Ellman’s method is still an appropriate method for measurement of cholinesterases activities

Shetab-Boushehri, Seyed Vahid

doi:10.17179/excli2018-1536

Cited by 8 publications

(4 citation statements)

References 6 publications

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“…The studies assessed the inhibition of AChE activity using the ITC and SD interaction model with hydroxybenzoic acids. The calorimetric method, like the colorimetric Ellman's method, allows the determination of the IC50 value of inhibition of enzyme activity [18], however, in Ellman's method, the important role of a "false-positive" blank sample in the routine analysis should be considered [19]. The ITC method is a useful tool for verifying the effectiveness of enzyme inhibitors using only a physiological substrate, without additional reagents or secondary reactions.…”

Section: Resultsmentioning

confidence: 99%

Hydroxybenzoic Acids as Acetylcholinesterase Inhibitors: Calorimetric and Docking Simulation Studies

et al. 2022

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One of the symptoms of Alzheimer’s disease (AD) is low acetylcholine level due to high acetylcholinesterase (AChE) activity. For this reason, AChE inhibitors are used in the treatment of AD, the prolonged use of which may cause a cholinergic crisis. There is a need to search for safe natural AChE inhibitors. The study analyzed 16 hydroxybenzoic acids using calorimetry and docking simulation as AChE inhibitors. All tested compounds were shown to inhibit the hydrolysis of ACh. The best properties were shown by methyl syringinate, which acted as competitive inhibitor at a catalytic site. The tested compounds also interacted with the anionic or peripheral binding site known to block β-amyloid plaques formation. The activity of the tested hydroxybenzoic acids IC50 ranged from 5.50 to 34.19 µmol/µmol of AChE, and the binding constant Ka from 20.53 to 253.16 L/mol, which proves their reversible, non-toxic effect, and activity at physiological concentrations.

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Section: Resultsmentioning

confidence: 99%

Hydroxybenzoic Acids as Acetylcholinesterase Inhibitors: Calorimetric and Docking Simulation Studies

et al. 2022

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“…The DTNB method was used to determine the reduced glutathione amount [19]. This method utilizes the spectroscopic measurement of the p-nitrophenol anion which is created in a 1:1 ratio for each thiol molecule created by the reaction of the 5,5 dithiobis-2-nitrobenzoic acid (DTNB, Ellman reactive) with the aliphatic thiol molecules in the samples, under mild-alkaline conditions.…”

Section: Colony Forming Assaymentioning

confidence: 99%

Combination therapy application of Abemaciclib with Doxorubicin in triple negative breast cancer cell line MDA-MB-231

Tugce Nur Eralp,

Akin Sevinc,

Banu Mansuroglu

2024

Cell Mol Biol (Noisy-le-grand)

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Due to lack of clinical biomarkers, Triple Negative Breast Cancer (TNBC) is more likely to have spread to other tissues at time of diagnosis and therapy planning generally involves use of cytotoxic chemotherapy agents, such as Doxorubicin. We aimed to investigate possible advantages of using combination strategy using Doxorubicin alongside Abemaciclib. After determining the IC50 values for Doxorubicin (DOX) and Abemaciclib (ABE); CompuSyn and ComBenefit software were used to reveal the effect resulting from the combination of two drugs. Following the determined effect, cell death was revealed by fluorescence microscopy and a colony forming assay was performed to see the potential of even a single cancer cell with adhesive character to survive over time and form a clone of itself. Detection of changing antioxidant activity following DOX, ABE and DOX+ABE combination therapy in MDAMB231 cells was determined by measuring MDA, SOD and GSH activities. The expression of Cleaved Caspase 3, PARP, Cleaved PARP, Cdk2 and Bax, which changed as a result of DOX, ABE and DOX+ABE application, was shown by Western Blotting.Cyclin-dependent kinase inhibitors appear as promising agents in therapy planning for breast cancer due to their prominent role in cell cycle regulation, where the number of studies interrogating its efficiency in the treatment of cancer such as TNBC is limited. For this reason, in this study, we aimed to determine the impact of the combined use of the CDK4/6 inhibitors ABE and DOX on the cytotoxicity, apoptotic homeostasis, alterations in antioxidative mechanisms, and the molecular pathways that they utilize. Our results showed that when used in combination, Doxorubicin and Abemaciclib showed a synergistic effect on TNBC cell line MDA-MB-231.

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“…The method described by Ellman (Ellman, 1959;Shetab-Boushehri, 2018) was used. A cuvette containing the nonreduced TrxR1 (1.31 μM) in PE buffer was mixed with 1.5 mM DTNB in a total volume of 205 μL.…”

Section: Identification Of Dtnb-reactive Group(s) In the Non-reduced ...mentioning

confidence: 99%

The C-terminal selenenylsulfide of extracellular/non-reduced thioredoxin reductase endows this protein with selectivity to small-molecule electrophilic reagents under oxidative conditions

Qin,

Guo,

Chen

et al. 2024

Front. Mol. Biosci.

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Mammalian cytosolic thioredoxin reductase (TrxR1) serves as an antioxidant protein by transferring electrons from NADPH to various substrates. The action of TrxR1 is achieved via reversible changes between NADPH-reduced and non-reduced forms, which involves C-terminal selenolthiol/selenenylsulfide exchanges. TrxR1 may be released into extracellular environment, where TrxR1 is present mainly in the non-reduced form with active-site disulfide and selenenylsulfide bonds. The relationships between extracellular TrxR1 and tumor metastasis or cellular signaling have been discovered, but there are few reports on small-molecule compounds in targeted the non-reduced form of TrxR1. Using eight types of small-molecule thiol-reactive reagents as electrophilic models, we report that the selenenylsulfide bond in the non-reduced form of TrxR1 functions as a selector for the thiol-reactive reagents at pH 7.5. The non-reduced form of TrxR1 is resistant to hydrogen peroxide/oxidized glutathione, but is sensitive to certain electrophilic reagents in different ways. With 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and S-nitrosoglutathione (GSNO), the polarized selenenylsulfide bond breaks, and selenolate anion donates electron to the dynamic covalent bond in DTNB or GSNO, forming TNB-S-Se-TrxR1 complex or ON-Se-TrxR1 complex. The both complexes lose the ability to transfer electrons from NADPH to substrate. For diamide, the non-reduced TrxR1 actually prevents irreversible damage by this oxidant. This is consistent with the regained activity of TrxR1 through removal of diamide via dialysis. Diamide shows effective in the presence of human cytosolic thioredoxin (hTrx1), Cys residue(s) of which is/are preferentially affected by diamide to yield disulfide, hTrx1 dimer and the mixed disulfide between TrxR1-Cys497/Sec498 and hTrx1-Cys73. In human serum samples, the non-reduced form of TrxR1 exists as dithiothreitol-reducible polymer/complexes, which might protect the non-reduced TrxR1 from inactivation by certain electrophilic reagents under oxidative conditions, because cleavage of these disulfides can lead to regain the activity of TrxR1. The details of the selective response of the selenenylsulfide bond to electrophilic reagents may provide new information for designing novel small-molecule inhibitors (drugs) in targeted extracellular/non-reduced TrxR1.

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Ellman’s method is still an appropriate method for measurement of cholinesterases activities

Cited by 8 publications

References 6 publications

Hydroxybenzoic Acids as Acetylcholinesterase Inhibitors: Calorimetric and Docking Simulation Studies

Hydroxybenzoic Acids as Acetylcholinesterase Inhibitors: Calorimetric and Docking Simulation Studies

Combination therapy application of Abemaciclib with Doxorubicin in triple negative breast cancer cell line MDA-MB-231

The C-terminal selenenylsulfide of extracellular/non-reduced thioredoxin reductase endows this protein with selectivity to small-molecule electrophilic reagents under oxidative conditions

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