Novel triazolopyridylbenzamides as potent and selective p38α inhibitors

A new class of p38α inhibitors based on a biaryl-triazolopyridine scaffold was investigated. X-ray crystallographic data of the initial lead compound cocrystallised with p38α was crucial in order to uncover a unique binding mode of the inhibitor to the hinge region via a pair of water molecules. Synthesis and SAR was directed towards the improvement of binding affinity, as well as ADME properties for this new class of p38α inhibitors and ultimately afforded compounds showing good in vivo efficacy.     

Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.     

Upregulation of KCNQ1/KCNE1 K+ channels by Klotho

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.     

Abnormal systolic blood pressure during treadmill test and brachial artery flow-mediated vasodilatation impairment

The aim of the study was to assess the relationship between systolic blood pressure during maximal treadmill test (SBP9mtt)) and flow-mediated vasodilation (FMD). Abnormal rise of SBP(mtt) is the phenomenon more frequent in hypertensive persons but it could be found in normotensive subjects too. 199 subjects referred to treadmill test were enrolled in the study. Four groups were formed: hypertensives with abnormal SBP(mtt) (group A), hypertensives with normal SBP(mtt) (group B), normotensives with abnormal SBP(mtt) (group C) and normotensives with normal SBP(mtt) (group D). Rise of SBP(mtt) above 200 mmHg was considered abnormal reaction. Simple linear regression analysis showed significant inverse relationship between SBP(mtt) and FMD (F = 20.2036, p < 0.001, R2 = 0.0956). Mean FMD index was worst in hypertensive subjects with abnormal SBP(mtt) (group A), followed by normotensives with abnormal SBP(mtt) (group C), hypertensives with normal SBP(mtt) (group B) and the best was in normotensives with normal SBP(mtt) (3.56 +/- 5.17, 4.19 +/- 5.14, 6.81 +/- 8.43 and 10.92 +/- 7.48%, respectively). In multivariate regression analysis FMD showed significant association with abnormal SBP(mtt) (p < 0.001) along with brachial artery diameter (p < 0.001), male gender (p < 0.001), but not with hypertension (p = 0.073), BMI (p = 0.137) and total cholesterol (p = 0.23) (coefficients: -0.26, -0.40, -0.27, -0.13, -0.11 and -0.07, respectively). There was a significant inverse relationship between SBP(mtt) and FMD. An impairment of FMD exists in normotensive subjects with abnormal SBP(mtt). In hypertensives with abnormal SBP(mtt) an additional impairment of FMD exists when compared to hypertensives with normal SBP(mtt). Abnormal SBP(mtt) should be taken into account in global cardiovascular risk assessment.     

Dasty3, a WEB framework for DAS

MOTIVATION: Dasty3 is a highly interactive and extensible Web-based framework. It provides a rich Application Programming Interface upon which it is possible to develop specialized clients capable of retrieving information from DAS sources as well as from data providers not using the DAS protocol. Dasty3 provides significant improvements on previous Web-based frameworks and is implemented using the 1.6 DAS specification. AVAILABILITY: Dasty3 is an open-source tool freely available at http://www.ebi.ac.uk/dasty/ under the terms of the GNU General public license. Source and documentation can be found at http://code.google.com/p/dasty/. CONTACT: hhe@ebi.ac.uk.     

Pyrazine-based Syk kinase inhibitors

A series of aminopyrazines as inhibitors of Syk kinase activity and showing inhibition of LAD2 cells degranulation is described. Optimization of the carboxamide motif with aminomethylpiperidines provided high potency inhibiting Syk but low cellular activity. Amides of cis and trans adamantanol showed good inhibitory activity against Syk as well as remarkable activity in LAD2 cells degranulation assay.     

Cancer genes hypermethylated in human embryonic stem cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.     

Elucidation of a monovalent cation dependence and characterization of the divalent cation binding site of the fosfomycin resistance protein (FosA).

The fosfomycin resistance protein FosA is a member of a distinct superfamily of metalloenzymes containing glyoxalase I, extradiol dioxygenases, and methylmalonyl-CoA epimerase. The dimeric enzyme, with the aid of a single mononuclear Mn2+ site in each subunit, catalyzes the addition of glutathione (GSH) to the oxirane ring of the antibiotic, rendering it inactive. Sequence alignments suggest that the metal binding site of FosA is composed of three residues: H7, H67, and E113. The single mutants H7A, H67A, and E113A as well as the more conservative mutants H7Q, H67Q, and E113Q exhibit marked decreases in the ability to bind Mn2+ and, in most instances, decreases in catalytic efficiency and the ability to confer resistance to the antibiotic. The enzyme also requires the monovalent cation K+ for optimal activity. The K+ ion activates the enzyme 100-fold with an activation constant of 6 mM, well below the physiologic concentration of K+ in E. coli. K+ can be replaced by other monovalent cations of similar ionic radii. Several lines of evidence suggest that the K+ ion interacts directly with the active site. Interaction of the enzyme with K+ is found to be dependent on the presence of the substrate fosfomycin. Moreover, the E113Q mutant exhibits a kcat which is 40% that of wild-type in the absence of K+. This mutant is not activated by monovalent cations. The behavior of the E113Q mutant is consistent with the proposition that the K+ ion helps balance the charge at the metal center, further lowering the activation barrier for addition of the anionic nucleophile. The fully activated, native enzyme provides a rate acceleration of >10(15) with respect to the spontaneous addition of GSH to the oxirane.