Inhibition of wortmannin activities by amino compounds

Wortmannin caused normal and strong inhibition on catecholamine secretion from bovine adrenal chromaffin cells and in vitro phosphoinositide 3-kinase activity in NaCl-, Na isethionate-, choline Cl-, Na acetate-, and N-acetyl glycine-based media. However, brief preincubation of wortmannin with the media containing amino compounds such as glutamate, aspartate, lysine, and glycine resulted in the prevention of the inhibitory effects of wortmannin on the above responses as two indexes of wortmannin activities. On the other hand, the amino compounds also caused several rapid changes in wortmannin medium; the changes in absorption spectrum of the medium; and the changes in the retention time of the peak on the HPLC chromatogram using a reverse-phase C-18 column and in the pattern of absorption spectrum of the peak. These changes were not observed in the cases of NaCl, Na isethionate, choline Cl, Na acetate or N-acetyl glycine. Another amino compound Tris, which was commonly used as a pH buffer, was unique in time course and induced the slow but parallel changes and reached maximal up to about 24h. These results taken together indicate that the amino compounds markedly inhibit the activities of wortmannin presumably through the binding of wortmannin to amino group.     

Inhibition of the n-formylmethionyl-leucyl-phenylalanine induced respiratory burst in human neutrophils by adrenergic agonists and prostaglandins of the E series

Exogenous prostaglandins E1 and E2 and L-isoproterenol potently inhibited the production of superoxide anions by human neutrophils activated in vitro by n-formylmethionyl-leucyl-phenylalanine (FMLP). An estimated ID50 of 50 nM was found for all three agents while L-epinephrine and prostaglandin F2 alpha were 10 and 100 fold, respectively, less active. Inhibition occurred whether these agents were added before, together with, or after the addition of the tripeptide to cell suspensions. Cells treated with dibutyryl adenosine 3',5'-monophosphate also expressed reduced rates of superoxide synthesis thus suggesting that the hormonal inhibitors acted indirectly by stimulating membrane bound adenylate cyclase.     

Endo-xylanase GH11 activation by the fungal metabolite eugenitin

Eugenitin, a chromone derivative and a metabolite of the endophyte Mycoleptodiscus indicus, at 5 mM activated a recombinant GH11 endo-xylanase by 40 %. The in silico prediction of ligand-binding sites on the three-dimensional structure of the endo-xylanase revealed that eugenitin interacts mainly by a hydrogen bond with a serine residue and a stacking interaction of the heterocyclic aromatic ring system with a tryptophan residue. Eugenitin improved the GH11 endo-xylanase activity on different substrates, modified the optimal pH and temperature activities and slightly affected the kinetic parameters of the enzyme.     

Inhibition of the neutrophil oxidative burst and degranulation by phenothiazines

Trifluoperazine inhibits superoxide production and O₂ uptake by guinea pig neutrophils incubated with arachidonic acid, N-formylmethionylphenylalanine, digitonin or ionophore A23187, with IC₅₀ values of 7–37uM. Since this inhibition is produced by concentrations of trifluoperazine which inhibit interaction of calmodulin with proteins, we examined the effects of two other phenothiazines which interact less effectively with calmodulin. Chlorpromazine, promethazine and trifluoperazine all inhibit N-formylmethionylphenylalanine-stimulated superoxide production with similar efficiency. Furthermore, degranulation stimulated by A23187 or N-formylmethionylphenylalanine is inhibited similarly by all three phenothiazines with IC₅₀ values of 18–45 uM. These results are consistent with the suggestion that phenothiazines may inhibit neutrophil function as a result of non-specific interactions with the cells' membranes rather than by specific interaction with calmodulin.     

Inhibition of fungal sporulation by aflatoxin

Summary The fungi Penicillium expansum, Aspergillus niger, Cladosporium herbarum, Thamnidium elegans, Mucor hiemalis and Rhizopus nigricans were cultivated on filter disks containing various concentrations of crude aflatoxin or coumarin. After 3 days the rate of sporulation was noted. 100 g/disk aflatoxin inhibits the sporulation of all fungi with the exception of P. expansum which was only slightly influenced. 10 g/disk aflatoxin also exerts a restricting influence on most of the fungi, whereas at 1 g/disk aflatoxin the quantity of newly formed spores was nearly equal to the toxin-free controls. In comparison with the action of coumarin it is evident that the influence of aflatoxin is specific for that toxin and not or only in a small degree dependent on an action of the coumarin nucleus itself.     

Protein phosphorylation and the respiratory burst

The exposure of 32P-loaded neutrophils to any of a variety of activating agents induces changes in the levels of phosphorylation of a large number of phosphoproteins. The uptake of phosphate by one set of phosphoproteins in particular, a family whose members migrate at Mr 48K with near neutral pI values, appears to be closely related to the activation of the respiratory burst oxidase, the O2--producing enzyme of phagocytes that is responsible for the generation of microbicidal oxidants by these cells. Evidence for the relationship between the phosphorylation of these proteins and the activation of the respiratory burst oxidase has been furnished by kinetic studies as well as by studies on protein phosphorylation in neutrophils from patients with chronic granulomatous disease, a group of inherited disorders affecting this oxidase. The details of this relationship are obscure, although the evidence suggests that these phosphoproteins act in substoichiometric amounts with respect to the oxidase.     

Turning on the respiratory burst

The respiratory burst is a distinguishing property of phagocytes. It is induced by chemotactic stimulation or phagocytosis and reflects the activation of a membrane-bound enzyme system that transfers electrons from cytosolic NADPH to extracellular oxygen, producing superoxide. The products of the burst are essential for the killing of microorganisms, but are also a cause of tissue damage and inflammation. Studies aimed at a better understanding of the regulation of the respiratory burst should help in the search for new ways to treat infections and inflammation.     

Inhibition of the respiratory burst by resveratrol in human monocytes: correlation with inhibition of PI3K signaling

trans-Resveratrol (t-RVT) has been shown to have a wide range of anti-inflammatory properties, some of which have been suggested to contribute to the molecular explanation of the French Paradox, a possible reason for the low incidence of heart disease in France. The ability of t-RVT to inhibit the production of reactive oxygen species (ROS) from monocytes (differentiated U937) was investigated using isoluminol, luminol, lucigenin, and 2',7'-dichlorofluorescein (DCF). t-RVT (0.1-50 microM) was found to significantly inhibit cellular ROS production stimulated by f-Met-Leu-Phe (fMLP), 12-phorbol 13-myristate, and arachidonic acid after a 1-h preincubation. The efficacy of t-RVT could be increased if it was added directly into the assay. NADPH-dependent superoxide production was measured in cell homogenates and t-RVT (10-50 microM) was found to have no effect on this activity. The majority of these redox probes require a peroxidase to be oxidized; therefore, the inhibitory effect of t-RVT on ROS measured by these probes is complicated by its ability to be oxidized by peroxidase enzymes and thus compete with the probe. t-RVT, known to be oxidized by the horseradish peroxidase (HRP)/H(2)O(2) system, was found to inhibit the HRP-dependent oxidation of the fluorescent probe DCF and the chemiluminescent probe isoluminol. However, using a redox probe that did not require oxidation by a peroxidase (lucigenin), significant inhibition was still observed. Moreover, the inhibitory effects of t-RVT on fMLP-induced ROS production correlated with significant inhibitory effects on fMLP-induced phosphatidylinositol 3-kinase (PI3K) activity at 50 microM and Akt phosphorylation (10-50 microM). Other known inhibitors of both PI3K and Akt were also found to inhibit this response. Therefore, inhibition of signaling through the PI3K to NADPH oxidase by t-RVT might represent an important anti-inflammatory mechanism.     

Inhibition of phosphatase and sulfatase by transition-state analogues

The inhibition constants for vanadate, chromate, molybdate, and tungstate have been determined with Escherichia coli alkaline phosphatase, potato acid phosphatase, and Helix pomatia aryl sulfatase. Vanadate was a potent inhibitor of all three enzymes. Inhibition of both phosphatases followed the order WO4(2-) greater than MoO4(2-) greater than CrO4(2-). The Ki values for potato acid phosphatase were about 3 orders of magnitude lower than those for alkaline phosphatase. Aryl sulfatase followed the reverse order of inhibition by group VI oxyanions. Phenol enhanced inhibition of alkaline phosphatase by vanadate and chromate but did not affect inhibition of acid phosphatase. Phenol enhanced inhibition of aryl sulfatase by metal oxyanions in all cases following the order H2VO4- greater than CrO4(2-) greater than MoO4(2-) greater than WO4(2-), and N-acetyltyrosine ethyl ester enhanced inhibition of aryl sulfatase by H2VO4- and CrO4(2-) more strongly than did phenol. It is apparent that the effectiveness of metal oxyanions as inhibitors of phosphatases and sulfatases can be selectively enhanced in the presence of other solutes. The relevance of these observations to the effects of transition metal oxyanions on protein phosphatases in vivo is discussed.     

Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide

The inhibitory effects of silymarin, its main constituent silibinin and the metabolite silibinin-glucuronide on UDP-glucuronosiltransferase (UGT) were evaluated in rat hepatic microsomes. Three substrates were chosen to cover both UGT1A and UGT2B family isozymes: bilirubin (substrate of UGT1A1), p-nitrophenol (UGT1A6) and ethinylestradiol (UGT2B1 and 2B3 for position C17 and UGT1A1 for position C3). The study of p-nitrophenol and bilirubin glucuronidation indicated that silymarin (SM) and silibinin glucuronide (SB-G) were enzyme inhibitors. The kinetic analysis showed that the type of inhibition was competitive in all cases and the Ki obtained were: for p-nitrophenol glucuronidation, KiSB-Gapp: 14+/-1 microg/ml and KiSMapp: 51+/-10 microg/ml and for bilirubin glucuronidation, KiSB-Gapp: 16+/-3 microg/ml. In turn, ethinylestradiol glucuronidation was not affected by any of the compounds studied suggesting that the inhibitory effect was restricted to UGT1A isozymes. Similar studies performed using human hepatic microsomes showed that SM and SB-G were also inhibitors of human UGT1A isozymes. In conclusion, administration of silymarin or its main constituent silibinin could lead to the decrease in the glucuronidation of substrates whose conjugation depends on UGT1A isozymes in a process mediated by silibinin-glucuronide, though their effect in humans needs further investigation.     

Regulation of the phagocyte respiratory burst by small GTP-binding proteins

Bacteria phagocytosed by leukocytes are killed and degraded by toxic oxygen metabolites produced in the phagosome via an NADPH oxidase. NADPH oxidase activity is regulated by small GTP-binding proteins in response to phagocytic stimuli. In this review, Gary Bokoch focuses on the role of Rac in regulating this important phagocytic process.     

Inhibition of monoamine oxidase by phthalide analogues

Based on recent reports that the small molecules, isatin and phthalimide, are suitable scaffolds for the design of high potency monoamine oxidase (MAO) inhibitors, the present study examines the MAO inhibitory properties of a series of phthalide [2-benzofuran-1(3H)-one] analogues. Phthalide is structurally related to isatin and phthalimide and it is demonstrated here that substitution at C6 of the phthalide moiety yields compounds endowed with high binding affinities to both human MAO isoforms. Among the nineteen homologues evaluated, the lowest IC₅₀ values recorded for the inhibition of MAO-A and -B were 0.096 and 0.0014 μM, respectively. In most instances, C6-substituted phthalides exhibit MAO-B specific inhibition. Among a series of 6-benzyloxyphthalides bearing substituents on the para position of the phenyl ring the general order of potency was CF₃ > I > Br > Cl > F > CH₃ > H. The results also show that the binding modes of representative phthalides are reversible and competitive at both MAO isoforms. Based on these data, C6-substituted phthalides may serve as leads for the development of therapies for neurodegenerative disorders such as Parkinson’s disease.     

Inhibition of firefly luciferase by alkane analogues

We reported that anesthetics increased the partial molal volume of firefly luciferase (FFL), while long-chain fatty acids (LCFA) decreased it. The present study measured the actions of dodecanol (neutral), dodecanoic acid (negatively charged), and dodecylamine (positively charged) hydrophobic molecules on FFL. The interaction modes are measured by (1) ATP-induced bioluminescence of FFL and (2) fluorescence of 2-(p-toluidino)naphthalene-6-sulfonate (TNS). TNS fluoresces brightly in hydrophobic media. It competes with the substrate luciferin on the FFL binding. From the Scatchard plot of TNS titration, the maximum binding number of TNS was 0.83, and its binding constant was 8.27 x 10(5) M(-1). Job's plot also showed that the binding number is 0.89. From the TNS titration of FFL, the binding constant was estimated to be 8.8 x 10(5) M(-1). Dodecanoic acid quenched the TNS fluorescence entirely. Dodecanol quenched about 25% of the fluorescence, whereas dodecylamine increased it. By comparing the fluorescence of TNS and bioluminescence of FFL, the binding modes and the inhibition mechanisms of these dodecane analogues are classified in three different modes: competitive (dodecanoic acid), noncompetitive (dodecylamine), and mixed (dodecanol).     

Inhibition of translation by the fungal toxin cyclopiazonic acid

Cyclopiazonic acid is a toxic metabolite of fungal origin that inhibits protein synthesis in intact HeLa cells and eukaryotic cell-free systems. It has been shown that cyclopiazonic acid blocks the GTP- and EF-1-dependent binding of (³H)Phe-tRNA to 80S ribosomes. Moreover the translocation of N-Ac-(³H)Phe-tRNA by 80S ribosomes that takes place in the presence of EF-2 and GTP is also halted by cyclopiazonic acid. It is concluded that this drug affects a ribosomal site involved in the alternative interaction of elongation factors EF-1 and EF-2.     

Inhibition of serine and proline racemases by substrate-product analogues

d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (K_i = 167 ± 21 mM, K_i′ = 661 ± 81 mM, cf. K_m = 19 ± 2 mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5 mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (K_i = 111 ± 15 mM, cf. K_m = 5.7 ± 0.5 mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible.     

Inhibition of telomerase by BIBR 1532 and related analogues

BIBR 1532 has been reported to be a potent, small molecule inhibitor of human telomerase, suggesting it as a lead for the development of anti-telomerase therapy. We confirm the ability of BIBR 1532 to inhibit telomerase and report the discovery of an equally potent analogue. Importantly, IC(50) values in cell extract are considerably higher than those previously reported using assays for purified enzyme, indicating that substantial improvement may be necessary.     

Inhibition of polyamine and spermine oxidases by polyamine analogues

Polyamine oxidase (PAO) and spermine oxidase (SMO) are involved in the catabolism of polyamines--basic regulators of cell growth and proliferation. The discovery of selective inhibitors of PAO and SMO represents an important tool in studying the involvement of these enzymes in polyamine homeostasis and a starting point for the development of novel antineoplastic drugs. Here, a comparative study on murine PAO (mPAO) and SMO (mSMO) inhibition by the polyamine analogues 1,8-diaminooctane, 1,12-diaminododecane, N-prenylagmatine (G3), guazatine and N,N1-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527) is reported. Interestingly, 1,12-Diaminododecane and G3 behave as specific inhibitors of mPAO, values of K(i) for mPAO inhibition being lower than those for mSMO inactivation by several orders of magnitude. The analysis of molecular models of mPAO and mSMO indicates a significant reduction of the hydrophobic pocket located in maize PAO (MPAO) at the wider catalytic tunnel opening. This observation provides a rationale to explain the lower affinity displayed by G3, guazatine and MDL72527 for mPAO and mSMO as compared to MPAO. The different behaviour displayed by 1,12-diaminododecane towards mPAO and mSMO reveals the occurrence of basic differences in the ligand binding mode of the two enzymes, the first enzyme interacting mainly with substrate secondary amino groups and the second one with substrate primary amino groups. Thus, the data reported here provide the basis for the development of novel and selective inhibitors able to discriminate between mammalian SMO and PAO activities.