Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.     

Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

High competitiveness of a resource demanding invasive acacia under low resource supply

Mechanisms controlling the successful invasion of resource demanding species into low-resource environments are still poorly understood. Well-adapted native species are often considered superior competitors under stressful conditions. Here we investigate the competitive ability of the resource demanding alien Acacia longifolia, which invades nutrient-poor Mediterranean sand dunes such as in coastal areas of Portugal. We explore the hypothesis that drought may limit invasion in a factorial competition experiment of the alien invasive versus two native species of different functional groups (Halimium halimifolium, Pinus pinea), under well-watered and drought conditions. Changes in biomass, allocation pattern, and N-uptake-efficiency (via ¹⁵N-labeling) indicated a marked drought sensitivity of the invader. However, highly efficient drought adaptations of the native species did not provide a competitive advantage under water limiting conditions. The competitive strength of H. halimifolium towards the alien invader under well-watered conditions turned into a positive interaction between both species under drought. Further, low resource utilization by native species benefited A. longifolia by permitting continued high nitrogen uptake under drought. Hence, the N-fixing invader expresses low plasticity by continuous high resource utilization, even under low resource conditions. The introduction of novel traits into a community like N-fixation and high resource use may promote A. longifolia invasiveness through changes in the physical environment, i.e., the water and nutrient cycle of the invaded sand dune system, thereby potentially disrupting the co-evolved interactions within the native plant community.     

DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.     

Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.     

Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE

Background

UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family.

Principal Findings

We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location – dimer interface, interlobar helices, protein surface, or within other secondary structural elements.

Conclusions

The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme.

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Temporal instability in tree-growth/climate response in the Lower Bavarian Forest region: implications for dendroclimatic reconstruction

This paper explores the temporal stability of growth/climate relationships in ring-width chronologies of Norway spruce [ Picea abies (L.) Karst] and silver fir ( Abies alba Mill) in the Lower Bavarian Forest region in southern Germany. These chronologies were compiled, using both historic and living tree-ring data, with the main aim of developing a dendroclimatic reconstruction for the region covering the last 500 years. Moving window correlation analysis shows that prior to the twentieth century, both species co-vary in a similar way (1480–1899 mean r =0.66). There is no significant correlation between the species chronologies since ca. 1930, which partly reflects anomalous growth trends in the fir chronology since ca. 1960. Multiple regression analysis was utilised to assess the ability of both species chronologies to model March–August precipitation. The precipitation signal of the spruce data was found to be both stronger than the fir data (1872–1930 calibration: r ²=0.45 vs 0.25) and more time stable. After ca. 1930, the fir chronology loses it ability to model March–August precipitation until there is no climate signal at all in the fir data in recent decades. The spruce data also express a later weakening in their climate signal in the mid 1970s. We present compelling evidence indicating that the anomalous trends observed in the fir data, since the mid 1960s, appear to be predominantly related to local SO₂ emissions from power plants and refineries. It is also likely that this local anthropogenic forcing is the cause of the weakening of the climate signal in the spruce data since the mid 1970s. The conclusions from this study are: (1) The fir tree-ring data cannot be used for traditional dendroclimatic calibration, although prior to the twentieth century the decadal variability in the fir data is very similar to spruce and so these data could be used to extend potential reconstructions in the future; (2) The recent decline and recovery event in the fir data appears to be unique to the twentieth century and is not part of a natural episodic phenomenon; (3) Traditional dendroclimatic calibration of March–August precipitation will be made using solely the spruce ring-width data. However, due to SO₂ forcing in recent decades, the calibration period will be shortened to the 1871–1978 period.