Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)-two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 ± 3.6 µM and a Ki of 23.2 ± 3.2 µM. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Ethacrynic acid inhibits STAT3 activity through the modulation of SHP2 and PTP1B tyrosine phosphatases in DU145 prostate carcinoma cells.

To identify signal transducer and activator of transcription factor 3 (STAT3) inhibitors, we generated STAT3-dependent gene expression signature by analyzing gene expression profiles of DU145 cancer cells treated with STAT3 inhibitor, piperlongumine and 2-hydroxycinnamaldehyde. Then we explored gene expression signature-based strategies using a connectivity map database and identified several STAT3 inhibitors, including ethacrynic acid (EA). EA is currently used as a diuretic drug. EA inhibited STAT3 activation in DU145 prostate cancer cells and consequently decreased the levels of STAT3 target genes such as cyclin A and MCL-1. Furthermore, EA treatment inhibited tumor growth in mice xenografted with DU145 cells and decreased p-STAT3 expression in tumor tissues. Knockdown of Src homology region 2 domain-containing phosphatase-2 (SHP2) or Protein tyrosine phosphatase 1B (PTP1B) gene expression by siRNA suppressed the ability of EA to inhibit STAT3 activation. When EA was combined with an activator of SHP2 or PTP1B, p-STAT3 expression was synergistically decreased; when EA was combined with an inhibitor of SHP2 or PTP1B, p-STAT3 expression was rescued. By using an affinity pulldown assay with biotinyl-EA, EA was shown to associate with SHP2 and PTP1B in vitro. Additionally, the drug affinity responsive target stability (DARTS) assay confirmed the direct binding of EA to SHP2 and PTP1B. SHP2 is activated by EA through active phosphorylation at Y580 and direct binding to SHP2. Collectively, our results suggest that EA inhibits STAT3 activity through the modulation of phosphatases such as SHP2 and PTP1B and may be a potential anticancer drug to target STAT3 in cancer progression.

Potent inhibitors of equine steroid isomerase EcaGST A3-3.

Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 µM concentration afforded at least 50% inhibition of EcaGST A3-3. The most potent inhibitors, anthralin, sennoside A, tannic acid, and ethacrynic acid, were characterized by IC50 values in the submicromolar range when assayed with the natural substrate Δ5-androstene-3,17-dione.

Isotope-reinforced polyunsaturated fatty acids protect mitochondria from oxidative stress.

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure-activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D1-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D2-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20-50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function.

Investigations on dendrimer space reveal solid and liquid tumor growth-inhibition by original phosphorus-based dendrimers and the corresponding monomers and dendrons with ethacrynic acid motifs.

The well-known reactive diuretic ethacrynic acid (EA, Edecrin), with low antiproliferative activities, was chemically modified and grafted onto phosphorus dendrimers and the corresponding simple branched phosphorus dendron-like derivatives affording novel nanodevices showing moderate to strong antiproliferative activities against liquid and solid tumor cell lines, respectively.

FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

OBJECTIVE: Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. METHODS: We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. RESULTS: We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. CONCLUSION AND PRACTICAL IMPLICATIONS: In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

Effects of novel ethacrynic acid derivatives on human trabecular meshwork cell shape, actin cytoskeletal organization, and transcellular fluid flow.

To determine efficacy and therapeutic index in the context of ocular hypotensive activity of the new ethacrynic acid (ECA) derivatives of the series (SA8,248 and SA8,389), 9,000 series (SA9,000, SA9,622 and SA9,995) and ticrynafen, we undertook a comparative evaluation of the dose-dependent effects of these compounds on human trabecular meshwork (HTM) cell shape, actin cytoskeletal organization, focal adhesions and transcellular fluid flow. Responses were either scored using an arbitrary scale of 1-5 or quantified. Compounds of the 9000 series (SA9,995>SA9,000>SA9,622) were found to be 14- to 20-fold more potent than ECA, ticrynafen or analogs from the 8,000 series (SA8,389>SA8,248) in terms of ability to induce cell shape alterations in HTM cells. Similarly, compounds of the 9,000 series (SA9,995>SA9,622>SA9,000) were found to be much stronger (2 to 20 fold) than ECA, ticrynafen or analogs of the 8000 series in terms of affecting decreases in actin stress fiber content in HTM cells. Analogs of the 9000 series (SA9,622>SA9,995>SA9,000) were also observed to be 8 to 10 fold more potent than ECA (SA8,389>ECA>SA8,248>ticrynafen) at eliciting decreases in cellular focal adhesions. Interestingly, analogs of the 9000 series (SA9,000>SA9,622>SA9,995) and SA8,248 demonstrated a huge increase (by many folds) in transcellular fluid flow of HTM cell monolayers as compared to ECA and ticrynafen. Collectively, these analyses revealed that the structural modification of ECA improves its ocular hypotensive efficacy, indicating that the SA9,000 series compounds might be promising novel ocular hypotensive drugs.

Coffee diterpenes prevent benzo[a]pyrene genotoxicity in rat and human culture systems.

The coffee-specific diterpenes cafestol and kahweol (C+K) have been identified as two important chemoprotective agents in coffee. In the present study, the potential preventive effects of C+K against the genotoxicity of B[a]P were investigated in rat primary hepatocytes and in human bronchial Beas-2B cells. Several independent mechanisms were identified and their respective contribution to the overall protective effects was determined. A marked dose-dependent inhibition by C+K of B[a]P DNA-binding was found in cells of both origins. However, data showed that the significant induction by C+K of the detoxifying enzyme GST-Yp subunit is the key mechanism of protection against B[a]P DNA-binding in rat liver. In contrast, the phase I-mediated mechanism where C+K produce an inhibition of CYP 1A1 induction by B[a]P is of key significance for the C+K protection in human Beas-2B cells. Moreover, this effect suggests a novel mechanism of chemoprotection by the coffee diterpenes cafestol and kahweol.

Bioelectrical cochlear noise and its contralateral suppression: relation to background activity of the eighth nerve and effects of sedation and anesthesia.

The bioelectrical activity of the cochlea, without any ipsilateral acoustic stimulation, was recorded in awake guinea pigs (GPs) between electrodes chronically implanted at the round window (RW) and the skull. Measuring its power in the band centered around 1.0 kHz (0.5-2.5 kHz) provided an indirect measure of the ensemble background (EBA) activity of the eighth nerve. Contralateral white-noise (CLWN) stimulation reduced this EBA, presumably by activation of medial olivocochlear fibers. The aim of the investigation was to validate measurements of EBA and of its contralateral suppression in order to study the medial efferent function. The first goal was to find the best conditions for recording the EBA in the absence of ipsilateral stimulation and for studying its suppression by contralateral acoustic stimulation, which implies that no noise was generated by the experimental animal. Thus recordings were compared in normal, awake GPs and in GPs under sedation with xylazine, anesthetized with a combination of xylazine and ketamine, and with and without temperature regulation. In order to monitor the effects of sedation and anesthesia, the recordings were analyzed not only in the 0.5- to 2.5-kHz frequency band but also in the other frequency bands, 5-50 Hz, 50-150 Hz, and 150-500 Hz, which presumably include general central and neuromuscular contributions. The results show that sedation with xylazine accompanied by regulation of body temperature does not affect the EBA value nor its contralateral suppression. Nevertheless, anesthesia should be avoided, even with control of body temperature. The second goal of this study was to identify the specific cochlear contribution to the raw RW signal. Thus recordings were performed in normal and deafened animals and analyzed in the frequency band 0.5-2.5 kHz and also in the other frequency bands of 5-50 Hz, 50-150 Hz, and 150-500 Hz. The results indicate that most of the cochlear activity lies in the frequency band 0.5-2.5 kHz, with also some minor contribution coming from the 150- to 500-Hz band. Analysis and comparison of power values in the different conditions indicate that specific cochlear EBA power was about 60 microV2. From a commonly accepted mean background discharge rate of 50 spikes/s (sp/s), the EBA power without CLWN should have been around 4.4 microV2 if the fibers' activity was random. This difference suggests that there is probably some degree of synchrony between individual fibers. There was a reduction of approximately 45% during CLWN stimulation. This suppression might correspond to a reduction in both discharge rate and synchrony of the fibers.

Neuroprotective effect of insulin-like growth factor I in immortalized hypothalamic cells.

The neuroprotective action of insulin-like growth factor I (IGF-I) was tested in immortalized hypothalamic GT1-7 cells exposed to reduced glutathione depleting agents, which cause oxidative stress and cell death. The extent of cell survival was assessed by either using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide cytotoxicity assay or counting at the fluorescence microscope GT1-7 cells prelabeled with fluorescent dyes selective for viable and dead cells. Treatments with buthionine sulfoximine (500 microns), diethylmaleate (1 mM), and ethacrynic acid (200 microns) caused diffuse GT1-7 cell death (40-60%). Exposure of the same cells to IGF-I (either before or concomitant to the toxic agent, depending on the drug used) significantly prevented neuronal death. This effect was rapid, concentration-dependent, maximal at concentrations of 25-50 ng/ml, and mimicked by IGF-II, fibroblast growth factor, and the potent antioxidant idebenone. In contrast, IGF-I, as well as idebenone, were completely ineffective in antagonizing the toxic effect produced by different concentrations of menadione. In conclusion, the present data demonstrate a protective role for IGF-I against glutathione depleting agents-induced damage in GT1-7 cells suggesting an antioxidant action of this growth factor in hypothalamic neurons.

Inhibition of NAD(P)H:quinone oxidoreductase1 in ethacrynic acid-resistant human colon carcinoma cells.

Human colon carcinoma HT29 cells sensitive (WT) and resistant (HT/M and HT/S) to ethacrynic acid (EA) were used to investigate the role of NAD(P)H:quinone oxidoreductase1 (NQO1) in drug resistance. Significant decreases in the levels of NQO1 activity were observed in resistant cells as compared with the sensitive cells. However, the decreased activities of NQO1 in resistant cells were found to be due to inhibition of the enzyme by EA. Human NQO1 cDNA-derived protein in monkey kidney COS1 cell extract was used to demonstrate that in vitro inhibition of NQO1 activity by EA was rapid, reversible and concentration dependent, with an IC50 value of 250 microM. These results suggest that NQO1 may not have a role in EA resistance of human colon carcinoma HT29 cells and that EA is an inhibitor of NQO1 activity.

Evidence for two discrete sources of 2f1-f2 distortion-product otoacoustic emission in rabbit. II: Differential physiological vulnerability.

In a previous report, it was shown that, in normal rabbit ears, the amplitude and phase of 2f1-f2 distortion-product otoacoustic emissions (DPOAEs) elicited by low-level (< 60-70 dB SPL) stimuli display a differential dependence on stimulus parameters to those evoked by high-level (> 60-70 dB SPL) stimuli, indicating differences in the underlying generation mechanisms. In the present study, the physiological vulnerability of DPOAEs in each of the two 2f1-f2 DPOAE-response regions identified on the basis of differential parametric properties, was characterized. Thus emissions evoked using stimulus levels from 45-75 dB SPL were measured over time upon: (1) induction of lethal anoxia, (2) acute injection of ethacrynic acid, and (3) acute injection of ethacrynic acid 2 h after a single administration of gentamicin. The DPOAEs evoked by low-level stimuli (45 dB SPL) were abolished within 3-4 min of induction of anoxia, whereas DPOAEs evoked by high-level stimuli (75 dB SPL) were unchanged in this period. The high-level emissions decreased with a complex time course postmortem, and demonstrated behaviors, including evidence of susceptibility to fatigue, suggesting a dependence upon a cochlear energy supply. Low-level DPOAEs could be temporarily abolished, with complete recovery, by an acute administration of ethacrynic acid that had little effect on high-level DPOAEs. Treatment with the gentamicin and ethacrynic-acid combination, which would be expected to produce widespread hair-cell damage, eliminated low-level DPOAEs, and greatly reduced high-level emissions. In combination with previously published data, these findings strongly suggest that low- and high-level 2f1-f2 DPOAEs arise from discrete sources. The data are consistent with the proposal that the low-level DPOAE source is an active, micromechanical process, but suggest that the proposed origin of high-level DPOAEs exclusively in the passive macromechanics of the cochlear partition may be incorrect. The elimination of both low- and high-level DPOAEs revealed the presence of a third, residual 2f1-f2 DPOAE component, approximately 75-80 dB below the stimulus-tone levels, that may reflect the true passive-distortion response of the cochlea.

Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.

Simultaneous investigations of elemental changes in individual cells of the stria vascularis and in endolymph.

Using the microprobe for energy dispersive X-ray microanalysis, the elemental compositions of both the individual cells of the stria vascularis and of the endolymph were followed simultaneously under normal conditions and after the administration of 120 mg/kg ethacrynic acid (EA). Marginal cells and intermediate cells showed reversible increases in potassium and decreases in sodium concentrations. Shifts in the ionic composition of endolymph occurred later than after elemental changes in the strial cells. The present results indicate that the marginal and the intermediate cells are the primary target for EA-induced ototoxicity. However, generalized toxic effects of EA are also indicated, with a general leakage of different elements occurring during the 30-60 min period after EA administration.

[Effect of diuretics on vascular permeability and proliferative inflammation in rats]. / Vliianie diuretikov na sosudistuiu pronitsaemost' i proliferativnoe vospalenie u krys.

It is shown that ethacrynic acid and dichlothiazide in 50 mg/kg dose reduce the dextran-induced increase of rat skin vascular permeability. Besides, durable administration of furosemide, dichlothiazide and ethacrynic acid in 50-80 mg/kg doses decreases the dry mass of inflammatory granuloma. This fact shows the ability of diuretics to suppress the development of proliferative inflammation.

Placental calcium and phosphorus transfer in the guinea pig: lack of effect of modulators of Ca-ATPase and alkaline phosphatase activity.

The effects of modulators of Ca-ATPase and alkaline phosphatase (AP) activity on placental calcium and phosphorus transfer were studied using the in situ perfused guinea pig placenta. The diuretics ethacrynic acid and furosemide had no significant effect on placental calcium and phosphorus transfer when injected into the mother (1.0 or 10.0 mg X kg-1) or added to the solution perfusing the fetal side of the placenta (0.25 or 2.0 mM). These two drugs have previously been shown to inhibit placental Ca-ATPase and enhance AP activity in vitro. D-Penicillamine, which inhibits placental AP but not Ca-ATPase activity in vitro, also had no significant effect on net calcium and phosphorus transfer from mother to fetus either when given to the mother (50 mg X kg-1) or added to the placental perfusion solution (0.25 or 2.0 mM). These results suggest that placental transfer of calcium and phosphorus in the guinea pig may not be directly related to placental Ca-ATPase and AP activities.

Placental transfer of calcium (Ca) and phosphorus (Pi) in the guinea pig: lack of correlation with placental Ca-ATPase and alkaline phosphatase (AP) activities.

In summary, it has been observed that in vitro inhibitors of placental Ca-ATPase and AP activities (EA, F, D-pen) and activators of placental AP (EA,F) are not associated with changes in Ca and Pi transfer across the in situ perfused guinea pig placenta. Assuming that the two enzyme activities were altered in vivo by these drugs, it may be that they are not directly related to active transport of Ca and Pi across the placenta.

[Relationship between the endocochlear potential and metabolism in the vascular stria of the inner ear]. / Zavisimost' éndokokhlearnogo potentsiala ot metabolizma v sosudistoi poloske vntrennego ukha.

Effects of dihydrostreptomycin, etacrine acid and ATPh on the endocochlear potential in guinea-pigs depended on the dosage and means of administration. Dihydrostreptomycin suppressed the potential only when perfusing the perilymphatic space or being microinjected into the endolymphatic duct. I. v. administration of etacrine acid suppressed the potential with its following partial restoration. I.v. administration of ATPh in large doses suppressed the potential for a short period but did not prevent the ototoxic effect of etacrine acid. ATPh in small doses did not suppress the potential but exerted the protective effect.

Energy requirement for the maintenance of membrane potential in rat liver cells in situ.

The transmembrane potential of female rat liver cells in situ was-52.4 mV. This was decreased to -42.9 mV by ethacrynic acid (4 mg/100 g), but not by ouabain (1 mg). DL-Ethionine (25-100 mg) caused a decrease in the membrane potential and tissue ATP content. A high dose ethionine (100 mg) increased tissue Na content and decreased K content. By applying adenine (25 mg) to the animals treated with ethionine (100 mg), the membrane potential, ATP content and K content were increased and the Na content was decreased. The repolarized membrane potential in the animals treated with adenine following the ethionine was again depolarized by the administration of ouabain, but not by the administration of ethacrynic acid. These results suggest that two kinds of active ion transport mechanisms, ethacrynic acid-sensitive and ouabain-sensitive mechanisms, may be involved in maintenance of the membrane potential of rat liver cells.