Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors

Structure–activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC₅₀ 0.7 nM, FMS cell IC₅₀ 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.     

Impacts of inundation and drought on eukaryote biodiversity in semi‐arid floodplain soils

Floodplain ecosystems are characterized by alternating wet and dry phases and periodic inundation defines their ecological character. Climate change, river regulation and the construction of levees have substantially altered natural flooding and drying regimes worldwide with uncertain effects on key biotic groups. In southern Australia, we hypothesized that soil eukaryotic communities in climate change affected areas of a semi‐arid floodplain would transition towards comprising mainly dry‐soil specialist species with increasing drought severity. Here, we used 18S rRNA amplicon pyrosequencing to measure the eukaryote community composition in soils that had been depleted of water to varying degrees to confirm that reproducible transitional changes occur in eukaryotic biodiversity on this floodplain. Interflood community structures (3 years post‐flood) were dominated by persistent rather than either aquatic or dry‐specialist organisms. Only 2% of taxa were unique to dry locations by 8 years post‐flood, and 10% were restricted to wet locations (inundated a year to 2 weeks post‐flood). Almost half (48%) of the total soil biota were detected in both these environments. The discovery of a large suite of organisms able to survive nearly a decade of drought, and up to a year submerged supports the concept of inherent resilience of Australian semi‐arid floodplain soil communities under increasing pressure from climatic induced changes in water availability.     

Patterns of diversity and adaptation in Glomeromycota from three prairie grasslands

Arbuscular mycorrhizal (AM) fungi are widespread root symbionts that often improve the fitness of their plant hosts. We tested whether local adaptation in mycorrhizal symbioses would shape the community structure of these root symbionts in a way that maximizes their symbiotic functioning. We grew a native prairie grass (Andropogon gerardii) with all possible combinations of soils and AM fungal inocula from three different prairies that varied in soil characteristics and disturbance history (two native prairie remnants and one recently restored). We identified the AM fungi colonizing A. gerardii roots using PCR amplification and cloning of the small subunit rRNA gene. We observed 13 operational taxonomic units (OTUs) belonging to six genera in three families. Taxonomic richness was higher in the restored than the native prairies with one member of the Gigaspora dominating the roots of plants grown with inocula from native prairies. Inoculum source and the soil environment influenced the composition of AM fungi that colonized plant roots. Correspondingly, host plants and AM fungi responded significantly to the soil–inoculum combinations such that home fungi often had the highest fitness and provided the greatest benefit to A. gerardii. Similar patterns were observed within the soil–inoculum combinations originating from two native prairies, where five sequence types of a single Gigaspora OTU were virtually the only root colonizers. Our results indicate that indigenous assemblages of AM fungi were adapted to the local soil environment and that this process occurred both at a community scale and at the scale of fungal sequence types within a dominant OTU.     

Competitive and demographic leverage points of community shifts under climate warming

Accelerating rates of climate change and a paucity of whole-community studies of climate impacts limit our ability to forecast shifts in ecosystem structure and dynamics, particularly because climate change can lead to idiosyncratic responses via both demographic effects and altered species interactions. We used a multispecies model to predict which processes and species'' responses are likely to drive shifts in the composition of a space-limited benthic marine community. Our model was parametrized from experimental manipulations of the community. Model simulations indicated shifts in species dominance patterns as temperatures increase, with projected shifts in composition primarily owing to the temperature dependence of growth, mortality and competition for three critical species. By contrast, warming impacts on two other species (rendering them weaker competitors for space) and recruitment rates of all species were of lesser importance in determining projected community changes. Our analysis reveals the importance of temperature-dependent competitive interactions for predicting effects of changing climate on such communities. Furthermore, by identifying processes and species that could disproportionately leverage shifts in community composition, our results contribute to a mechanistic understanding of climate change impacts, thereby allowing more insightful predictions of future biodiversity patterns.     

The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

Monocarboxylate transporters MCT1-MCT4 require basigin (CD147) or embigin (gp70), ancillary proteins with a glutamate residue in their single transmembrane (TM) domain, for plasma membrane (PM) expression and activity. Here we use site-directed mutagenesis and expression in COS cells or Xenopus oocytes to investigate whether this glutamate (Glu₂₁₈ in basigin) may charge-pair with a positively charged TM-residue of MCT1. Such residues were predicted using a new molecular model of MCT1 based upon the published structure of the E. coli glycerol-3-phosphate transporter. No evidence was obtained for Arg₃₀₆ (TM 8) of MCT1 and Glu₂₁₈ of basigin forming a charge-pair; indeed E218Q-basigin could replace WT-basigin, although E218R-basigin was inactive. No PM expression of R306E-MCT1 or D302R-MCT1 was observed but D302R/R306D-MCT1 reached the PM, as did R306K-MCT1. However, both were catalytically inactive suggesting that Arg₃₀₆ and Asp₃₀₂ form a charge-pair in either orientation, but their precise geometry is essential for catalytic activity. Mutation of Arg₈₆ to Glu or Gln within TM3 of MCT1 had no effect on plasma membrane expression or activity of MCT1. However, unlike WT-MCT1, these mutants enabled expression of E218R-basigin at the plasma membrane of COS cells. We propose that TM3 of MCT1 lies alongside the TM of basigin with Arg₈₆ adjacent to Glu₂₁₈ of basigin. Only when both these residues are positively charged (E218R-basigin with WT-MCT1) is this interaction prevented; all other residue pairings at these positions may be accommodated by charge-pairing or stabilization of unionized residues through hydrogen bonding or local distortion of the helical structure.     

Novel inhibitors of bacterial virulence: Development of 5,6-dihydrobenzo[h]quinazolin-4(3H)-ones for the inhibition of group A streptococcal streptokinase expression

Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of new modes of treating infection. We recently identified 1 (CCG-2979) as an inhibitor of the expression of streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with fibrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identified structural modifications that improve the solubility and metabolic stability of the scaffold. The efficacy of two new compounds 12c (CCG-203592) and 12k (CCG-205363) against biofilm formation in Staphylococcus aureus represents a promising additional mode of action for this novel class of compounds.     

Y-Family Polymerase Conformation Is a Major Determinant of Fidelity and Translesion Specificity

1. Download : Download high-res image (246KB)
2. Download : Download full-size image

Highlights? Y-family polymerases differ in fidelity and translesion synthesis specificity ? Polymerase fidelity and specificity are controlled by the interdomain linker ? Three amino acids in the interdomain linker are sufficient to determine conformation ? Differences in polymerase conformation determine differences in polymerase activity     

Determining the mechanical properties of hazel forks by testing their component parts

The ability to accurately predict the load-bearing capacity of tree forks would improve tree surveying and tree surgery techniques and assist with the biomechanical modelling of a tree’s structure. In this study, the bending strength of forks of hazel (Corylus avellana L.) was investigated by assessing the mechanical contributions from three component parts of each fork. Intact forks and ones in which either central or peripheral xylem lying under the branch bark ridge at the apex of the forks had been removed were subjected to tensile tests. The bending strength of these forks was compared with that of the arising branches by carrying out a three-point bending test on the smaller arising branches of the intact specimens. All forks failed in tension, splitting between the arising branches. By removing the centrally placed xylem, constituting approximately a fifth of the width of the fracture surface, the forks’ bending strength was reduced by around 32 %, while removing the outer four-fifths reduced the forks’ bending strength by 49 %. Intact forks had around 74 % of the maximum bending strength of the smaller arising branch. It is concluded that the tensile strength of the centrally placed xylem at the apex of a tree fork is a critical strengthening component. This helps to explain the weakness of forks with included bark, which lack this component. This study concludes that tree forks should not by default be considered flaws in a tree’s structure.     

Enzymatic Synthesis of s-Substituted l-Cysteines with Tryptophan Synthase of Escherichia coli

The α₂β₂ complex of tryptophan synthase from Escherichia coli catalyzes β-replacement reactions of l-serine and its derivatives (e.g., β-chloro-l-alanine and O-methyl-Dl-serine) with various alkanethiols. The products from thiobenzyl alcohol and ethanethiol were isolated to demonstrate the enzymatic synthesis of the corresponding S-substituted l-cysteines. Reactivities of various S-substituent donors were examined, and thiols such as thiobenzyl alcohol, 1-propanethiol and 1-butanethiol were found to be much more efficient substituent donors than the physiological substrate, indole. In addition, tryptophan synthase catalyzes β-replacement reactions of l-threonine with thiols to form the corresponding S-substituted β-methylcysteines, which are also produced by β-addition reactions of l-vinylglycine with thiols. These enzymatic reactions facilitate the synthesis of various sulfur-containing amino acids.