Binding of xanthine oxidase to glycosaminoglycans limits inhibition by oxypurinol

Although the binding of xanthine oxidase (XO) to glycosaminoglycans (GAGs) results in significant alterations in its catalytic properties, the consequence of XO/GAG immobilization on interactions with clinically relevant inhibitors is unknown. Thus, the inhibition kinetics of oxypurinol for XO was determined using saturating concentrations of xanthine. When XO was bound to a prototypical GAG, heparin-Sepharose 6B (HS6B-XO), the rate of inactivation for uric acid formation from xanthine was less than that for XO in solution (k(inact) = 0.24 versus 0.39 min(-1)). Additionally, the overall inhibition constant (K(i)) of oxypurinol for HS6B-XO was 2-5-fold greater than for free XO (451 versus 85 nm). Univalent electron flux (O(2)(.) formation) was diminished by the binding of XO to heparin from 28.5% for free XO to 18.7% for GAG-immobilized XO. Similar to the results obtained with HS6B-XO, the binding of XO to bovine aortic endothelial cells rendered the enzyme resistant to inhibition by oxypurinol, achieving approximately 50% inhibition. These results reveal that GAG immobilization of XO in both HS6B and cell models substantially limits oxypurinol inhibition of XO, an event that has important relevance for the use of pyrazolo inhibitors of XO in clinical situations where XO and its products may play a pathogenic role.     

Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC₅₀=0.600±0.009 µM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC₅₀=1.326±0.013 µM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC₅₀=1.564±0.065 µM) also showed inhibitory activity comparable to that of allopurinol (IC₅₀ = 0.776 ± 0.012 µM). All three compounds showed competitive type of inhibition with comparable K_i values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophe nolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound inhibited enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.     

Transition metal complexes (M = Cu, Ni and Mn) of Schiff-base ligands: Syntheses, crystal structures, and inhibitory bioactivities against urease and xanthine oxidase

Six new transition metal complexes (M = Cu(II), Ni(II) and Mn(III)) of tridentate (H₂L¹, HL²) and/or bidentate (HL³, HL⁴) Schiff-base ligands, obtained from the condensation of salicylaldehyde with glycine, N-(2-aminoethyl)morpholine, 4-(2-aminoethyl)phenylic acid and 4-(2-aminoethyl)benzsulfamide, respectively, were synthesized and structurally determined by single-crystal X-ray analysis. Complexes 1-6 were evaluated for their effect on the jack bean urease and xanthine oxidase (XO). Copper(II) complexes 1-3 (IC₅₀ = 0.43-2.25 μM) showed potent inhibitory activity against jack bean urease, comparable with acetohydroxamicacid (IC₅₀ = 42.12 μM), which is a positive reference. And these copper(II) complexes (IC₅₀ = 10.26-15.82 μM) also exhibited strong ability to inhibit activity of XO, comparable to allopurinol (IC₅₀ = 10.37 μM), which was used as a positive reference. Nickel(II) and manganese(III) complexes 4-6 showed weak inhibitory activity to jack bean urease (IC₅₀ = 4.36-8.25 μM) and no ability to inhibit XO (IC₅₀ > 100 μM).     

Relationship correlation of antioxidant and antiproliferative capacity of Cyperus rotundus products towards K562 erythroleukemia cells

A Total Oligomers Flavonoids (TOFs) and ethyl acetate extracts of Cyperus rotundus were analyzed, in vitro, for their antioxidant activity using several biochemical assays: the xanthine (X)/xanthine oxidase (XO), the lipid peroxidation induced by H₂O₂ in K562 human chronic myelogenous leukemia cells and the DNA damage in pKS plasmid DNA assay induced by H₂O₂/UV-photolysis and for their apoptotic effect. TOF and ethyl acetate extracts were found to be efficient in inhibiting xanthine oxidase with IC₅₀ values of 240 and 185 μg/ml and superoxide anion with IC₅₀ values of 150 and 215 μg/ml, respectively. Also, all the extracts tested were effective in reducing the production of thiobarbituric acid reactive substances (TBARS) and were able to protect against H₂O₂/UV-photolysis induced DNA damage. The highest activity, measured as equivalents of MDA concentration, was observed in the ethyl acetate extract (MDA = 2.04 nM). In addition, the data suggest that only TOF enriched extract exerts growth inhibition on K562 cells through apoptosis induction. Therefore, these extracts were subjected to further separation by chromatographic methods. Thus, three major compounds (catechin, afzelechin and galloyl quinic acid) were isolated from the TOF enriched extract and five major compounds (luteolin, ferulic acid, quercetin, 3-hydroxy, 4-methoxy-benzoic acid and 6,7-dimethoxycoumarin) from ethyl acetate extract. Their structures were determined by spectroscopic data analysis and comparison with the literature. In addition, we evaluate the biological activities of the catechin, ferulic acid and luteolin. This investigation has revealed that the luteolin was the most active in reducing the production of TBARS (MDA = 1.5 nM), inhibiting significantly the proliferation of K562 cells (IC₅₀ = 25 μg/ml) and protecting against H₂O₂/UV-photolysis induced DNA damage. In conclusion, the study reveals that the ability of C. rotundus to inhibit the enzyme xanthine oxidase (XO), the lipid peroxidation and to exert apoptotic effect, may explain possible mechanisms by which C. rotundus exhibits its health benefits.     

Design strategies to target crystallographic waters applied to the Hsp90 molecular chaperone

A series of novel and potent small molecule Hsp90 inhibitors was optimized using X-ray crystal structures. These compounds bind in a deep pocket of the Hsp90 enzyme that is partially comprised by residues Asn51 and Ser52. Displacement of several water molecules observed crystallographically in this pocket using rule-based strategies led to significant improvements in inhibitor potency. An optimized inhibitor (compound 17) exhibited potent Hsp90 inhibition in ITC, biochemical, and cell-based assays (K_d = 1.3 nM, K_i = 15 nM, and cellular IC₅₀ = 0.5 μM).     

Discovery of N-methyl-1-(1-phenylcyclohexyl)methanamine, a novel triple serotonin, norepinephrine, and dopamine reuptake inhibitor

The current work discloses a novel cyclohexylarylamine chemotype with potent inhibition of the serotonin, norepinephrine, and dopamine transporters and potential for treatment of major depressive disorder. Optimized compounds 1 (SERT, NET, DAT, IC₅₀ = 169, 85, 21 nM) and 42 (SERT, NET, DAT IC₅₀ = 34, 295, 90 nM) were highly brain penetrant, active in vivo in the mouse tail suspension test at 30 mpk po and were not general motor stimulants.     

Renierol from marine sponge Haliclona.SP.: A natural inhibitor of xanthine oxidase with hypouricemic effects

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC₅₀ value was 1.85 μg·ml^{? 1} through the measuring of uric acid and was 1.36 μg.ml^{? 1} through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg^{? 1}, there was a significant decrease in the serum urate level (4.08 ± 0.09 mg.dl^{? 1}, P < 0.01), (3.47 ± 0.11 mg.dl^{? 1}, P < 0.01) and (3.12 ± 0.08 mg.dl^{? 1}, P < 0.01), when compared to the hyperuricaemic control (6.74 ± 0.23 mg.dl^{? 1}). Renierol was a potent XO inhibitor with hypouricemic activity in mice.     

Detection of myosin light chain phosphorylation--a cell-based assay for screening Rho-kinase inhibitors

Here, we describe the first example of a cell-based myosin light chain phosphorylation assay in 96-well format that allows for the rapid screening of novel Rho-kinase inhibitors. We obtained IC₅₀ values for the prototypic Rho-kinase inhibitors Y-27632 (1.2 ± 0.05 μM) and Fasudil (3.7 ± 1.2 μM) that were similar to those previously published utilizing electrophoresis-based methodologies. H-1152P, a Fasudil analog showed an IC₅₀ value of 77 ± 30 nM. Data derived from a set of 21 novel Rho-kinase inhibitors correlate with those generated by a well-established cell-based phenotypic Rho-kinase inhibition assay (R² = 0.744). These results show that imaging technology measuring changes in myosin light chain phosphorylation can be used to rapidly generate quantitative IC₅₀ values and to screen a larger set of small molecule Rho-kinase inhibitors and suggests that this approach can be broadly applied to other cell lines and signaling pathways.     

Non-genomic effects of ginsenoside-Re in endothelial cells via glucocorticoid receptor

We demonstrated that ginsenoside-Re (Re), a pharmacological active component of ginseng, is a functional ligand of glucocorticoid receptor (GR) using competitive ligand-binding assay (IC₅₀ = 156.6 nM; K_d = 49.7 nM) and reporter gene assay. Treatment with Re (1 μM) raises intracellular Ca²⁺ ([Ca²⁺]_i) and nitric oxide (NO) levels in human umbilical vein endothelial cells as measured using fura-2 and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, respectively. Western blot analysis shows that Re increased phosphorylation of endothelial nitric oxide synthase. These effects were abolished by GR antagonist RU486, siRNA targeting GR, non-selective cation channel blocker 2-aminoethyldiphenylborate, or in the absence of extracellular Ca²⁺, indicating Re is indeed an agonistic ligand for the GR and the activated GR induces rapid Ca²⁺ influx and NO production in endothelial cells.     

Skeletal muscle glycogen phosphorylase is irreversibly inhibited by mercury: Molecular,cellular and kinetic aspects

Muscle glycogen phosphorylase (GP) plays an important role in muscle functions. Mercury has toxic effects in skeletal muscle leading to muscle weakness or cramps. However, the mechanisms underlying these toxic effects are poorly understood. We report that GP is irreversibly inhibited by inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury (IC₅₀ = 380 nM and k_inact = 600 M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 43 μM and k_inact = 13 M⁻¹ s⁻¹ for CH₃Hg⁺) through reaction of these compounds with cysteine residues of the enzyme. Our data suggest that the irreversible inhibition of GP could represent one of the mechanisms that contribute to mercury-dependent muscle toxicity.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Design and synthesis of potent macrocyclic renin inhibitors

Two types of P1-P3-linked macrocyclic renin inhibitors containing the hydroxyethylene isostere (HE) scaffold just outside the macrocyclic ring have been synthesized. An aromatic or aliphatic substituent (P3sp) was introduced in the macrocyclic ring aiming at the S3 subpocket (S3sp) in order to optimize the potency. A 5-6-fold improvement in both the K_i and the human plasma renin activity (HPRA)IC₅₀ was observed when moving from the starting linear peptidomimetic compound 1 to the most potent macrocycle 42 (K_i = 3.3 nM and HPRA IC₅₀ = 7 nM). Truncation of the prime side of 42 led to 8-10-fold loss of inhibitory activity in macrocycle 43 (K_i = 34 nM and HPRA IC₅₀ = 56 nM). All macrocycles were epimeric mixtures in regard to the P3sp substituent and X-ray crystallographic data of the representative renin macrocycle 43 complex showed that only the S-isomer buried the substituent into the S3sp. Inhibitory selectivity over cathepsin D (Cat-D) and BACE-1 was also investigated for all the macrocycles and showed that truncation of the prime side increased selectivity of inhibition in favor of renin.     

Trisubstituted thiophene analogues of 1-thiazolyl-2-pyrazoline,super oxidase inhibitors and free radical scavengers

Xanthine oxidase (XO) generates superoxide anions and H₂O₂ for the self-defence system of organism. Abnormal production of this superoxide’s (reactive oxygen species) is responsible for a number of complications including inflammation, metabolic disorder, cellular aging, reperfusion damage, atherosclerosis and carcinogenesis. Series of novel trisubstituted thiophenyl-1-thiazolyl-2-pyrazoline libraries are synthesized containing 2,5-dichloro thiophene, 5-chloro-2-(benzylthio) thiophene and 5-chlorothiophene-2-sulphonamide, from chalcones in PEG-400 as green solvent. Superoxide (XO) inhibitory and free radical scavenging activities were also figured out with molecular modeling analysis, bearing in mind their possible future for super oxide inhibitor (Gout) therapeutics, compound 3k shows interesting superoxide inhibitory and free radical scavenger activity with IC₅₀ = 6.2 μM, in comparison with allopurinol.     

α-Glucosidase inhibition and antihyperglycemic activity of prenylated xanthones from Garcinia mangostana

An ethanol extract of the fruit case of Garcinia mangostan, whose most abundant chemical species are xanthones, showed potent α-glucosidase inhibitory activity (IC₅₀ = 3.2 μg/ml). A series of isolated xanthones (1-16) demonstrated modest to high inhibition of α-glucosidase with IC₅₀ values of 1.5-63.5 μM. In particular, one hitherto unknown xanthone 16 has a very rare 2-oxoethyl group on C-8. Kinetic enzymatic assays with a p-nitrophenyl glucopyranoside indicated that one of them, compound (9) exhibited the highest activity (K_i = 1.4 μM) and mixed inhibition. Using, a physiologically relevant substrate, maltose, as substrate, many compounds (6, 9, 14, and 15) also showed potent inhibition which ranged between 17.5 and 53.5 μM and thus compared favorably with deoxynojirimycin (IC₅₀ = 68.8 μM). Finally, the actual pharmacological potential of the ethanol extract was demonstrated by showing that it could elicit reduction of postprandial blood glucose levels. Furthermore, the most active α-glucosidase inhibitors (6, 9, and 14) were proven to be present in high quantities in the native seedcase by a HPLC chromatogram.     

Microwave assisted synthesis of naphthopyrans catalysed by silica supported fluoroboric acid as a new class of non purine xanthine oxidase inhibitors

A series of naphthopyrans was synthesized employing silica supported fluoroboric acid under solvent free conditions in a microwave reactor. The catalytic influence of HBF₄–SiO₂ was investigated in detail to optimize the reaction conditions. The synthesised compounds were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure–activity relationship analyses have also been presented. Among the synthesised compounds, NP-17, NP-19, NP-20, NP-23, NP-24, NP-25 and NP-26 were the active inhibitors with an IC₅₀ ranging from 4 to 17 μM. Compound NP-19 with a thiophenyl ring at position 1 emerged as the most potent xanthine oxidase inhibitor (IC₅₀ = 4 μM) in comparison to allopurinol (IC₅₀ = 11.10 μM) and febuxostat (IC₅₀ = 0.025 μM). The basis of significant inhibition of xanthine oxidase by NP-19 was rationalized by its molecular docking at MTE binding site of xanthine oxidase.     

Characterization of NADPH–cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera

A complete cDNA encoding the NADPH–cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4 kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis–Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC₅₀ = 1.69 μM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC₅₀ value ranged from 15 to 90 μM) was also observed.     

Inhibition of xanthine oxidase — xanthine — iron mediated lipid peroxidation by eugenol in liposomes

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe⁺³-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 µm concentration and hence formation of O₂ also However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O₂ generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe²⁺ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe⁺³ ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. (Mol Cell Biochem 166: 65-71, 1997)     

Exploration of SAR regarding glucose moiety in novel C-aryl glucoside inhibitors of SGLT2

In order to investigate SAR regarding glucose moiety in novel C-aryl glucoside SGLT2 inhibitors containing a thiazole motif, a series of chemical modifications on glucose was conducted to explore potential utility as a suitable replacement of glucose per se. Among the compounds prepared, deshydroxy 29 (IC₅₀ = 7.01 nM) demonstrated the best in vitro inhibitory activity against SGLT2 in this series to date. But, none of the compounds were better than the parent molecule 5 (IC₅₀ = 1.75 nM).     

Heteromerization of angiotensin receptors changes trafficking and arrestin recruitment profiles

The cardiovascular hormone angiotensin II (AngII) exerts its actions via two G protein-coupled receptor (GPCR) subtypes, AT₁ and AT₂, which often display antagonistic functions. Methodological constraints have so far precluded detailed analyses of the ligand-dependency, cellular localization, and functional relevance of AngII receptor interactions in live cells. In this study, we utilize a protein-fragment complementation assay (PCA) and GPCR-Heteromer Identification Technology (GPCR-HIT) to provide the first detailed investigation of the ligand-dependency and cellular localization of AngII receptor interactions in human embryonic kidney 293 cells. Fluorescent-tagged receptor constructs for PCA and GPCR-HIT displayed normal affinity and selectivity for AngII (AT₁: IC₅₀ = 1.0-1.6 nM; AT₂: IC₅₀ = 2.0-3.0 nM). Well-characterized angiotensin receptor interactions were used as positive and negative controls to demonstrate the sensitivity and specificity of these fluorescence-based assays. We report that AT₁-AT₂ receptor heteromers form constitutively, are localized to the plasma membrane and perinuclear compartments, and do not internalize following AngII stimulation despite arrestin being recruited specifically to the heteromer. Our findings using novel fluorescence-based technologies reveal a previously unrecognized mechanism of angiotensin receptor cross-talk involving cross-inhibition of AT₁ receptor internalization through heteromerization with the AT₂ receptor subtype.