Inhibition of rat and human glutathione S-transferase isoenzymes by ethacrynic acid and its glutathione conjugate

doi:10.1016/0006-2952(90)90465-W

Biochemical Pharmacology

Volume 40, Issue 7, 1 October 1990, Pages 1631-1635

https://doi.org/10.1016/0006-2952(90)90465-W Get rights and content

Abstract

Ethacrynic acid, a potent inhibitor of glutathione S-transferases (GST), has been shown to enhance the cytotoxicity of chlorambucil in drug resistant cell lines, but a definite mechanism has not been established. Both covalent binding to GST and reversible inhibition of GST have been reported. In the present study no irreversible inhibition was observed: for all rat GST tested, inactivation was complete within 15 sec at 0°, and dialysis of GST after incubation with ethacrynic acid gave complete recovery of enzyme activity for all isoenzymes tested. Moreover, the inhibition was competitive towards 1-chloro-2,4-dinitrobenzene and non-competitive towards glutathione for rat isoenzyme 1-1. Strong inhibition of both human and rat GST of the α-, μ- and π-classes was obtained with ethacrynic acid, while conjugation of ethacrynic acid with glutathione did not abolish its inhibiting properties. For the α-, μ- and π-class i₅₀ values (μM) were 4.6–6.0, 0.3–1.9 and 3.3–4.8, respectively for ethacrynic acid, and 0.8–2.8, < 0.1–1.2 and 11.0, respectively for its glutathione conjugate. Of all isoenzymes tested the human isoenzyme μ is most sensitive to the action of both ethacrynic acid and its glutathione conjugate.

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Selective inhibition of leukotriene C4 synthesis in human neutrophilis by ethacrynic acid

Biochem Biophys Res Commun

(1986)

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The present work focused on molecular structure and various spectroscopic studies of 2-[2,3-dichloro-4-(2-methylidenebutanoyl)phenoxy] acetic acid is also called as Ethacrynic acid (EA). The B3LYP/6-311G(d,p) level of theory was adopted to compute the molecular geometry (monomer and dimer) for EA molecule, the computed geometrical parameters are well coincided with related XRD data. The Natural Bond Orbital (NBO) was calculated to find donor-acceptor interaction. The vibrational modes of EA molecule was computed by B3LYP/6-311G(d,p) method and assignments were carried out by finding Potential Energy Distribution (PED) using VEDA software. The deviation in wavenumber of O-H vibrations (in COOH) group evident the intermolecular hydrogen bond interaction is possible between nearby molecules. The Gauge Independent Atomic Orbital (GIAO) method is used to find ¹H and ¹³C NMR chemical shift and correlated with recorded NMR spectrum. The Time-Dependent density functional theory (TD-DFT) is used to determine the various electronic parameters. The intra, inter molecular and non covalent interactions of the EA molecule are examined by Atoms in Molecules (AIM), ELF, LOL and RDG analysis. From AIM analysis the EA molecule form C-H…...Cl hydrogen bond of energies 1.2863 and 1.1922 Kcal/mol in both monomer and dimer were determined. Mulliken atomic charge analysis is used find the electronegativity equalization and charge transfer in the molecular system. Fukui function carried out to identify electrophilic and nucleophilic attack in the EA molecule. Thermal (DTA) and thermogravimetric (TG) analyzes adopted to determine the thermal behavior of compound which denote the compound dissociate around 122.5 °C. The thermodynamic parameters for different temperature were also calculated by B3LYP/6-311G(d,p) method. The EA compound was monitor to the antibacterial activity by agar well diffusion method. Molecular docking analysis carried out using Autodock software with protein inhibit thyroid and breast cancer show binding energies of -4.79 and -3.99 Kcal/mol.
Changing human hair fibre colour and shape from the follicle
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Natural hair curvature and colour are genetically determined human traits, that we intentionally change by applying thermal and chemical treatments to the fibre. Presently, those cosmetic methodologies act externally and their recurrent use is quite detrimental to hair fibre quality and even to our health.
This work represents a disruptive concept to modify natural hair colour and curvature. We aim to model the fibre phenotype as it is actively produced in the follicle through the topical delivery of specific bioactive molecules to the scalp.
Transcriptome differences between curly and straight hairs were identified by microarray. In scalp samples, the most variable transcripts were mapped by in situ hybridization. Then, by using appropriate cellular models, we screened a chemical library of 1200 generic drugs, searching for molecules that could lead to changes in either fibre colour or curvature. A pilot-scale, single-centre, investigator-initiated, prospective, blind, bilateral (split-scalp) placebo-controlled clinical study with the intervention of cosmetics was conducted to obtain a proof of concept (RNEC n.92938).
We found 85 genes transcribed significantly different between curly and straight hair, not previously associated with this human trait. Next, we mapped some of the most variable genes to the inner root sheath of follicles, reinforcing the role of this cell layer in fibre shape moulding. From the drug library screening, we selected 3 and 4 hits as modulators of melanin synthesis and gene transcription, respectively, to be further tested in 33 volunteers. The intentional specific hair change occurred: 8 of 14 volunteers exhibited colour changes, and 16 of 19 volunteers presented curvature modifications, by the end of the study.
The promising results obtained are the first step towards future cosmetics, complementary or alternative to current methodologies, taking hair styling to a new level: changing hair from the inside out.
An overview on the synthesis and anticancer properties of ethacrynic acid and their analogues
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Ethacrynic acid (EA, Edecrin) is a well-known diuretic drug used in the treatment of high blood pressure and the swelling caused by diseases such as congestive heart failure, liver failure, and kidney failure. EA has an α,β-unsaturated carbonyl unit which acts as a Michael acceptor involved in the inhibition of the major classes of glutathione S-transferases (GSTs) (α, μ, and π) that plays a crucial role in regulating the multidrug resistance. Moreover, EA was used as an adjuvant to improve the therapeutic efficacy of several anticancer chemotherapeutic agents. EA and their derivatives have received considerable attention in the past decades due to their interesting biological properties. This review highlights the recent development in this area, focusing on their synthesis and their antitumor activities.
A Switch-On Affinity-Based Iridium(III) Conjugate Probe for Imaging Mitochondrial Glutathione S-Transferase in Breast Cancer Cells
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Glutathione S-transferase is heterogeneously expressed in breast cancer cells and is therefore emerging as a potential diagnostic biomarker for studying the heterogeneity of breast cancers. However, available fluorescent probes for GSTs depend heavily on GSTs-catalyzed glutathione (GSH) nucleophilic substitution reactions, making them susceptible to interference by the high concentration of nucleophilic species in the cellular environment. Moreover, the functions of subcellular GSTs are generally overlooked due to the lack of suitable luminescence probes. Herein, we report a highly selective affinity-based luminescence probe 1 for GST in breast cancer cells through tethering a GST inhibitor, ethacrynic acid, to an iridium(III) complex. Compared to activity-based probes which require the use of GSH, this probe could image GST-pi in the mitochondria by directly adducting to GST-pi (or potentially GST-pi/GS) in living cells. Probe 1 possesses desirable photophysical properties including a lifetime of 911 ns, a Stokes shift of 343 nm, and high photostability. The “turn on” luminescence mode of the probe enables highly selective detection of the GST with a limit of detection of 1.01 μM, while its long emission lifetime allows sensitive detection in organic dye-spiked autofluorescence samples by a time-resolved mode. The probe was further applied to specifically and quantitatively visualize MDA-MB-231 cells via specific binding to mitochondrial GST, and could differentiate breast cell lines based on their expression levels of GST. To the best of our knowledge, this probe is the first affinity-based iridium(III) imaging probe for the subcellular GST. Our work provides a valuable tool for unmasking the diverse roles of a subcellular GST in living systems, as well as for studying the heterogeneity of breast cancers.
Benoxacor is enantioselectively metabolized by microsomes and cytosol from the human liver
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Citation Excerpt :
Benzil (CES inhibitor [11,12]) was obtained from ACROS Organics (≥99% purity; InChIKey: WURBFLDFSFBTLW-UHFFFAOYSA-N; New Jersey, NJ, USA). Ethacrynic acid (GST inhibitor [13,14]) was purchased from Enzo Life Sciences (≥98%; InChIKey: AVOLMBLBETYQHX-UHFFFAOYSA-N; Farmingdale, NY, USA). Pesticide-grade ethyl acetate was purchased from Fisher Chemical (Fair Lawn, NJ, USA).
Benoxacor is a safener added to current-use herbicide formulations to protect the target crop from herbicidal toxicity. It is an emerging environmental contaminant that has been detected in surface waters, raising the possibility of human exposure via drinking water. Because it is not subject to the same regulations as active pesticide ingredients, its metabolism and toxicity in humans have not been studied. Here we investigate the enantioselective metabolism of benoxacor in human subcellular fractions. Pooled human liver microsomes (pHLM) and cytosol (pHLC) were incubated with racemic benoxacor for up to 30-min. Gas chromatographic analyses were used to measure the enantioselective depletion of benoxacor. pHLMs with and without nicotinamide adenine dinucleotide phosphate (NADPH, co-factor for cytochrome P450 enzymes [CYPs]) and pHLC with glutathione (GSH, co-factor for glutathione S-transferases [GSTs]) metabolized benoxacor. These results demonstrate that microsomal CYPs, microsomal carboxylesterase (CESs), and cytosolic GSTs metabolize benoxacor. Females were predicted to have a higher clearance of benoxacor by GSTs than males. Male and female pHLM incubations with NADPH showed enrichment of the first eluting benoxacor enantiomer (E₁-benoxacor). pHLM incubations without NADPH and pHLC incubations with GSH showed an enrichment of the second eluting enantiomer of benoxacor (E₂-benoxacor). Our results indicate that human hepatic microsomal and cytosolic enzymes enantioselectively metabolize benoxacor, a fact that needs to be considered when investigating human exposures and toxicities of benoxacor.
Improving reactivity of naphthalimide-based GST probe by imparting TPP cation: Development and application for live cell imaging
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Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes comprising multiple classes and subtypes. This paper describes the synthesis and characterization of TPPBN-1, a naphthalimide derivative conjugated with a triphenylphosphonium (TPP) cation. When 4-bromonaphthalimide (BrNaph), a previously characterized GST substrate, was conjugated to a TPP cation, the conjugate showed increased reactivity towards most alpha- and mu-class GSTs, particularly the GSTA2 subtype, compared to the parent compound, but hardly towards Pi-class GSTs. Using this probe with enhanced reactivity, the enzymatic activity of endogenous GSTA1/2 in HepG2 cells was visualized by confocal fluorescence microscopy. The results demonstrated that modification with TPP cations, which are often used as tags for targeting mitochondria, can be used to enhance the reactivity of probes for specific GST subtypes.

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Biochemical Pharmacology

Abstract

References (22)

Glutathione transferases as markers of preneoplasia and neoplasia

Adv Cancer Res

Characterization of melphalanglutathione adducts whose formation is catalyzed by glutathione transferases

Biochem Pharmacol

Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells

J Biol Chem

Determination of intracellular glutathione and glutathione related enzyme activities in cisplatin-sensitive and resistant experimental ovarian carcinoma cell lines

Cancer Lett

Inhibition of purified rat liver glutathione S-transferase isoenzymes by diuretic drugs

Biochem Pharmacol

Characterization of the activation of rat liver glutathione S-transferases by nonsubstrate ligands

Tox Appl Pharmacol

Glutathione S-transferases. The first step in mercapturic acid formation

J Biol Chem

Binding of ethacrynic acid to hepatic glutathione S-transferases in vivo in the rat

Biochem Pharmacol

Differential induction of rat hepatic glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate: comparison with induction by phenobarbital and 3-methylcholanthrene

Biochem Pharmacol

An improved method for the separation and quantification of glutathione S-transferase subunits in rat tissue using high-performance liquid chromatography

J Chromatog

Selective inhibition of leukotriene C4 synthesis in human neutrophilis by ethacrynic acid

Biochem Biophys Res Commun

Cited by (151)

Exploring molecular structure (monomer & dimer), spectroscopic (FT-IR, FT-Raman, UV-Vis, NMR), TG/DTA, antibacterial and molecular docking investigation on 2-[2,3-dichloro-4-(2-methylidenebutanoyl)phenoxy] acetic acid by DFT and wavefunction analysis

Changing human hair fibre colour and shape from the follicle

An overview on the synthesis and anticancer properties of ethacrynic acid and their analogues

A Switch-On Affinity-Based Iridium(III) Conjugate Probe for Imaging Mitochondrial Glutathione S-Transferase in Breast Cancer Cells

Benoxacor is enantioselectively metabolized by microsomes and cytosol from the human liver

Improving reactivity of naphthalimide-based GST probe by imparting TPP cation: Development and application for live cell imaging