Isatin, an endogenous monoamine oxidase inhibitor, triggers a dose- and time-dependent switch from apoptosis to necrosis in human neuroblastoma cells

Isatin is an endogenous indole that is increased in stress, inhibits monoamine oxidase (MAO) B and improves bradykinesia and striatal dopamine levels in rat models of Parkinson's disease. Consequently, it has been suggested that isatin might be a possible treatment for Parkinson's disease although little is known about its effects on neural cell growth and survival. The aim of this study was to investigate the survival of dopaminergic human neuroblastoma (SH-SY5Y) cells following treatment with increasing concentrations of isatin. SH-SY5Y cells were exposed to isatin for defined time points, after which cell survival was determined by MTT assay. A combination of Annexin V binding and propidium iodide (PI) exclusion was used to distinguish apoptosis from necrosis in flow cytometry experiments and FACS profiles of permeabilised PI-labelled cells were employed for the assessment of cell cycle distribution. Isatin treatment (1-400 microM) for 24h induced a significant dose-dependent increase in MTT metabolism by SH-SY5Y cells in culture, but this was not due to an increase in cell division. At the higher concentrations (200-400 microm) isatin triggered cell death, although MTT metabolism was still increased in the culture, suggesting that surviving cells were hypermetabolic. Following a longer (48 h) exposure, isatin was found to cause cell death in a dose-dependent manner; at lower concentrations (50 microM), the predominant mode of cell death was apoptosis while at the highest concentration (400 microm) increasing numbers of necrotic cells were also evident. Thus, in dopaminergic SH-SY5Y cells isatin induces cell death in dose- and time-dependent manner. This death occurred as a continuum of survival, apoptosis and necrosis. Our results re-emphasise that caution should be exercised when considering high doses of isatin as a putative anti-Parkinson's disease therapeutic.     

The effect of isatin (tribulin) on metabolism of indoles in the rat brain and pineal: In vitro and in vivo studies

Isatin (Tribulin) produced a dose-dependent inhibition of both MAO A and MAO B in broken cell preparations from rat brain and pineal. However, isatin administered in vivo (80–160 mg/kg) to the intact animal significantly increased brain, but not pineal, serotonin and did not affect 5HIAA or other indoles in either brain or pineal. Further, in vivo administration did not produce detectable MAO inhibition in either tissue. In pineal organ culture, addition of isatin up to 1mM had no influence on the concentrations of pineal indoles or the activities of monoamine oxidase or serotonin N-acetyltransferase. However, the diazepam augmentation of beta adrenergic induction of serotonin N-acetyltransferase activity was blocked by isatin. The results of these studies call into question the proposed role of isatin as an endogenous monoamine oxidase inhibitor but support a possible role as a benzodiazepine receptor blocker.     

Effect of isatin on the electrical activity in the rat hippocampus cells

In vitro superfusion of rat hippocampal slices with isatin changed the population spikes. Isatin perfusion produced two clear effects. 50 microM isatin it increased the amplitude of the population spike in the CA1 evoked by stimulation of stratum radiatum. This effect was readily reversible. 100 microM isatin decreased the population spike amplitude with minimal effect on its latency. High initial response were more suppressed. This effect on the population spike amplitude was not eliminated even after 1 h of washing with saline. The data obtained suggest that isatin-induced electrophysiological changes are involved into the anticonvulsant effect of isatin.     

Inhibition of monoamine oxidase by C5-substituted phthalimide analogues

Literature reports that isatin as well as C5- and C6-substituted isatin analogues are reversible inhibitors of human monoamine oxidase (MAO) A and B. In general, C5- and C6-substitution of isatin leads to enhanced binding affinity to both MAO isozymes compared to isatin and in most instances result in selective binding to the MAO-B isoform. Crystallographic and modeling studies suggest that the isatin ring binds to the substrate cavities of MAO-A and -B and is stabilized by hydrogen bond interactions between the NH and the C2 carbonyl oxygen of the dioxoindolyl moiety and water molecules present in the substrate cavities of MAO-A and -B. Based on these observations and the close structural resemblances between isatin and its phthalimide isomer, a series of phthalimide analogues were synthesized and evaluated as MAO inhibitors. While phthalimide and N-aryl-substituted phthalimides were found to be weak MAO inhibitors, phthalimide homologues containing C5 substituents were potent reversible inhibitors of recombinant human MAO-B with IC(50) values ranging from 0.007 to 2.5 μM and moderately potent reversible inhibitors of recombinant human MAO-A with IC(50) values ranging from 0.22 to 9.0 μM. By employing molecular docking the importance of hydrogen bonding between the active sites of MAO-A and -B and the phthalimide inhibitors are highlighted.     

Inhibition of NO-dependent soluble human platelet guanylate cyclase by isatin

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. Physiologically relevant concentrations of isatin (ranged from 1 nM to 10 μM) did not influence basal activity of soluble human platelet guanylate cyclase (sGC), but caused a bell-shaped inhibition of the NO-activated enzyme. Inhibition of the NO-dependent activation by isatin did not depend on a chemical nature of the NO donors. The inhibitory effects of ODC (a heme-dependent inhibitor of sGC) and isatin were non-additive suggesting that the inhibitory effect of isatin may involve the heme binding domain (possibly heme iron) and experiments with hemin revealed some isatin-dependent changes in its spectrum. Isatin also inhibited sGC activation by the allosteric activator YC-1. It is suggested that the bell shaped inhibition of the NO-dependent activation of sGC by isatin may be attributed to complex interaction of isatin with the heme binding domain and the allosteric YC-1-binding site of sGC.     

Demonstration of isoleucine 199 as a structural determinant for the selective inhibition of human monoamine oxidase B by specific reversible inhibitors

Several reversible inhibitors selective for human monoamine oxidase B (MAO B) that do not inhibit MAO A have been described in the literature. The following compounds: 8-(3-chlorostyryl)caffeine, 1,4-diphenyl-2-butene, and trans,trans-farnesol are shown to inhibit competitively human, horse, rat, and mouse MAO B with K(i) values in the low micromolar range but are without effect on either bovine or sheep MAO B or human MAO A. In contrast, the reversible competitive inhibitor isatin binds to all known MAO B and MAO A with similar affinities. Sequence alignments and the crystal structures of human MAO B in complex with 1,4-diphenyl-2-butene or with trans,trans-farnesol provide molecular insights into these specificities. These inhibitors span the substrate and entrance cavities with the side chain of Ile-199 rotated out of its normal conformation suggesting that Ile-199 is gating the substrate cavity. Ile-199 is conserved in all known MAO B sequences except bovine MAO B, which has Phe in this position (the sequence of sheep MAO B is unknown). Phe is conserved in the analogous position in MAO A sequences. The human MAO B I199F mutant protein of MAO B binds to isatin (K(i) = 3 microM) but not to the three inhibitors listed above. The crystal structure of this mutant demonstrates that the side chain of Phe-199 interferes with the binding of those compounds. This suggests that the Ile-199 "gate" is a determinant for the specificity of these MAO B inhibitors and provides a molecular basis for the development of MAO B-specific reversible inhibitors without interference with MAO A function in neurotransmitter metabolism.     

Isatin‐binding proteins of rat and mouse brain: Proteomic identification and optical biosensor validation

Isatin (indole‐2,3‐dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X‐100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5‐aminocaproyl‐isatin‐Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin‐binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5‐aminocarproyl‐isatin, and 5‐aminoisatin as the affinity ligands. The K_d values (of 0.1–20 μM) obtained during the optical biosensor experiments were consistent with the range of K_d values recently reported for [³H]isatin binding to brain sections. Although the number of isatin‐binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one‐third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.     

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid peroxide formation and monoamine oxidase inhibition

Iron-dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)-B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinson's disease, Alzheimer's disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO-B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO-B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO-B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron-dependent lipid peroxidation in rat brain homogenates with IC50 values (12-16 microM), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 microM were able to attenuate cell death induced by serum deprivation and by 6-hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti-Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non-selective MAO-A and MAO-B inhibitor (IC50 < 0.1 microM). However, HLA20 was more selective for MAO-B but had poor MAO inhibition, with an IC50 value of 64.2 microM. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.     

Conversion of isatin to isatate as related to growth promotion in Avena coleoptile and pisum stem sections

Isatin, (indole 2,3-dione), which promotes elongation of Pisum stem sections at concentrations exceeding 0.1 mm, promotes elongation of Avena coleoptile sections only at higher concentrations, exceeding 1 mm. Aged isatin solutions are more active than fresh solutions, due to the slow, spontaneous conversion to isatate (o-aminophenylglyoxylate). A concentration of 0.1 mm aged isatin is as active in Avena coleoptile sections as in peas. Isatate has been independently synthesized and its auxin activity in both Avena coleoptile and Pisum stem sections confirmed. The synthetic isatate is more effective than isatin in both systems. This suggests that the auxin activity of isatin is due to its conversion to isatate.     

Isatin-enzyme interactions. VII. Mechanism of inhibition of rat kidney alkaline phosphatase.

Isatin has been found to inhibit rat kidney alkaline phosphatase (EC 3.1.3.1). The inhibition is dependent on isatin concentration and is of un-competitive type. The hydrolysis of disodium phenyl phosphate by the enzyme at different temperatures (17--37 degrees C) obeys the Arrhenius equation. Energy of activation in the absence and presence of isatin has been found to be 9.84 and 10.24 kCal/mol. The hyperbolic profile of isatin inhibition; the lowering of both Km and Vmax in the presence of isatin, and, small changes in enthalpy, free energy and entropy in the presence of isatin suggest a non-allosteric un-competitive inhibition of the enzyme.     

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.     

Isatin inhibition of rat testicular alkaline phosphatase. A non-allosteric phenomenon.

Isatin has been found to inhibit rat testicular alkaline phosphatase (EC 3.1.3.1) uncompetitively. The hyperbolic curve relating inhibition as a function of substrate concentration; the persistence of inhibition after the tertiary structure of the enzyme has been altered by heat denaturation, exposure to urea or papain digestion; the small changes in entropy, free energy and enthalpy in the presence of isatin, and the number of isatin molecules (n = 1.29) combining with one molecule of enzyme indicate the non-allosteric nature of inhibition.     

Characterization of a major form of human isatin reductase and the reduced metabolite.

Isatin, an endogenous indole, has been shown to inhibit monoamine oxidase, and exhibit various pharmacological actions. However, the metabolism of isatin in humans remains unknown. We have found high isatin reductase activity in the 105,000 g supernatants of human liver and kidney homogenates, and have purified and characterized a major form of the enzyme in the two tissues. The hepatic and renal enzymes showed the same properties, including an M(r) of 31 kDa, substrate specificity for carbonyl compounds and inhibitor sensitivity, which were also identical to those of recombinant human carbonyl reductase. The identity of the isatin reductase with carbonyl reductase was immunologically demonstrated with an antibody against the recombinant carbonyl reductase. About 90% of the soluble isatin reductase activity in the liver and kidney was immunoprecipitated by the antibody. The Km (10 microm) and k(cat)/K(m) (1.7 s(-1) x microm(-1)) values for isatin at pH 7.0 were comparable to those for phenanthrenequinone, the best xenobiotic substrate of carbonyl reductase. The reduced product of isatin was chemically identified with 3-hydroxy-2-oxoindole, which is also excreted in human urine. The inhibitory potency of the reduced product for monoamine oxidase A and B was significantly lower than that of isatin. The results indicate that the novel metabolic pathway of isatin in humans is mediated mainly by carbonyl reductase, which may play a critical role in controlling the biological activity of isatin.     

Synthesis and evaluation of isatin derivatives as effective SARS coronavirus 3CL protease inhibitors

N-Substituted isatin derivatives were prepared from the reaction of isatin and various bromides via two steps. Bioactivity assay results (in vitro tests) demonstrated that some of these compounds are potent and selective inhibitors against SARS coronavirus 3CL protease with IC50 values ranging from 0.95 to 17.50 microM. Additionally, isatin 4o exhibited more potent inhibition for SARS coronavirus protease than for other proteases including papain, chymotrypsin, and trypsin.     

Synthesis and evaluation of isatins and thiosemicarbazone derivatives against cruzain, falcipain-2 and rhodesain

While commercial isatins were practically inactive against the target proteases, thiosemicarbazone derivatives were found to be active. The most active compound from the series displayed an inhibitory IC(50) value of 1 microM against rhodesain. One thiosemicarbazone was found to be active against all three proteases with inhibitory IC(50) values of 10 microM or less. A combination of N-benzylation and appropriate substitution on the aromatic portion of the isatin scaffold was generally found to be beneficial especially against cruzain for ketone inhibitors.     

Inhibition of D-glucose uptake by isatin in rat intestine: effect of harmaline and various sulfhydryl reagents

The effect of isatin (indole-2,3-dione) on D-glucose uptake has been studied in rat intestine. Isatin at 6 mM concentration significantly inhibited both the sugar uptake and transmural (mucosal to serosal side) transport in the intestine. The suppression of glucose uptake by isatin was irreversible. Similar to the action of various SH-group-reacting agents, isatin inhibited the sugar uptake, presumably by binding to membrane sulfhydryl groups through a covalent linkage. Isatin-induced reduction in glucose uptake was unaffected by pH (between 5.5 and 8.4) and by DTT addition to incubation medium. Inhibition of sugar uptake by isatin and harmaline was additive in nature; this suggested that these compounds interact at different sites on the microvillus membrane surface.     

Specific inhibitors of Plasmodium falciparum thioredoxin reductase as potential antimalarial agents

Plasmodium falciparum thioredoxin reductase (PfTrxR: NADPH+Trx(S)2+H+<-->NADP++Trx(SH)2) is a high Mr flavin-dependent TrxR that reduces thioredoxin (Trx) via a CysXXXXCys pair located penultimately to the C-terminal Gly. In this respect, PfTrxR differs significantly from its human counterpart which bears a Cys-Sec redox pair at the same position. PfTrxR is essentially involved in antioxidant defense and redox regulation of the parasite and has been previously validated by knock-out studies as a potential drug target for malaria chemotherapy. Moreover, human TrxR is present in most cancer cells at levels tenfold higher than in normal cells. Here we report the discovery of a series of potent inhibitors of PfTrxR. The three most promising inhibitors, 3(IC50(PfTrxR)=2 microM and IC50(hTrxR)=50 microM), 7(IC50(PfTrxR)=2 microM and IC50(hTrxR)=140 microM), and 11(IC50(PfTrxR)=0.5 microM and IC50(hTrxR)=4 microM) were selective for the parasite enzyme. Detailed mechanistic characterization of the effects of these compounds on the PfTrxR-catalyzed reaction showed clear uncompetitive inhibition with respect to both substrate and cofactor. For the most specific PfTrxR inhibitor 7, an alkylation mechanism study based on a thiol conjugation model was performed. Furthermore, all three compounds were active in the lower micromolar range on the chloroquine-resistant P. falciparum strain K1 in vitro.     

Design and synthesis of 2,6-diprenyl-4-iodophenol TX-1952 with a novel and potent anti-peroxidative activity

The structure-based elucidation of 2,4,6-tri-substituted phenols for their antioxidative and anti-peroxidative effects has been investigated using TX-1952 (2,6-diprenyl-4-iodophenol), TX-1961, TX-1980, BTBP and BHT. In the inhibition of mitochondrial lipid peroxidation, the inhibitory activity of 2,6-di-tert-butyl-4-bromophenol (BTBP) (IC(50)=0.17 microM) was twice as high as that of 2,6-di-tert-butyl-4-methylphenol (BHT) (IC(50)=0.31 microM). This result shows that the 4-halogen group increases inhibitory activity for mitochondrial lipid peroxidation. Besides, TX-1952 (IC(50)=0.60 microM) was the highest inhibitor among 2,6-diprenyl-4-halophenols, followed by TX-1961 (IC(50)=0.93 microM) and TX-1980 (IC(50)=1.2 microM). In 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging experiments, the activity of TX-1952 (IC(0.200)=53.1 microM) was lower than that of BHT (IC(0.200)=33.7 microM) and BTBP (IC(0.200)=16.0 microM), but TX-1952 and BHT showed the same HOMO energy (-8.991 eV). These results suggest that the two prenyl groups at ortho position hinder the phenolic hydrogen abstraction by DPPH radical. These findings demonstrated that TX-1952 was a novel and potent inhibitor for lipid peroxidation.     

Isatin causes a rapid accumulation of ATP in synaptosomes: Implication for stress and regulation of natriuretic peptide receptors

Isatin is an endogenous indole, which is increased in mammalian brain and peripheral tissues under conditions of stress. Physiological concentrations of isatin inhibit natriuretic peptide (NPR) receptor binding and NPR-dependent signalling. The inhibition of NPR signalling by isatin is attenuated by a nonhydrolyzable ATP analogue. In this study we have demonstrated that short term incubation of rat brain synaptosomes with a physiological concentration of isatin caused a rapid 3-fold accumulation of ATP. The additional increase of ATP in the presence of tyrphostin, an inhibitor of tyrosine kinase, which uses ATP for phosphorylation of some proteins, suggests the dependence of this phenomenon on the activity of ATP-consuming systems. ATP inhibited binding of [³H]isatin to both particulate and soluble fractions of the rat brain. These results suggest that isatin induces accumulation of ATP, which in turn may displace isatin from both membrane-bound and soluble binding sites. Since natriuretic peptides are known to decrease stress hormone release this regulatory loop may be involved in the maintenance of natriuretic peptide signalling under conditions of stress and thus contribute to the control of stress responses.     

D-mannonolactam amidrazone. A new mannosidase inhibitor that also inhibits the endoplasmic reticulum or cytoplasmic alpha-mannosidase.

The amidrazone of D-mannonolactam (see compound 5, Fig. 1) was synthesized chemically as a mimic of the mannopyranosyl cation and tested as a potential inhibitor of mannosidases. In this study compound 5 is shown to be a more general mannosidase inhibitor than other currently known compounds and exhibits properties not previously observed with any other mannosidase inhibitors. Thus D-mannonolactam amidrazone not only inhibits the Golgi mannosidase I (IC50 = 4 microM) and mannosidase II (IC50 = 90-100 nM), but it is the first inhibitor that has been shown to be a potent inhibitor of the soluble or endoplasmic reticulum alpha-mannosidase (IC50 = 1 microM). This compound also inhibited the aryl-mannosidases regardless of anomeric configuration although it was much more effective on enzymes recognizing alpha-linked mannose, i.e. jack bean and mung bean alpha-mannosidases (IC50 = 400 nM) as compared with fungal beta-mannosidase (IC50 = 150 microM). Mannonoamidrazone was tested in animal cell cultures using influenza virus-infected Madin-Darby canine kidney cells as a model system, and was found to prevent almost completely the formation of complex types of N-linked oligosaccharides with the formation of about equal amounts of Man9(GlcNAc)2 and Man8(GlcNAc)2 structures. Thus D-mannonolactam amidrazone is a potent but broad spectrum mannosidase inhibitor whose structure and properties should provide valuable insight into the design of other useful glycosidase inhibitors.