'-N-Butylcarbamates

Biphenyl-4-acyoxylate-4'-N-butylcarbamates 1-8 are synthesized from 4,4'-biphenol and are characterized as the pseudosubstrate inhibitors of acetylcholinesterase. In other words, the inhibitors bind to the enzyme and react with the enzyme to form the tetrahedral intermediates for the K(i) steps, and then the tetrahedral intermediates exclude the leaving groups to form a common N-butycarbamyl enzyme intermediate for the k(c) steps. Due to a linear character of the 4,4'-biphenyl moiety, the 4'-N-butylcarbamate moieties of the inhibitors react with the Ser200 residue of the enzyme while the 4-acyoxylate moieties of the inhibitors, on the other hand, should fit in the peripheral anionic site of the enzyme, which is located at the mouth of the deep active site gorge. Thus, carbamates with varied acyl substituents at the 4-position of the biphenyl ring are good candidates for probing the quantitative structure activity relationships for the peripheral anionic site of the enzyme. The fact that the pK(i), log k(c), and log K(i) values are correlated with neither the Taft substituent constant (sigma*) nor the Taft steric constant (E(s)) indicates that the 4-acyoxylate moieties of the inhibitors are too far away from the reaction center. However, the pK(i), log k(c), and log K(i) values are linearly correlated with the Hansch hydrophobicity constant, pi. The intensity constants (psi) for these correlations are 0.16, -0.035, and 0.13, respectively. These results indicate that interactions between the 4-acyoxylate groups of the inhibitors and the peripheral anionic site of the enzyme are mainly hydrophobic ones. The correlation results are slightly improved by using the two-parameter correlations with the Taft substituent steric constant, E(s), and pi. For pK(i), log k(c), and log K(i)-E(s)-pi correlations, the psi values are 0.21, -0.021, and 0.19, respectively; the intensity constants for steric effect (delta) are 0.08, 0.022, and 0.10, respectively. Besides hydrophobic interactions, the two-parameter correlations also suggest that little steric hindrance occurs for the bulkier inhibitors to pass by the peripheral anionic site of the enzyme.     

贝类血细胞硫代黄素T荧光染色方法

硫代黄素T( thioflavin T,TFT)是一种用于组织学的苯并噻唑荧光染料,因其对淀粉样蛋白有高亲和性而主要被用于淀粉样病变的荧光显微检测.本研究分别以软体动物门双壳纲的栉孔扇贝(Chlamys farreri)和中国蛤蜊(Mactra chinensis)、腹足纲的拟紫口玉螺(Natica janthosto...     

Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and ultraviolet detection

An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid β-sheet structure of these proteins and quantifying them. In this study, the property of amyloids to incorporate the fluorescent dye Thioflavin T has been exploited to propose a method of quantification. The experimental protocol includes the preparation of amyloids from commercial κ-casein (κCN) and their fractionation through size exclusion chromatography (SEC) to provide calibration curves from fluorescence and absorbance signals. Finally, a bacterial biofilm extract was injected into SEC, and the amyloid fibrils could be expressed as equivalent κCN, representing approximately 21% of the total proteins.     

The inhibition enzymatic hydrolysis of acetylthiocholine by acetylcholinesterase using principal alkaloids isolated from celandine and macleya and their derivatives

A study was made of a possible inhibitory action on the enzymatic hydrolysis of acetylthiocholine by human erythrocyte acetylcholinesterase of principal alkaloids isolated from Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa (namely sanguinarine, chelidonine, berberine), and of drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Chelidonium majus L.) and "Sanguirythrine" (a mixture of unseparated closely related to benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine, isolated from Chelidonium majus L. and other plants of Papaveraceae family). All agents under study have been shown to be reversible inhibitors of the enzymatic hydrolysis of acetylthiocholine. On the basis of the kinetic data it has been determined that chelidonine belonged to reversible inhibitors of a competitive type. All other examined agents have been demonstrated to be inhibitors of a mixed competitive-noncompetitive type, and a greater contribution to the inhibition was made by the competitive constituent. Among all examined agents berberine, sanguinarine and "Sanguirythrine" were the strongest inhibitors of this reaction (the values of generalized inhibitory constants being 0.23, 0.23 and 0.29 microM, respectively) and cheliodonine and "Ukrain" were much weaker (2.0 and 2.5 microM, respectively). Judging from the data obtained, sanguinarine and chelerythrine exert similar inhibitory effects on the reaction of enzymatic hydrolysis of acetylthiocholine, since sanguinarine and "Sanguirythrine" have nearly equal generalized inhibitory constants.     

Inhibition of acetylcholinesterase hydrolysis of cationic substrates by the reaction product

The kinetics of acetylcholinesterase-catalyzed hydrolysis of the two cationic substrates (I and II in Russian text) was analyzed by means of the integrated Michaelis equation (3). The constants kII, kcat Km and the enzyme-product complex dissociation constant Ki were determined. (Table 1). It was shown that acetylcholine (II) binds to to the enzyme active center more effectively than the alcohol product of its hydrolysis. In case of the pipecholine derivative (I) reversed situation occurs. The different dependence of the ester substrate and appropriate alcohol binding effectiveness upon the reagent structure indicates the dissimilar location of the molecules in the active center of acetylcholinesterase. Some structural implications of the enzyme active center were discussed.     

Antioxidant and acetylcholinesterase inhibition properties of novel bromophenol derivatives

In this study, series of novel bromophenol derivatives were synthesized and investigated for their antioxidant and AChE inhibition properties. Novel brominated diarylmethanones were obtained from the acylation reactions of benzoic acids with substituted benzenes. One of the bromodiarylmethanone was synthesized from the bromination of diarylmethanone with molecular bromine. All diarylmethanones were converted into their bromophenol derivatives with BBr₃. The antioxidant activities of all synthesized compounds were elucidated by using various bioanalytical assays. Radical scavenging activities of compounds 10–24 were evaluated by means of DPPH and ABTS⁺ scavenging activities. In addition, reducing ability of 10–24 were determined by Fe³⁺, Cu²⁺, and [Fe³⁺-(TPTZ)₂]³ reducing activities. α-Tocopherol, trolox, BHA, and BHT were used as positive antioxidant and radical scavenger molecules. On the other hand, IC₅₀ values were calculated for DPPH, ABTS⁺ scavenging, and AChE inhibition effects of novel compounds. The results obtained from the current studies clearly show that novel bromophenol derivatives 20–24 have considerable antioxidant, antiradical, and AChE inhibition effects.     

The inactivation of acetylcholinesterase by alpha-terthienyl and ultraviolet light Studies in vitro and in larvae of the mosquito Aedes aegypti

Acetylcholinesterase (EC 3.1.1.7) was inactivated photochemically in solution, in the presence of dissolved terthiophene sensitizers. Alpha-terthienyl (2,2′:5,2″-terthiophene) and its isomers 3,2′:5′,2″- and 3,2′:5′,3″-terthiophenes showed very similar sensitizing properties. With all three terthiophenes, the photosensitization was completely suppressed under anaerobic conditions, and therefore the inactivation process required the presence of oxygen. The enzyme was inactivated in vivo when fourth instar larvae of the mosquito Aedes aegypti were treated with alpha-terthienyl in the presence of long-wavelength ultraviolet light. No inactivation was observed when the organisms were treated with the ultraviolet light alone, with the chemical alone, or with a previously irradiated sample of the chemical. This paper represents the first example of acetylcholinesterase inactivation in vivo by a photoactive insecticide.     

Quantum chemical modeling of the GTP hydrolysis by the RAS–GAP protein complex

We present results of the modeling for the hydrolysis reaction of guanosine triphosphate (GTP) in the RAS–GAP protein complex using essentially ab initio quantum chemistry methods. One of the approaches considers a supermolecular cluster composed of 150 atoms at a consistent quantum level. Another is a hybrid QM/MM method based on the effective fragment potential technique, which describes interactions between quantum and molecular mechanical subsystems at the ab initio level of the theory. Our results show that the GTP hydrolysis in the RAS–GAP protein complex can be modeled by a substrate-assisted catalytic mechanism. We can locate a configuration on the top of the barrier corresponding to the transition state of the hydrolysis reaction such that the straightforward descents from this point lead either to reactants GTP+H₂O or to products guanosine diphosphate (GDP)+H₂PO₄^?. However, in all calculations such a single-step process is characterized by an activation barrier that is too high. Another possibility is a two-step reaction consistent with formation of an intermediate. Here the Pγ-O(Pβ) bond is already broken, but the lytic water molecule is still in the pre-reactive state. We present arguments favoring the assumption that the first step of the GTP hydrolysis reaction in the RAS–GAP protein complex may be assigned to the breaking of the Pγ-O(Pβ) bond prior to the creation of the inorganic phosphate.     

Temporospatial localization of acetylcholinesterase activity in the dental epithelium during mouse tooth development

Acetylcholinesterase (AChE), a principal modulator of cholinergic neurotransmission, also has been demonstrated to be involved in the morphogenetic processes of neuronal and non-neuronal tissues. This study shows that AChE exhibits temporospatial activity in the dental epithelium of the developing mouse tooth. To identify the AChE activity in the mouse tooth during development, we performed enzyme histochemistry on the mouse embryos from embryonic day 13 (E13) to E18 and on the incisors and molars of the neonatal mouse at 10 days after birth (P10). In the developing molars of mouse embryos, AChE activity was not found in the dental epithelium at E13 (bud stage). AChE activity first appeared in the developing cervical loops of the enamel organ at E14 (cap stage), but was not found in the enamel knot. At E18 (bell stage), AChE activity was localized in the inner enamel epithelium except the cervical-loop area. In the incisors and molars of neonatal mice (P10), AChE activity was localized in the inner enamel epithelium of the cervical-loop and enamel-free area. Overall, AChE activity was localized in the differentiating dental epithelium while the activity of butyrylcholinesterse, another cholinesterase, was located primarily in the cells of the dental follicle. The results suggest that AChE may play a role in the histo- and cytodifferentiation of dental epithelium during tooth development.     

Synthesis,biological evaluation and molecular docking of novel pyrazole derivatives as potent carbonic anhydrase and acetylcholinesterase inhibitors

A series of substituted pyrazole compounds (1–8 and 9a, b) were synthesized and their structure was characterized by IR, NMR, and Mass analysis. These obtained novel pyrazole derivatives (1–8 and 9a, b) were emerged as effective inhibitors of the cytosolic carbonic anhydrase I and II isoforms (hCA I and II) and acetylcholinesterase (AChE) enzymes with K_i values in the range of 1.03 ± 0.23–22.65 ± 5.36 µM for hCA I, 1.82 ± 0.30–27.94 ± 4.74 µM for hCA II, and 48.94 ± 9.63–116.05 ± 14.95 µM for AChE, respectively. Docking studies were performed for the most active compounds, 2 and 5, and binding mode between the compounds and the receptors were determined.     

The neuropeptide TRH has a minor effect on the enzymatic activity of acetylcholinesterase in vitro

The neuropeptide thyrotropin-releasing hormone (TRH) elicits a variety of physiological effects of which some are due to cholinergic mechanisms. TRH modulates in vivo the effects of compounds affecting acetylcholinesterase (AChE). In the present study the in vitro effects of TRH on the activity of AChE were explored. TRH has no effect at physiologically relevant concentrations. At unphysiologically high concentrations (>5 mM) a slight inhibition was found. This was noticed also when the enzyme was exposed to the amide-free tripeptide analog p-Glu-His-Pro. We conclude that any cholinergic effect of TRH observed in vivo is unlikely to be due to a direct interaction of the peptide with AChE.     

Investigation of kinetic interactions between approved oximes and human acetylcholinesterase inhibited by pesticide carbamates

Carbamates are widely used for pest control and act primarily by inhibition of insect and mammalian acetylcholinesterase (AChE). Accidental or intentional uptake of carbamates may result in typical signs and symptoms of cholinergic overstimulation which cannot be discriminated from those of organophosphorus pesticide poisoning. There is an ongoing debate whether standard treatment with atropine and oximes should be recommended for human carbamate poisoning as well, since in vitro and in vivo animal data indicate a deleterious effect of oximes when used in combination with the N-methyl carbamate carbaryl. Therefore, we performed an in vitro kinetic study to investigate the effect of clinically used oximes on carbamoylation and decarbamoylation of human AChE. It became evident that pralidoxime and obidoxime in therapeutic concentrations aggravate the inhibition of AChE by carbaryl and propoxur, with obidoxime being substantially more potent compared to 2-PAM. However, obidoxime had no impact on the decarbamoylation kinetics. Hence, the administration of 2-PAM and especially of obidoxime to severely propoxur and carbaryl poisoned humans cannot be recommended.     

The active site and partial sequence of cobra venom acetylcholinesterase

About 30% of the primary structure of acetylcholinesterase (AchE) from the cobraNaja naja oxiana has been determined. The sequence around the serine residue labeled by diisopropylfluorophosphate (DFP) was found to be TVTLFGESAGAASVGM which is similar to the active sites of AChE from other tissues. The part of the primary structure determined shows 76% identity with AChE from Torpedo and 42% identity with the Drosophila enzyme. A surprisingly large identity (42% in the sequence determined) was found with lysophospholipase from rat (Hanet al., 1987).     

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.