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.     

Isatin-enzyme interactions. VI. Inhibition of rat kidney alkaline phosphatase.]

Interaction of isatin with rat kidney alkaline phosphatase has been studied. Mode of attachment of isatin with the enzyme protein is most likely through amino group(s), which is also imperative for catalysis. Sulphydryl group(s) do not seem to be involved in enzyme action. Zinc is also needed for enzyme activity. Use of sulphydryl compounds suggests that isatin inhibition of the enzyme is through attachment at the metal site. However, this inhibition may not only be due to simple chelation of the metal by isatin.     

Interaction of pyruvate kinase with isatin and deprenyl

The key glycolytic enzyme, pyruvate kinase, exhibits moderate affinity [³H]isatin binding (K_D ～10 μM) which is inhibited by ATP (IC₅₀ 25 μM) and deprenyl (IC₅₀ 5 μM). Interaction of pyruvate kinase with isatin and its inhibition by ATP and deprenyl has also been confirmed using an independent biosensor technique and the immobilized isatin analogue, aminoisatin. This effect has some specificity because the enzyme, creatine phosphokinase, does not exhibit specific isatin-binding. It is suggested that interaction of pyruvate kinase with isatin may reflect some non-glycolytic functions of this enzyme.     

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.     

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 analogs as caspase-3 inhibitors: introduction of a hydrophilic group increases potency in a whole cell assay

A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group, and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both in vitro (i.e., recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells compared to the corresponding unsubstituted N-phenyl isatin analogs.     

Glycosides of hydroxylamine derivatives. I. phase transfer synthesis and study of isatine-3-oximes glucosaminides influence on bacterial luminescence

In the phase transfer system solid calcium carbonate-acetonitrile, per acetate alpha-D-glucosaminilchloride glycosilate easily deprotoned isatine-3-oximes hydroxyl groups. It was found that the presence in the reaction mixture a catalytic amounts of 15-crown-5 accelerated the process twice. Obtained O-beta-D-glucosaminides were identified with 1H-NMR spectroscopy. Features of synthesized compound's NMR spectra are discussed in comparison with those of another N-acetylglucosamine 1-O-derivatives. The biological activity of the some oximes with different substituents in isatin residuum has been studied in a test of inhibition of bioluminescence of marine luminescent bacteria Photobacterium leiognathi Sh12. The nature of N-substituent of isatin fragment and 5-substituent of isatin main structure is compared with glycosides ability to suppress bacterial luminescence.     

Alkaline phosphatase from Echinococcus multilocularis: purification and characterization.

1. Kinetic and physical parameters of purified alkaline phosphatase from Echinococcus multilocularis metacestodes, livers of infected gerbils and control animals were determined. 2. Km value for p-nitrophenyl phosphate was about 0.05 +/- 0.02 mM for the three enzymes. 3. Vmax values were 357 +/- 67 nmol/min/mg proteins for metacestode enzyme, and 6.7 +/- 1.1 and 6.7 +/- 0.8 nmol/min/mg proteins for liver enzyme of infected and control animals, respectively. 4. Mr and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation of temperature than hepatic enzyme. 6. The isatin inhibition was a competitive inhibition type for parasite and uncompetitive type for host liver enzyme.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

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.     

A novel fungal enzyme, NADPH-dependent carbonyl reductase, showing high specificity to conjugated polyketones. Purification and characterization

A novel enzyme which specifically catalyzes the reduction of conjugated polyketones was purified to homogeneity from cells of Mucor ambiguus AKU 3006. The enzyme has a strict requirement for NADPH and irreversibly reduces a number of quinones such as p-benzoquinone, alpha-naphthoquinone and acenaphthenequione. The enzyme also reduces polyketones such as isatin and ketopantoyl lactone, and their derivatives. The apparent Km values for isatin and ketopantoyl lactone are 49.9 microM and 714 microM, respectively. The reduction of ketopantoyl lactone proceeds stereospecifically to yield L-(+)-pantoyl lactone. The pro-S (A) hydrogen at C-4 of NADPH is transferred to the substrate. The enzyme is not a flavoprotein and consists of two polypeptide chains with an identical relative molecular mass of 27,500. Quercetin, dicoumarol and some SH reagents inhibit the enzyme activity. 3-Methyl-1,2-cyclopentanedione and 1,3-cyclohexanedione are uncompetitive inhibitors with Ki values of 80.9 microM and 64.5 microM, respectively, to ketopantoyl lactone.     

Isatin: a privileged scaffold for the design of carbonic anhydrase inhibitors

The isatin scaffold is the constitutive fragment of several natural and synthetic bioactive molecules. Albeit several benzene sulphonamide-based carbonic anhydrase inhibitors (CAIs) have been reported, only recently isatin benzene sulphonamides have been studied and proposed as CAIs. In this study we have designed, synthesised, and evaluated the biological activity of a series of differently substituted isatin-based benzene sulphonamides which have been designed for the inhibition of carbonic anhydrase isoforms. The activity of all the synthesised compounds was evaluated towards human carbonic anhydrase I, II, IX, and XII isozymes. Our results indicate that the nature and position of substituents on the isatin ring can modulate both activity and isozyme selectivity.     

Synthesis,SAR elucidations and molecular docking study of newly designed isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase

Thymidine phosphorylase is an enzyme involved in pyrimidine salvage pathway that is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and gliostatin. It is enormously up regulated in a variety of solid tumors. Furthermore, surpassing of TP level protects tumor cells from apoptosis and helps cell survival. Thus TP is identified as a prime target for developing novel anticancer therapies. A new class of exceptionally potent isatin based oxadiazole (1–30) has been synthesized and evaluated for thymidine phosphorylase inhibitory potential. All analogs showed potent thymidine phosphorylase inhibition when compared with standard 7-Deazaxanthine, 7DX (IC₅₀ = 38.68 ± 1.12 µM). Molecular docking study was performed in order to determine the binding interaction of these newly synthesized compounds, which revealed that these synthesized compounds established stronger hydrogen bonding network with active site of residues as compare to the standard compound 7DX.     

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.     

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.     

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.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by 5-nitroisatin and the antiviral agent arbidol

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. The effect of isatin derivatives, 5-nitroisatin and arbidol (an antiviral agent) on spermine NONO-induced activation of human platelet soluble guanylate cyclase has been investigated. 5-Nitroisatin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylate cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. However, both compounds did not influence the activation of guanylate cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylate cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylate cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of the spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.     

The action of inhibitors (histidine and AMP) on the ATP phosphoribosyltransferase of E. coli.

The inhibitors histidine and AMP cause the enzyme ATP phosphoribosyltransferase of E. coli to associate into a hexamer from its initial dimeric form. The behaviour of these inhibitors has been studied by three different methods. I) Equilibrium dialysis studies have shown that one mole of dimeric enzyme (67,000 g) binds one mole of histidine. II) By kinetic inhibition of the reaction studied at 21, 25 and 38 degrees C the enthalpy changes in the process of histidine and of AMP inhibition have been deduced. The inhibition has also been studied in function of enzyme concentration and temperature. The inhibition appears to be slightly negatively cooperative for histidine and positively cooperative for AMP. In neither case is it possible to obtain 100% maximal inhibition. III) By microcalorimetric analysis the values obtained for the enthalpies of histidine and of AMP interaction with the enzyme are similar.     

Inhibition of enzymes by metal ion-chelating reagents. The action of copper-chelating reagents on diamine oxidase

1. The inhibition of diamine oxidase has been studied by using the following copper-chelating reagents: 1,10-phenanthroline; 2,2'-bipyridyl; 8-hydroxyquinoline (oxine); diethyldithiocarbamate and dithio-oxamide (rubeanic acid). 2. Addition of chelating reagent caused a rapid inhibition of enzyme to a degree dependent solely on the final inhibitor concentration. Addition of substrate gave linear initial rates of reaction showing that under these conditions the inhibition was not being rapidly reversed. 3. The inhibition has been investigated by using new graphical methods and has been found in all cases to involve the chelating agents completely removing two Cu(2+) ions from the enzyme. An alternative possibility, involving ligand substitution, was eliminated. 4. A value of K=8.0x10(-33)m(-2) has been found for the enzyme in equilibrium with 2 Cu(2+) ions (i.e. beta(2), the stability constant for diamine oxidase/two Cu(2+), is 32.1).     

Purification and Characterization of Oxopantoyl Lactone Reductase from Higher Plants: Role in Pantothenate Synthesis

Oxopantoyl lactone reductase has been purified to homogeneity from a crude extract of spinach leaves (Spinacia oleracea L.) using affinity chromatography on Red-Agarose and several subsequent ion exchange steps. The enzyme is monomeric with a relative molecular mass between 33,000 to 36,000. Affinity-purified antibodies directed against the homogenous enzyme have been used to determine the amount of oxopantoyl lactone reductase in the crude leaf extract as well as the chloroplast stroma. The overall purification factor has been determined to be 22,000. The subcellular location of the enzyme is chloroplastic. The final specific activity (strictly NADPH-dependent) is 4.5 μmole . min^?1 . mg^?1. The enzyme is also able to reduce isatin, bornanedione and acenaphthenequinone. The enzyme activity is strongly and uncompetitively inhibited by 2-keto-4-hydroxybutyrolactone and substituted 4,5-dioxopyrrolidines. An oxopantoate reductase associated with acetohydroxy acid isomeroreductase could be detected in the plant extract. Using a specific inhibitor of this latter enzyme or oxopyrrolidines, complementation studies with branched chain amino-acids and pantothenate have shown that oxopantoyl lactone reductase is likely to be involved in pantothenate biosynthesis. Furthermore, pantoyl lactone, the putative product of the reaction, together with β-alanine and ATP, has been shown to be the substrate of pantothenate synthase using a novel assay for pantothenate.