α-Carbonic anhydrases are sulfatases with cyclic diol monosulfate esters

Carbonic anhydrases (CA) catalyze activated ester hydrolysis in addition to the hydration of CO₂ to bicarbonate. They also show phosphatase activity with 4-nitrophenyl phosphate as substrate but not sulfatase with the corresponding sulfate. Here we prove that the enzyme is catalyzing the synthesis of cyclic diols from sulfate esters. 5-, 6- and 8-membered ring cyclic sulfates incorporating a neighboring secondary alcohol moiety were treated with CA II and yielded the corresponding cyclic diols. Inhibitory properties of obtained cyclic and original sulfate esters were then investigated on human carbonic anhydrase I (hCA I), hCA II, hCA IV and hCA VI (h?=?human isoform). K_I-s of these compounds ranged between 32.7–423 μM against hCA I, 2.13–32.4 μM against hCA II, 13.7–234 μM against hCA IV and 76–278 μM against CA VI, respectively. The sulfatase activity of CA with such esters is amazing considering the fact that 4-nitrophenyl-sulfate is not a substrate of these enzymes.     

α-Carbonic anhydrases are sulfatases with cyclic diol monosulfate esters

Carbonic anhydrases (CA) catalyze activated ester hydrolysis in addition to the hydration of CO(2) to bicarbonate. They also show phosphatase activity with 4-nitrophenyl phosphate as substrate but not sulfatase with the corresponding sulfate. Here we prove that the enzyme is catalyzing the synthesis of cyclic diols from sulfate esters. 5-, 6- and 8-membered ring cyclic sulfates incorporating a neighboring secondary alcohol moiety were treated with CA II and yielded the corresponding cyclic diols. Inhibitory properties of obtained cyclic and original sulfate esters were then investigated on human carbonic anhydrase I (hCA I), hCA II, hCA IV and hCA VI (h?=?human isoform). K(I)-s of these compounds ranged between 32.7-423 μM against hCA I, 2.13-32.4 μM against hCA II, 13.7-234 μM against hCA IV and 76-278 μM against CA VI, respectively. The sulfatase activity of CA with such esters is amazing considering the fact that 4-nitrophenyl-sulfate is not a substrate of these enzymes.     

Burkholderia pseudomallei γ-carbonic anhydrase is strongly activated by amino acids and amines

Activation of the γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei (BpsγCA) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. The best BpsγCA activators were d-His, l-DOPA, d-Trp, 4-amino-l-Phe, dopamine, 2-(2-aminoethyl)pyridine, 2-aminoethyl-piparazine/morpholine and l-adrenaline, which showed activation constants ranging between 9 and 86 nM. The least effective activators were l-His, l-Phe and 2-pyridyl-methylamine, with K_As in the range of 1.73–24.7 μM. As little is known about the role of γ-CAs in the lifecycle and virulence of this saprophytic bacterium, this study may shed some light on such phenomena. This is the first CA activation study of a γ-CA from a pathogenic bacterium, the only other such study being on the enzyme discovered in the archaeon Methanosarcina thermophila, Cam.     

Dithiocarbamates strongly inhibit the β-class carbonic anhydrases from Mycobacterium tuberculosis

A series of N-mono- and N,N-disubstituted dithiocarbamates have been investigated as inhibitors of two β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Mycobacterium tuberculosis, mtCA 1 (Rv1284) and mtCA 3 (Rv3273). Both enzymes were inhibited with efficacies between the subnanomolar to the micromolar one, depending on the substitution pattern at the nitrogen atom from the dithiocarbamate zinc-binding group. Aryl, arylalkyl-, heterocyclic as well as aliphatic and amino acyl such moieties led to potent mtCA 1 and 3 inhibitors in both the N-mono- and N,N-disubstituted dithiocarbamate series. This new class of β-CA inhibitors may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug/extensive multi-drug resistance.     

Carbonic anhydrase inhibitors. The β-carbonic anhydrases from the fungal pathogens Cryptococcus neoformans and Candida albicans are strongly inhibited by substituted-phenyl-1H-indole-5-sulfonamides

A series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides and 1-({[5-(aminosulfonyl)-3-phenyl-1H-indol-2-yl]carbonyl}amino)-2,4,6 trimethylpyridinium perchlorates possessing various 2-, 3- or 4-substituted phenyl groups with methyl-, halogeno- and methoxy-functionalities, as well as the perfluorophenyl moiety, have been evaluated as inhibitors of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (CaNce103). Both enzymes were potently inhibited by these sulfonamides, K_Is in the range of 4.4–118 nM against Can2, and of 5.1–128 against CaNce103, respectively. Minor structural changes in the 3-substituted phenyl moiety contribute significantly to the inhibitory activity. Some of the investigated sulfonamides showed promising selectivity ratios for inhibiting Can2 over the host, human enzymes CA I and II.     

Intestinal intraepithelial TCRγδ+ T cells are activated by normal commensal bacteria

TCR???⁺ T cells play a critical role in protecting the intestinal mucosa against pathogenic infection. In the absence of infection, TCR???⁺ T cell activation must be continuously regulated by T regulatory cells (Treg) to prevent the development of colitis. However, the activation of intestinal TCR???⁺ T cells under normal conditions has not been clearly resolved. In order to determine TCR???⁺ T cell activation in vivo, we designed an NF-??B based reporter system. Using the recombinant lentiviral method, we delivered the NF-??B reporter to isolated TCR???⁺ T cells, which were then adoptively transferred into normal mice. Our data indicate that the NF-??B activation level in TCR???⁺ T cells is higher in the intestinal intraepithelial layer than in the lamina propria region. In addition, the surface expression level of lymphocyte activation marker CD69 in TCR???⁺ T cells is also higher in the intestinal intraepithelial layer and this activation was reduced by Sulfatrim treatment which removes of commensal bacteria. Collectively, our data indicate that the TCR???⁺ T cell population attached to the intestinal lumen is constitutively activated even by normal commensal bacteria.     

Plant tyrosine decarboxylase can be strongly inhibited by l-α-aminooxy-β-phenylpropionate

The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of DNA and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1–1 g · ml^-1 tunicamycin, cell division and DNA synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear DNA content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells.Abbreviations TCA trichloroacetic acid - TM tunicamycin     

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.     

Synthesis ofα-triazolylα-amino acid derivatives

Summary We report the synthesis of-triazolyl-amino esters by 1,3 dipolar cycloaddition of acetylenic compounds and-azido-amino esters.     

N-Sulfamoylphenyl- and N-sulfamoylphenyl-N-thiazolyl-β-alanines and their derivatives as inhibitors of human carbonic anhydrases

A series of N-substituted and N,N-disubstituted β-amino acids and their derivatives bearing benzenesulfonamide moiety were designed and synthesized in search of compounds that would be high-affinity and selective inhibitors of human carbonic anhydrases (CA). There are 12 catalytically active human CA isoforms, the cytosolic CA I, CA II, CA III, CA VII, and CA XIII, secreted CA VI, the mitochondrial CA VA and CA VB, membrane-associated CA IV, and transmembrane CA IX, CA XII, and CA XIV. The di-bromo meta-substituted compounds exhibited low nanomolar dissociation constants and over 10-fold selectivity for mitochondrial isozyme CA VB, implicated in diseases of the central nervous system and obesity. These compounds can be used for further development as inhibitors of significant binding affinity and selectivity towards CA VB isozyme.     

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA), an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II) as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO₂ substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.     

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic “X” shape. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the “intrinsic structure” of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology.     

The liver-enriched transcription factor CREBH is nutritionally regulated and activated by fatty acids and PPARα

To elucidate the physiological role of CREBH, the hepatic mRNA and protein levels of CREBH were estimated in various feeding states of wild and obesity mice. In the fast state, the expression of CREBH mRNA and nuclear protein were high and profoundly suppressed by refeeding in the wild-type mice. In ob/ob mice, the refeeding suppression was impaired. The diet studies suggested that CREBH expression was activated by fatty acids. CREBH mRNA levels in the mouse primary hepatocytes were elevated by addition of the palmitate, oleate and eicosapenonate. It was also induced by PPARα agonist and repressed by PPARα antagonist. Luciferase reporter gene assays indicated that the CREBH promoter activity was induced by fatty acids and co-expression of PPARα. Deletion studies identified the PPRE for PPARα activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay confirmed that PPARα directly binds to the PPRE. Activation of CREBH at fasting through fatty acids and PPARα suggest that CREBH is involved in nutritional regulation.     

Vα14 NKT cells activated by alpha-galactosylceramide augment lipopolysaccharide-induced nitric oxide production in mouse intra-hepatic lymphocytes

Vα14 natural killer T (Vα14 NKT) cells activated by α-galactosylceramide (α-GalCer) secrete a large amount of Th1 and Th2 cytokines. IFN-γ plays a crucial role in the inflammation response, and is also known as an activator of nitric oxide (NO) production. We previously reported that lipopolysaccharide (LPS)-induced NO production is augmented by α-GalCer in mouse peritoneal cells. Since the liver is susceptible to LPS stimulation via the portal vein, we examined the effect of α-GalCer on LPS-induced NO production in murine intra-hepatic lymphocytes (IHLs). Although IHLs augmented LPS-induced NO production by α-GalCer administration, such an augmentation was not observed in non-treated mice. Furthermore, α-GalCer did not augment LPS-induced NO production in IHLs from IFN-γ knockout mice. In flow cytometry analysis of IHLs from α-GalCer-treated mice, the ratio and number of F4/80- and TLR4-positive cells rose as compared with non-treated mice. The liver injury may be induced by LPS and NO under the condition where Vα14 NKT cells were activated.     

Heterotrimeric Gα(i) proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis

Previous studies have implicated a role of heterotrimeric Gα(i) proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gα(i) proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gα(i)-GTP protein complex was immunoprecipitated with a Gα(i) protein activation assay. In subsequent in vivo studies, the Gα(i) protein inhibitor pertussis toxin (PTx) or G(i) protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gα(i2) in sepsis, Gα(i2) (-/-) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gα(i) proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα(i) proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gα(i2) (-/-) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gα(i2) (-/-) compared to WT mice. Gα(i2) (-/-) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα(i) proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gα(i2) protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.     

Heterotrimeric Gαi proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis

Previous studies have implicated a role of heterotrimeric Gα_i proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gα_i proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gα_i-GTP protein complex was immunoprecipitated with a Gα_i protein activation assay. In subsequent in vivo studies, the Gα_i protein inhibitor pertussis toxin (PTx) or G_i protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gα_i2 in sepsis, Gα_i2 (−/−) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gα_i proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα_i proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gα_i2 (−/−) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gα_i2 (−/−) compared to WT mice. Gα_i2 (−/−) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα_i proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gα_i2 protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.     

Biotransformation of 7α-hydroxy- and 7-oxo-ent-atis-16-ene derivatives by the fungus Gibberella fujikuroi

The microbiological transformation of 7α,19-dihydroxy-ent-atis-16-ene by the fungus Gibberella fujikuroi gave 19-hydroxy-7-oxo-ent-atis-16-ene, 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene, 7α,11β,19-trihydroxy-ent-atis-16-ene and 7α,16β,19-trihydroxy-ent-atis-16-ene, while the incubation of 19-hydroxy-7-oxo-ent-atis-16-ene afforded 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene and 16β,17-dihydroxy-7-oxo-ent-atisan-19-al. The biotransformation of 7-oxo-ent-atis-16-en-19-oic acid gave 6β-hydroxy-7-oxo-ent-atis-16-en-19-oic acid, 6β,16β,17-trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene and 3β,7α-dihydroxy-6-oxo-ent-atis-16-en-19-oic acid.     

α-Tetralone derivatives as inhibitors of monoamine oxidase

In the present study, a series of fifteen α-tetralone (3,4-dihydro-2H-naphthalen-1-one) derivatives were synthesised and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The α-tetralone derivatives examined are structurally related to a series of chromone (1-benzopyran-4-one) derivatives which has previously been shown to act as MAO-B inhibitors. The results document that the α-tetralones are highly potent MAO-B inhibitors with all compounds exhibiting IC₅₀ values in the nanomolar range (<78 nM). Although most compounds are selective inhibitors of MAO-B, the α-tetralones are also potent MAO-A inhibitors with ten compounds exhibiting IC₅₀ values in the nanomolar range (<792 nM). The most potent MAO-B inhibitor, 6-(3-iodobenzyloxy)-3,4-dihydro-2H-naphthalen-1-one, exhibits an IC₅₀ value of 4.5 nM with a 287-fold selectivity for MAO-B over the MAO-A isoform, while the most potent MAO-A inhibitor, 6-(3-cyanobenzyloxy)-3,4-dihydro-2H-naphthalen-1-one, exhibits an IC₅₀ value of 24 nM with a 3.25-fold selectivity for MAO-A. Analyses of the structure–activity relationships for MAO inhibition show that substitution on the C6 position of the α-tetralone moiety is a requirement for MAO-A and MAO-B inhibition, and that a benzyloxy substituent on this position is more favourable for MAO-A inhibition than phenylethoxy and phenylpropoxy substitution. For MAO-B inhibition, alkyl and halogen substituents on the meta and para positions of the benzyloxy ring enhance inhibitory potency. It may be concluded that α-tetralone derivatives are promising leads for design of therapies for Parkinson’s disease and depression.