Inhibition of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases by nonsteroidal anti-inflammatory drugs

A number of nonsteroidal anti-inflammatory drugs are non-competitive or mixed inhibitors of human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases. Cis- and trans-sulindac sulfide and cis- and trans-sulindac inhibit the NAD-linked enzyme as well or better than they inhibit various cyclooxygenases in vitro. The remainder of the compounds tested are at least one order of magnitude less effective as inhibitors of the 15-hydroxyprostaglandin dehydrogenases than they are as inhibitors of cyclooxygenases. Cis- and trans-sulindac sulfide are sufficiently strong inhibitors of the NAD-linked enzyme (Kis of 7.8 microM and 6.8 microM respectively) to raise the possibility that they might also inhibit this enzyme in vivo.     

Expression, Purification, and Characterization of Histidine-Tagged Rat and Human Flavoenzyme Dihydroorotate Dehydrogenase

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in thede novosynthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system inTrichoplusia nicells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria ofSpodoptera frugiperdacells infected with the recombinant virus using Triton X-100 were loaded onto Ni²⁺-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of ourpreviously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130– 150 μmol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8–1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 μmol/min/mg and the rat enzyme with 83 μmol/min/mg, respectively, at pH 8.0–8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate,K_m= 14.6 μM; electron acceptor decylubiquinone,K_m= 9.5 μM) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8–1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.     

Detoxification of promutagenic aldehydes derived from methylpyrenes by human aldehyde dehydrogenases ALDH2 and ALDH3A1

Methylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulpho conjugation to reactive esters, which can induce mutations and tumours. Yet, further oxidation of the alcohol may compete with this toxification. We previously demonstrated that several human alcohol dehydrogenases (ADH1C, 2, 3 and 4) oxidise various benzylic alcohols (derived from alkylated pyrenes) to their aldehydes with high catalytic efficiency. However, all these ADHs also catalysed the reverse reaction, the reduction of the aldehydes to the alcohols, with comparable or higher efficiency. Thus, final detoxification requires elimination of the aldehydes by further biotransformation. We have expressed two human aldehyde dehydrogenases (ALDH2 and 3A1) in bacteria. All pyrene aldehydes studied (1-, 2- and 4-formylpyrene, 1-formyl-6-methylpyrene and 1-formyl-8-methylpyrene) were high-affinity substrates for ALDH2 (K_m = 0.027–0.9 μM) as well as ALDH3A1 (K_m = 0.78–11 μM). Catalytic efficiencies (k_cat/K_m) were higher for ALDH2 than ALDH3A1 by a moderate to a very large margin depending on the substrate. Most important, they were also substantially higher than the catalytic efficiencies of the various ADHs for the reduction the aldehydes to the alcohols. These kinetic properties ensure that ALDHs, and particularly ALDH2, can complete the ADH-mediated detoxification.     

Inhibitory Activity of Quaternary Isoquinoline Alkaloids on Soluble Epoxide Hydrolase

The quaternary isoquinoline alkaloids of palmatine (1), berberine (2), and jatrorrhizine (3) were evaluated in terms of their ability to inhibit soluble epoxide hydrolase (sEH). They had similar inhibitory activities, with IC₅₀ values of 29.6 ± 0.5, 33.4 ± 0.8, and 27.3 ± 0.4 μM, respectively. Their respective Ki values of 26.9, 46.8, and 44.5 μM—determined by enzyme kinetics—indicated that they inhibited the catalytic reaction by binding noncompetitively with sEH. The application of computational chemistry to the in vitro results revealed the site of the receptor to which the ligand would likely bind. Accordingly, three alkaloids were identified as having a suitable basic skeleton for lead compound development of sEH inhibitors.     

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α₁-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.     

Comparison of substrate specificities of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases

A study of the relative activity of the purified placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases with various prostaglandins and thromboxane B₂(TxB₂) suggests that most, if not all, oxidation in the placenta of the 15-hydroxyl group of prostaglandins of the A, E, and F series as well as PGI₂ (prostacyclin) and 6-keto PGF_1α is catalyzed by the NAD-linked enzyme. Prostaglandin B₁ is an excellent substrate for the NADP-linked enzyme. Despite the conformational similarities between PGB₁ and PGI₂, the latter molecule is a poor substrate for the NADP-linked enzyme. Thromboxane B₂ is not oxidized by the NAD-linked enzyme and is oxidized slowly by the NADP-linked enzyme.     

Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonim of leukotriene (LT) C₄-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cycloocygenase inhibitor indomethacin, NDGA (30 μM) as well as idomethacin (5 μM) inhibited LTC₄-iduced contraction. But in the presence of indomethacin NDGA was ineffective to inhibit the LTC₄ response, whereas two other lipoxygenase inhibitors, phenidone (3–30 μM) and 5,8,11,14-eicostatetraynoic acid (ETYA, 10 μM), markedly inhibited it. The antagonist action of an LTD₄ receptor antagonist FPL55712 against LTC₄-induced contractions was significantly reduced by NDGA (10–30 μM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect n the LTC₄ antagonism in the presence of indomethacin, but 0.3 μM phenidone and 1 μM ETYA which did not inhibit the LTC₄ response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC₄, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LCT₄ response was not inhibited by indomethacin, 0.3 μM phenidone and 1 μM ETYA. In the presence of a γ-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC₄ antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 μM histamine- and 30 μM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H₁-blocker pyrilamine. These results suggest that some putative nonprostanoids are involved in LTC₄-induced contractions of guinea-pig trachea and which regulate the effects of LTD₄ antagonism and β-adrenoceptor activation.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.     

α-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of α-cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

Mechanism and Mechanism-Based Inactivation of 4-Hydroxyphenylpyruvate Dioxygenase

Six substrate analogs of 4-hydroxyphenylpyruvate, specifically pentafluorophenylpyruvate, 4-hydroxytetrafluorophenylpyruvate,2-thienylpyruvate, 3-thienylpyruvate, thiophenol oxalate, and p-thiocresoloxalate were synthesized and their interactions with porcine liver 4-hydroxyphenylpyruvate dioxygenase investigated. Both pentafluorophenylpyruvate and thiophenol oxalate are competitive inhibitors of the enzyme with K_I values of 14 and 150 μM, respectively, but p-thiocresol oxalate has no effect on the enzymic activity. The other three substrate analogs are both substrates and mechanism-based inactivators of the enzyme with the following kinetic characteristics (compound, K_m, V_max, k_inact, K′, partition ratio) at pH 6.0, 37°C, and an air atmosphere: 4-hydroxytetrafluorophenylpyruvate, 50 μM, 1.9 mkat/kg, 1.5/min, 70 μM 4.2; 2-thienylpyruvate, 500 μM, 7.8 mkat/kg, 0.6/min, 400 μM, 41; 3-thienylpymvate, 250 μM, 2 9 mkat/kg, 0.6/min, 300 μM, 22. When inactivated, the dioxygenase was found to contain per mole of active enzyme, 0.78 mol of label from 3-thienyl-3[³H]pyruvate and 0.85 mol of label from 4-hydroxytetrafluorophenyl-3 [³H]pyruvate. The product formed from the enzyme-catalyzed oxidation of 3-thienylpyruvate was determined to be 3-carboxymethyl-3-thiolene-2-one. The implication of these results to the mechanism of the dioxygenase is considered,     

Metabolism of icosa-5,11,14-trienoic acid in human platelets and the inhibition of arachidonic acid metabolism in human platelets by icosa-5,8,14-triynoic and icosa-5,11,14-triynoic acids

Two fatty acids differing from arachidonic acid in lacking one of the internal double bonds (20:35,8,14 and 20:35,11,14) and their 1-C¹⁴ and acetylenic analogues were synthesized. 20:35,8,14 was not metabolized by human platelets but 20:35,11,14 yielded a small amount (1.5% conversion) of two hydroxy fatty acids in a three (11-hydroxy-5,12,14-icosatrienoic acid) to one (15-hydroxy-5,11,13-icosatrienoic acid) proportion. Indomethacin inhibited formation of both hydroxy fatty acids indicating that they are produced via cyclooxygenase. Both ethylenic acids were weak inhibitors of cyclooxygenase (substrate 20 μM arachidonic acid) (ID₅₀: 8.8 μM 20:3^{5,8,14; 11.2 μM} 20:3^5,11,14) but were inactive against lipoxygenase (RD₅₀ > 100 μM). Similarly, both acetylenic analogues were poor inhibitors of lipoxygenase (ID₅₀: 23.4 μM 20:3^5,8,14; 47.8 μM 20:3^5,11,14) but although 20:3^5,8,14 was inactive against cyclooxygenase (ID₅₀ > 100 μM) the 20:3^5,11,14 was a potent inhibitor (ID₅₀: 0.35 μM). The results are interpreted on the basis that hydrogen removal by the lipoxygenase is from C10 and by the cyclooxygenase from C13 but only in 20:3^5,11,14 are these hydrogens (C13) located at the center of a 1,4 pentadiene system (ethylenic) or a 1,4 pentadiyne system (acetylenic).     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis

Fatty acids from natural sources (mostly seed oils) were isolated and assayed for their effect on the bioconversio of arachidonic acid into prostaglandin E₂, using sheep vesicular gland microsomes. Homologues and isomers of the naturally occurring fatty acids, obtained by chemical modification and/or organic synthetic methods, were also tested. Two very active cyclooxygenase inhibitors were discovered, namely jacarandic acid (8 , 10 , 12 -octadecatrienoic acid), isolated from , the concentration which gives 50% inhibition ([I]₅₀) being 2.4 μM and the synthetic 8 , 10 , 12 -octadecarienoic acid, having an [I]₅₀ of 1.0 μM. Under the conditions of the assay (75 μM substrate), earlier described potent inhibitors showed the following [I]₅₀′s: indomethacin: 1.3 μM; 9,12-octadecadiynoic acid: 1.3 μM, 8 , 12 , 14 -eicosatrienoic acid: 2.7 μM; 5,8,11,14-eicosatetraynoic acid: 4.4 μM. At a concentration of about half that of the substrate, the following naturally occurring fatty acids revealed inhibition ([I]₅₀): columbinic acid (29 μM), calendulic acid (31 μM), liagoric acid (31 μM), ximenynic acid (39 μM), crepenynic acid (40 μM) and timnodonic acid (43 μM). Other fatty acids, and some of the above acids, were converted themselves more or less rapidly, mostly into conjugated monohydroxy fatty acids.     

Nitrate Reduction in a Sulfate-Reducing Bacterium, Desulfovibrio desulfuricans, Isolated from Rice Paddy Soil: Sulfide Inhibition, Kinetics, and Regulation

From the second-highest dilution in a most-probable-number dilution series with lactate and sulfate as substrates and rice paddy soil as the inoculum, a strain of Desulfovibrio desulfuricans was isolated. In addition to reducing sulfate, sulfite, and thiosulfate, the strain also reduced nitrate to ammonia. The latter process was studied in detail, since the ability to reduce nitrate was strongly influenced by the presence of sulfide. Sulfide inhibited both growth on nitrate and nitrate reduction. A 70% inhibition of the nitrate reduction rate was obtained at 127 μM sulfide, and growth was inhibited by 50% at approximately 320 μM sulfide and was not detectable above 700 μM sulfide. In contrast, sulfate reduction was not affected at concentrations of up to 5 mM. After growth with sulfate, an induction period of 2 to 4 days was needed before nitrate reduction started. When nitrate and sulfate were present simultaneously, only sulfate was reduced, except when sulfate was present at very low concentrations (4 μM). At higher sulfate concentrations (500 μM), nitrate reduction was temporarily halted. The affinity for nitrate uptake was extremely high (K_m = 0.05 μM) compared with that for sulfate uptake (K_m = 5 μM). Thus, at low nitrate concentrations this bacterium is favored relative to denitrifiers (K_m = 1.8 to 13.7 μM) or other nitrate ammonifiers (e.g., Clostridium spp. [K_m = 500 μM]).     

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

Background

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.

Methodology/Principal Findings

COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC₅₀ values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D₂ and E₂ in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).

Conclusions/Significance

Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.     

Development and exploitation of CK2 inhibitors

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (K_i = 0.4 μM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (K_i = 0.040 μM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (K_i = 0.22 μM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (K_i = 0.17 μM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs.     

Glutathione mixed disulfide inhibitors of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase

Six glutathione-containing inhibitors of the human NADP-linked 15-hydroxyprostaglandin dehydrogenase have been isolated from placental homogenates. Glutathione disulfide is one of these inhibitors. Although the structures of the other five have not been fully elucidated, all are disulfides. Studies with these compounds and with other mixed disulfides have shown that the glutathione mixed disulfides of β-mercaptopyruvate, mercaptoacetate, and β-mercaptolactate are more effective inhibitors of the enzyme than are the glutathione-containing mixed disulfides isolated from placental homogenates. β-Mercaptolactate is particularly noteworthy because of its low K_j (0.13 μM). The results reported here suggest that the activity of the prostaglandin dehydrogenase may be influenced by various glutathione mixed disulfides.     

Motility of Marichromatium gracile in Response to Light, Oxygen, and Sulfide

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an ~500-μm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at ~10 μM O₂. Flux calculations yielded a molar conversion ratio S_tot/O₂ of 2.03:1 (S_tot = [H₂S] + [HS⁻] + [S²⁻]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 μM O₂. The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 μM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.     

Synthesis and evaluation of AKR1C inhibitory properties of A-ring halogenated oestrone derivatives

Enzymes AKR1C regulate the action of oestrogens, androgens, and progesterone at the pre-receptor level and are also associated with chemo-resistance. The activities of these oestrone halides were investigated on recombinant AKR1C enzymes. The oestrone halides with halogen atoms at both C-2 and C-4 positions (13β-, 13α-methyl-17-keto halogen derivatives) were the most potent inhibitors of AKR1C1. The lowest IC₅₀ values were for the 13α-epimers 2_2I,4Br and 2_2I,4Cl (IC₅₀, 0.7 μM, 0.8 μM, respectively), both of which selectively inhibited the AKR1C1 isoform. The 13α-methyl-17-keto halogen derivatives 2_2Br and 2_4Cl were the most potent inhibitors of AKR1C2 (IC₅₀, 1.5 μM, 1.8 μM, respectively), with high selectivity for the AKR1C2 isoform. Compound 1_2Cl,4Cl showed the best AKR1C3 inhibition, and it also inhibited AKR1C1 (Ki: AKR1C1, 0.69 μM; AKR1C3, 1.43 μM). These data show that halogenated derivatives of oestrone represent a new class of potent and selective AKR1C inhibitors as lead compounds for further optimisations.     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID₅₀'s for prostaglandin (PG) E₂ synthesis in these cells were 0.1 μM for 2,6-xylenol, 5 μM for tricresol, 6 μM for -cresol, 7 μM for -cresol, 15 μM for 3,5-xylenol, 30 μM for -cresol and 100 μM for phenol. The corresponding values for aspirin and indomethacin were 4 μM and 0.02 μM, respectively.The substituted phenols also inhibited serotonin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG₂.