Molecular modelling, synthesis, and biological evaluations of a 3,5-disubstituted isoxazole fatty acid analogue as a PPARα-selective agonist.

The peroxisome proliferator activated receptors (PPARs) are important drug targets in treatment of metabolic and inflammatory disorders. Fibrates, acting as PPARα agonists, have been widely used lipid-lowering agents for decades. However, the currently available PPARα targeting agents show low subtype-specificity and consequently a search for more potent agonists have emerged. In this study, previously isolated oxohexadecenoic acids from the marine algae Chaetoceros karianus were used to design a PPARα-specific analogue. Herein we report the design, synthesis, molecular modelling studies and biological evaluations of the novel 3,5-disubstituted isoxazole analogue 6-(5-heptyl-1,2-oxazol-3-yl)hexanoic acid (1), named ADAM. ADAM shows a clear receptor preference and significant dose-dependent activation of PPARα (EC50 = 47 µM) through its ligand-binding domain (LBD). Moreover, ADAM induces expression of important PPARα target genes, such as CPT1A, in the Huh7 cell line and primary mouse hepatocytes. In addition, ADAM exhibits a moderate ability to regulate PPARγ target genes and drive adipogenesis. Molecular modelling studies indicated that ADAM docks its carboxyl group into opposite ends of the PPARα and -γ LBD. ADAM interacts with the receptor-activating polar network of amino acids (Tyr501, His447 and Ser317) in PPARα, but not in PPARγ LBD. This may explain the lack of PPARγ agonism, and argues for a PPARα-dependent adipogenic function. Such compounds are of interest towards developing new lipid-lowering remedies.

Synthesis and biological evaluations of marine oxohexadecenoic acids: PPARα/γ dual agonism and anti-diabetic target gene effects.

Obesity and associated disorders such as metabolic syndrome and type 2 diabetes (T2D) have reached epidemic proportions. Several natural products have been reported as Peroxisome Proliferator-Activated Receptor (PPAR) agonists, functioning as lead compounds towards developing new anti-diabetic drugs due to adverse side effects of existing PPAR drugs. We recently isolated and identified (7E)-9-oxohexadec-7-enoic acid (1) and (10E)-9-oxohexadec-10-enoic acid (2) from the marine algae Chaetoceros karianus. Herein we report the total synthesis, pharmacological characterization, and biological evaluations of these naturally occurring oxo-fatty acids (oFAs). The syntheses of 1 and 2 afforded sufficient material for extensive biological evaluations. Both oFAs show an appreciable dose-dependent activation of PPARα and -γ, with EC50 values in the micromolar range, and an ability to regulate important PPAR target genes in hepatocytes and adipocytes. Moreover, both 1 and 2 are able to drive adipogenesis when evaluated in the Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell model, but with lowered expression of adipocyte markers and reduced lipid accumulation compared to the drug rosiglitazone. This seems to be caused by a transient upregulation of PPARγ and C/EBPα expression. Importantly, whole transcriptome analysis shows that both compounds induce anti-diabetic gene programs in adipocytes by upregulating insulin-sensitizing adipokines and repressing pro-inflammatory cytokines.

Two Isomeric C16 Oxo-Fatty Acids from the Diatom Chaetoceros karianus Show Dual Agonist Activity towards Human Peroxisome Proliferator-Activated Receptors (PPARs) α/γ.

The peroxisome proliferator-activated receptors (PPARs) function as ligand-activated transcription factors that convert signals in the form of lipids to physiological responses through the activation of metabolic target genes. Due to their key roles in lipid and carbohydrate metabolism, the PPARs are important drug targets. However, for several of the PPAR drugs currently in use, adverse side effects have been reported. In an effort to identify compounds from marine organisms that may serve as molecular scaffolds for the development of novel and safer PPAR-targeting drugs, we performed a bioassay-guided screening of organic extracts made from organisms supplied by the Norwegian Biobank of Arctic Marine Organisms (Marbank). Among several interesting hits, we identified two poorly described isomeric oxo-fatty acids from the microalgae Chaetoceros karianus for which we provide the first evidence that they might display dual specificity towards human PPARα and PPARγ. Principal component analysis showed that C. karianus stood out from other Chaetoceros species, both with respect to the metabolic profile and the PPAR activity. The isolation of these compounds holds the potential of uncovering a PPAR pharmacophore with tunable activity and specificity.

Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

Prediction, microarray and northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida.

Bacterial small RNAs (sRNAs) are trans-encoded regulatory RNAs that typically bind mRNAs by short-sequence complementarities and change the expression of the corresponding proteins. Some of the well-characterized sRNAs serve critical steps in the regulation of important cellular processes, such as quorum sensing (Qrr), iron homeostasis (RyhB), oxidative stress (OxyS), and carbon metabolism (Spot 42). However, many sRNAs remain to be identified, and the functional roles of sRNAs are known for only a small fraction. For example, of the hundreds of candidate sRNAs from members of the bacterial family Vibrionaceae, the function is known for only 9. We have in this study significantly contributed to the discovery and verification of new sRNAs in a representative of Vibrionaceae, i.e. the Aliivibrio salmonicida, which causes severe disease in farmed Atlantic salmon and other fishes. A computational search for intergenic non-coding (nc) RNAs in the 4.6-Mb genome identified a total of 252 potential ncRNAs (including 233 putative sRNAs). Depending on the set threshold value for fluorescence signal in our microarray approach, we identified 50-80 putative ncRNAs, 12 of which were verified by Northern blot analysis. In total, we identified 9 new sRNAs.

Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome.

Aliivibrio salmonicida is the aetiological agent of cold water vibriosis affecting farmed fish species, a disease that today is fully controlled by vaccination. However, the molecular mechanisms behind the successful vaccine are largely unknown. In order to gain insight into the possible mechanisms of A. salmonicida vaccines, we report here the profiles of both the outer membrane and secreted subproteomes of A. salmonicida LFI315. The 2 subproteomes were resolved by 2-dimensional electrophoresis that identified a total of 82 protein entries. Monoclonal antibodies specific to an unidentified protein antigen were utilized in the immunoproteomic analysis of both outer membrane proteins and extracellular proteins. The immunogenic protein was located in both subproteomes and identified as a 20 kDa peptidoglycan-associated lipoprotein (Pal). The identity of the antigen was verified by heterologous expression of the cloned A. salmonicida pal gene (VSAL_I1899). It is likely that the immunogenic Pal-like protein is among the constituents that act as a protective antigen in the successful vaccine used today. In view of this, it may be considered a potentially useful component in future vaccine development and pathogenicity studies.

Global responses of Aliivibrio salmonicida to hydrogen peroxide as revealed by microarray analysis.

Aliivibrio salmonicida causes "cold-water vibriosis" (or "Hitra disease") in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and microarray to study how A. salmonicida responds to hydrogen peroxide (H(2)O(2)). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H(2)O(2) response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis and microarray analysis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down regulated genes are distributed among all functional gene categories, but a striking number of ≥2 fold up regulated genes encode proteins involved in detoxification and DNA repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADPH. Our predictions and -omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.

Motility and flagellin gene expression in the fish pathogen Vibrio salmonicida: effects of salinity and temperature.

The success of several Vibrio species, including Vibrio cholerae, Vibrio anguillarum and Vibrio fischeri in colonizing their symbiont, or causing infection is linked to flagella-based motility. It is during early colonization or the initial phase of infection that motility appears to be critical. In this study we used Vibrio salmonicida, a psychrophilic and moderate halophilic bacterium that causes cold-water vibriosis in seawater-farmed Atlantic salmon (Salmo salar), to study motility and expression of flagellins under salt conditions mimicking the initial and later phases of an infection. Our results, which are based on motility in semi-solid agar, membrane protein proteomics, quantitation of flagellin gene expression, challenge infection of fish, and microscopy, show that V. salmonicida is highly motile, expresses elevated levels of flagellins, and typically contains several polar flagella under salt conditions that are seawater-like. In contrast, V. salmonicida cells are non-motile and express significantly lower levels of flagellins under physiological-like salt conditions.

Effect of fish skin mucus on the soluble proteome of Vibrio salmonicida analysed by 2-D gel electrophoresis and tandem mass spectrometry.

Vibrio salmonicida is the causative agent of cold-water vibriosis in farmed marine fish species. Adherence of pathogenic bacteria to mucosal surfaces is considered to be the first steps in the infective processes, and proteins involved are regarded as virulence factors. The global protein expression profile of V. salmonicida, grown with and without the presence of fish skin mucus in the synthetic media, was compared. Increased levels of proteins involved in motility, oxidative stress responses, and general stress responses were demonstrated as an effect of growth in the presence of mucus compared to non-mucus containing media. Enhanced levels of the flagellar proteins FlaC, FlaD and FlaE indicate increased motility capacity, while enhanced levels of the heat shock protein DnaK and the chaperonin GroEL indicate a general stress response. In addition, we observed that peroxidases, TPx.Grx and AhpC, involved in the oxidative stress responses, were induced by mucus proteins. The addition of mucus to the culture medium did not significantly alter the growth rate of V. salmonicida. An analysis of mucus proteins suggests that the mucus layer harbours a protein species that potentially possesses catalytic activity against DNA, and a protein with iron chelating activity. This study represents the first V. salmonicida proteomic analysis, and provides specific insight into the proteins necessary for the bacteria to challenge the skin mucus barrier of the fish.

Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli.

Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.

In vivo effects of beta-glucan and LPS on regulation of lysozyme activity and mRNA expression in Atlantic salmon (Salmo salar L.).

The present study was undertaken to compare the effects of intraperitoneally injected bacterial lipopolysaccharide (LPS) and yeast beta-glucan on lysozyme activity in Atlantic salmon, and to explore what organ(s) are responsible for the increase in plasma lysozyme activity induced by the compounds. The results indicated that LPS stimulates plasma lysozyme activity at least as efficiently as beta-glucan. The lysozyme gene was shown to be transcribed in head kidney, spleen, liver and intestine, and accumulation of transcript was demonstrated in response to both beta-glucan and LPS in all of these organs. Intracellular lysozyme activity was detected in the same organs and in isolated blood polymorphonuclear cells (PMN) and lymphocytes. Increased lysozyme activity in response to both beta-glucan and LPS was demonstrated in blood PMN and cells isolated from head kidney and intestine. In spleen and liver on the other hand, there was no increase in lysozyme activity in response to the stimulants. Based on previous work and the present results it is suggested that plasma lysozyme induced by LPS and beta-glucan originate from macrophages in the different organs. The head kidney is likely to be the main supplier of plasma lysozyme considering its high contents of macrophages. This work supports the notion that microbial compounds containing phylogenetically conserved structures (beta-glucan and LPS) are able to stimulate the non-specific defence of animals against infection by enhancing the lysozyme expression.