Synthesis and biological evaluation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors

The synthesis and biological evaluation of a number of differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives are reported. From the inhibition results on a selection of disease-relevant protein kinases [IC₅₀ (μM) DYRK1A = 11; CDK5 = 0.41; GSK-3 = 1.5] we have observed that 3,6-diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4) constitutes a potential new and simple lead compound in the search of drugs for the treatment of Alzheimer’s disease.     

Interferon-gamma increases monocyte HLA-DR expression without effects on glucose and fat metabolism in postoperative patients.

Tissue injury is associated with decreased cellular immunity and enhanced metabolism. Immunodepression is thought to be counteracted by interferon (IFN)-gamma, which increases human leukocyte antigen (HLA)-DR expression. Hypermetabolism could be enhanced by IFN-gamma because cytokines induce a hypermetabolic response to stress. In healthy humans, IFN-gamma enhanced HLA-DR expression without effects on glucose and fat metabolism. In the present study, we evaluated whether IFN-gamma lacks potential harmful side effects on metabolic and endocrine pathways while maintaining its beneficial effects on the immune system under conditions in which the inflammatory response system is activated. In 13 patients scheduled for major surgery, we studied HLA-DR expression on peripheral blood monocytes before surgery and postoperatively randomized the patients into an intervention and a placebo group. Subsequently, we evaluated the effects of a single dose of IFN-gamma vs. saline on short-term monocyte activation, glucose and lipid metabolism, and glucose and lipid regulatory hormones. HLA-DR expression on monocytes was restored from postoperative levels of 54% (42-60%; median and interquartiles) to 92% (91-96%) 24 h after IFN-gamma administration but stayed low in the placebo-treated patients. IFN-gamma did not affect glucose metabolism (plasma glucose, rate of appearance and disappearance of glucose) and lipid metabolism (plasma glycerol, plasma free fatty acids, and rates of appearance and disappearance of glycerol). IFN-gamma had no effect on plasma cortisol, adrenocorticotropic hormone, growth hormone, insulin, C-peptide, glucagon, epinephrine, and norepinephrine concentrations. We conclude that IFN-gamma exerts a favorable effect on cell-mediated immunity in patients after major surgery without effects on glucose and lipid metabolism.     

Temporal and spatial genetic structure in Vitellaria paradoxa (shea tree) in an agroforestry system in southern Mali

Ten microsatellite loci were used to investigate the impact of human activity on the spatial and temporal genetic structure of Vitellaria paradoxa (Sapotaceae), a parkland tree species in agroforestry systems in southern Mali. Two stands (forest and fallow) and three cohorts (adults, juveniles and natural regeneration) in each stand were studied to: (i) compare their levels of genetic diversity (gene diversity, HE; allelic richness, Rs; and inbreeding, FIS); (ii) assess their genetic differentiation (FST); and (iii) compare their levels of spatial genetic structuring. Gene diversity parameters did not vary substantially among stands or cohorts, and tests for bottleneck events were nonsignificant. The inbreeding coefficients were not significantly different from zero in most cases (FIS = -0.025 in forest and 0.045 in fallow), suggesting that the species is probably outbreeding. There was a weak decrease in F(IS) with age, suggesting inbreeding depression. Differentiation of stands within each cohort was weak (FST = 0.026, 0.0005, 0.010 for adults, juveniles and regeneration, respectively), suggesting extensive gene flow. Cohorts within each stand were little differentiated (FST = -0.001 and 0.001 in forest and fallow, respectively). The spatial genetic structure was more pronounced in fallow than in forest where adults showed no spatial structuring. In conclusion, despite the huge influence of human activity on the life cycle of Vitellaria paradoxa growing in parkland systems, the impact on the pattern of genetic variation at microsatellite loci appears rather limited, possibly due to the buffering effect of extensive gene flow between unmanaged and managed populations.     

Total syntheses of iso-, neuro- and phytoprostanes: new insight in lipid chemistry

Isoprostanes (IsoPs) are a complex family of compounds produced, in vivo, from peroxidation of polyunsaturated fatty acids (AA, DHA, EPA, alpha-linolenic) via a free-radical-catalyzed mechanism. Carbocyclic annulations are extremely important reactions and the stereocontrolled intramolecular free-radical cyclization has emerged as a powerful tool for carbon-carbon bond formation in synthetic chemistry. The hex-5-enyl radical cyclization is the most well-known for the synthesis of cyclopentane rings. After a short review of the literature, concerning the total synthesis of isoprostanes and intermediates, we will present our own contributions on the preparation of chiral cyclopentane rings from glucose leading to new isoprostanes. This study allowed us to control the cyclization outcome to yield the all-syn and/or syn-anti-syn precursors which permit us to the total synthesis of a large set of iso-, neuro-, and phytoprostanes.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria

Background

Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.

Methodology/Principal Findings

Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.

Conclusions

We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.     

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.     

Spatial Scales of Pollen and Seed-Mediated Gene Flow in Tropical Rain Forest Trees

Gene flow via seed and pollen is a primary determinant of genetic and species diversity in plant communities at different spatial scales. This paper reviews studies of gene flow and population genetic structure in tropical rain forest trees and places them in ecological and biogeographic context. Although much pollination is among nearest neighbors, an increasing number of genetic studies report pollination ranging from 0.5–14 km for canopy tree species, resulting in extensive breeding areas in disturbed and undisturbed rain forest. Direct genetic measures of seed dispersal are still rare; however, studies of fine scale spatial genetic structure (SGS) indicate that the bulk of effective seed dispersal occurs at local scales, and we found no difference in SGS (Sp statistic) between temperate (N?=?24 species) and tropical forest trees (N?=?15). Our analysis did find significantly higher genetic differentiation in tropical trees (F _ST?=?0.177; N?=?42) than in temperate forest trees (F _ST?=?0.116; N?=?82). This may be due to the fact that tropical trees experience low but significant rates of self-fertilization and bi-parental inbreeding, whereas half of the temperate tree species in our survey are wind pollinated and are more strictly allogamous. Genetic drift may also be more pronounced in tropical trees due to the low population densities of most species.     

The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B" regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.