Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease

NO is an essential cytotoxic agent in host defense, yet can be autotoxic if overproduced, as evidenced in inflammatory lesions and tissue destruction in experimental arthritis models. Treatment of streptococcal cell wal1-induced arthritis in rats with N:(G)-monomethyl-L-arginine (L-NMMA), a competitive nonspecific inhibitor of both constitutive and inducible isoforms of NO synthase (NOS), prevents intraarticular accumulation of leukocytes, joint swelling, and bone erosion. Because increased inducible NOS (iNOS) expression and NO generation are associated with pathogenesis of chronic inflammation, we investigated whether a selective inhibitor of iNOS, N:-iminoethyl-L-lysine (L-NIL), would have more directed anti-arthritic properties. Whereas both L-NMMA and L-NIL inhibited nitrite production by streptococcal cell wall-stimulated rat mononuclear cells in vitro and systemic treatment of arthritic rats with L-NMMA ablated synovitis, surprisingly L-NIL did not mediate resolution of inflammatory joint lesions. On the contrary, daily administration of L-NIL failed to reduce the acute response and exacerbated the chronic inflammatory response, as reflected by profound tissue destruction and loss of bone and cartilage. Although the number of iNOS-positive cells within the synovium decreased after treatment with L-NIL, immunohistochemical analyses revealed a distinct pattern of endothelial and neuronal NOS expression in the arthritic synovium that was unaffected by the isoform-specific L-NIL treatment. These studies uncover a contribution of the constitutive isoforms of NOS to the evolution of acute and chronic inflammation pathology which may be important in the design of therapeutic agents.     

Selective inhibition of histidine-modified pancreatic alpha-amylase by proteinaceous inhibitor from Phaseolus vulgaris

Chemical modification of two histidine residues of porcine pancreatic alpha-amylase (EC 3.2.1.1) by diethyl pyrocarbonate in the presence of a high concentration of maltotriose caused a decrease of amylase activity and an increase of maltosidase activity (hydrolysis of p-nitrophenyl-alpha-maltoside). By binding a proteinaceous inhibitor from Phaseolus vulgaris (white kidney bean) with the modified enzyme, the amylase activity was further decreased but the maltosidase activity was retained to about 100% that of the native enzyme. Both amylase and maltosidase activities of the native enzyme were almost completely inhibited by the proteinaceous inhibitor. The increase of maltosidase activity by histidine modification was due to an increase of kcat, whereas the Km value was not changed; but binding of the proteinous inhibitor affected mainly the Km value of the modified enzyme.     

NO-synthase inhibitor prevents flight-induced activation of agressiveness and courtship in male

Previous experience of flying enhances the aggressiveness (Hofmann and Stevenson, 2000, Nature, 403: 613) and accelerates the courtship behaviour (Dyakonova and Krushinski, 2003, DAN, 390: 709-712) of crickets Gryllus bimacultus. We present evidence that these effects may be mediated by activation of nitric oxide synthesis. The effects of flying on fighting and courtship were largely abolished in crickets who received haemocoel injections of a nonspecific NO-synthase inhibitor LNNA. Unlike this, LNNA exerted no significant effects on aggressive and courtship behaviour of nonflown males.     

Selective inhibition of RNase H by dextran

Ordinarily, ribonuclease H hydrolyzes poly(rA) . poly(dT) and phiX174DNA-RNA at equal rates. Here we show that in the presence of dextran, the degradation of poly(rA) . poly(dT) is inhibited, while that of phi 174DNA-RNA is not. A similar inhibition by sucrose is found to be due to trace contamination of dextran in the sucrose. Ribose, deoxyribose, and a number of other saccharides fail to inhibit RNase H. In experiments where the two substrates are presented in the presence of the inhibitor, the kinetics indicates that both molecules are recognized by the enzyme, but only the phi X174DNA-RNA is degraded. That is, dextran does not interfere with the recognition site, but rather blocks hydrolysis. It is proposed that the ability of dextran to confer selectivity toward different substrates reveals a potential regulatory mechanism for RNase H activity which may represent a control step in the initiation of DNA synthesis.     

Selective inhibition of chlorophyll biosynthesis by nicotinamide

Rhodobacter sphaeroides grown in the presence of nicotinamide excreted bacteriochlorophyll precursors, 2,4-divinyl protochlorophyllide (DV-Pchlide) and a small amount of 2-monovinyl protochlorophyllide (MV-Pchlide). Accumulation of these pigments indicates that nicotinamide inhibited the bacteriochlorophyll biosynthetic pathway site-specifically between DV-Pchlide and MV-Pchlide. This phenomenon is also observed in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114. Among 12 nicotinamide derivatives and isomers tested, only nicotinamide was effective, indicating that in addition to the completeness of the pyridine ring skeleton at positions 1 to 3, the carboxylic acid amide group is essential for this inhibition. The technique described in this report permits the simple preparation of large quantities of DV-Pchlide.     

Selective inhibition of platelet lipoxygenase by esculetin

The effects of coumarin and its derivatives on rat platelet lipoxygenase and cyclooxygenase activities were studied. Esculetin (6,7-dihydroxycoumarin) was found to inhibit the lipoxygenase more strongly than the cyclooxygenase; its concentration for 50% inhibition (IC50) was 0.65 microM for platelet lipoxygenase and 0.45 mM for platelet cyclooxygenase. Esculin (the 6-glucoside of esculetin) and umbelliferone (7-hydroxy-coumarin) also selectively inhibited the lipoxygenase, though less strongly (IC50 = 290 and 500 microM, respectively). 4-Hydroxycoumarin and coumarin had no inhibitory effect on either enzyme at concentrations up to 1 mM. The mechanism of the lipoxygenase inhibition by esculetin was non-competitive. Other antioxidants (hydroquinone, gallic acid and ascorbic acid) were less inhibitory to both enzymes and showed little selectivity.     

Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.     

Selective inhibition of protein disulfide isomerase by estrogens

Protein disulfide isomerase (PDI) is a multifunctional microsomal enzyme that participates in the formation of protein disulfide bonds. PDI catalyzes the reduction of protein disulfide bonds in the presence of excess reduced glutathione and has been implicated in the reductive degradation of insulin; E. coli thioredoxin is homologous to two regions in PDI and can also degrade insulin. PDI activity, measured by 125I-insulin degradation or reactivation of randomly oxidized RNase in the presence of reduced glutathione, is non-competitively inhibited by estrogens; half-maximal inhibition was observed at approximately 100 nM estrogen. Other steroid hormones at 1 microM had little or no effect. PDI segment 120-163 (which corresponds to exon 3 of the PDI gene) and 182-230 have significant similarity with estrogen receptor segments 350-392 and 304-349, respectively, located in the estrogen binding domain but not with the steroid domains of the progesterone and glucocorticoid receptors or with thioredoxin, which is insensitive to estrogens. We propose the hypothesis that enzymes can acquire sensitivity to a hormone via exon shuffling to the enzyme gene from the DNA region coding for the hormone binding domain of the hormone's receptor.     

Selective growth inhibition of Porphyromonas gingivalis by bestatin

Abstract Recent work in our laboratory indicates that selected protease/peptidase inhibitors interfere with the growth of Porphyromonas gingivalis . The aim of the present study was to further investigate the inhibitory effect of bestatin on the growth of P. gingivalis . Complete growth inhibition of P. gingivalis (11 strains) was observed when bestatin was incorporated at 2.5 μg ml⁻¹ in a complex broth medium. Fifty percent inhibition was still obtained with bestatin at a final concentration of 0.5 μg ml⁻¹. The inhibitory effect of bestatin was highly specific as the growth of 20 different oral bacterial species, including Gram-positive and Gram-negative as well as saccharolytic and asacharolytic bacteria, was not affected even at bestatin concentrations up to 50 μg ml⁻¹. Bestatin did not significantly affect the viability of P. gingivalis indicating that it has a bacteriostatic rather than a bactericidal effect. Growth assays using other specific inhibitors suggested that the effect of bestatin on the growth of P. gingivalis was unlikely to be related to its aminopeptidase inhibitor activity. Cultivation of P. gingivalis with a subinhibitory concentration of bestatin did not modify the cell envelope protein profile, as determined by SDS-PAGE analysis, but significantly decreased the number of extracellular vesicles produced. The present study indicated that bestasin is a highly effective inhibitor of cell growth of P. gingivalis . Additional studies will indicate whether bestatin should be considered as a potential drug in the control of P. gingivalis , a suspected pathogen in adult chronic periodontitis.     

Selective inhibition of glycosyltransferases by bivalent imidazolium salts

Galactosyltransferases (GalTs) extend the glycan chains of mammalian glycoproteins by adding Gal to terminal GlcNAc residues, and thus build the scaffolds for biologically important glycan structures. We have shown that positively charged bivalent imidazolium salts in which the two imidazolium groups are linked by an aliphatic chain of 20 or 22 carbons form potent inhibitors of purified human β3-GalT5, using GlcNAcβ-benzyl as acceptor substrate. The inhibitors are not substrate analogs and also inhibited a selected number of other glycosyltransferases. These bis-imidazolium compounds represent a new class of glycosyltransferase inhibitors with potential as anti-cancer and anti-inflammatory drugs.     

Selective inhibition of monoamine oxidase A by hispidol

Hispidol, an aurone, isolated from Glycine max Merrill, was found to potently and selectively inhibit an isoform of recombinant human monoamine oxidase-A (MAO-A), with an IC₅₀ value of 0.26?µM, and to inhibit MAO-B, but with lower potency (IC₅₀?=?2.45?µM). Hispidol reversibly and competitively inhibited MAO-A with a K_i value of 0.10?µM with a potency much greater than toloxatone (IC₅₀?=?1.10?µM), a marketed drug. It also reversibly and competitively inhibited MAO-B (K_i?= 0.51?µM). Sulfuretin, an analog of hispidol, effectively inhibited MAO-A (IC₅₀?=?4.16?µM) but not MAO-B (IC₅₀?>?80?µM). A comparison of their chemical structures showed that the 3′-hydroxyl group of sulfuretin might reduce its inhibitory activities against MAO-A and MAO-B. Flexible docking simulation revealed that the binding affinity of hispidol for MAO-A (?9.1?kcal/mol) was greater than its affinity for MAO-B (?8.7?kcal/mol). The docking simulation showed hispidol binds to the major pocket of MAO-A or MAO-B. The findings suggest hispidol is a potent, selective, reversible inhibitor of MAO-A, and that it be considered a novel lead compound for development of novel reversible inhibitors of MAO-A.     

Selective inhibition of proline hydroxylation by 3,4-dehydroproline

The effect of proline analogs on peptidyl proline hydroxylation has been studied in vivo using aerated root slices of Daucus carota. One analog, 3,4-dehydroproline, acted at micromolar concentrations to rapidly and selectively inhibit peptidyl proline hydroxylation. A structurally altered hydroxyproline-rich cell wall glycoprotein was synthesized and secreted by dehydroproline-treated tissue. The capacity to hydroxylate proline recovered slowly following a short pulse treatment with the analog, with a halftime for recovery of about 24 hours. Recovery was not altered by supplying exogenous proline. Dehydroproline had little effect on the induction of nitrate reductase by nitrate, nor on wound-induced increases in amino acid uptake and protein synthesis. In contrast, other proline analogs inhibit proline hydroxylation only at millimolar concentrations. It is hypothesized that dehydroproline acts as an enzyme-activated suicide inhibitor of prolyl hydroxylase. This analog should become a useful tool for elucidating the functional significance of hydroxyproline-rich glycoproteins.