Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties

Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 ​amino acids), and the H. pylori ‘26695 type’ (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding

Moderate exercise may attenuate some aspects of immunosenescence

BACKGROUND: Immunosenescence is related to the deterioration of many immune functions, which may be manifested in increased susceptibility to infection, cancer, and autoimmunity. Lifestyle factors, such as diet or physical activity, may influence the senescence of the immune system. It is widely accepted that moderate physical activity may cause beneficial effects for physical and psychological health as well as for the immune system activity in aged people. METHODS: Thirty elderly women aged 62 to 86 were subjected to a two-years authorized physical activity program. Peripheral blood lymphocytes distribution and the production of cytokines involved in the immune response development and regulation (IL-2, IL-4 and IFN-γ) were investigated. The same parameters were evaluated in two control groups of women: a sedentary group of 12 elderly women selected for the second round of the physical activity program and in a group of 20 sedentary young women. Flow cytometry methods were used for the examination of surface markers on peripheral blood lymphocytes and intracellular cytokines expression. RESULTS: The distribution of the main lymphocytes subpopulations in the peripheral blood of elderly women did not show changes after long-term moderate physical training. The percentage of lymphocytes expressing intracellular IL-2 was higher in the group of women attending 2-years physical activity program than in the control group of elderly sedentary women, and it was similar to the value estimated in the group of young sedentary women. There was no difference in the intracellular expression of IL-4 and IFN-γ between the active and elderly sedentary women. CONCLUSIONS: Our results suggest that moderate, long-term physical activity in elderly women may increase the production of IL-2, an important regulator of the immune response. This may help ameliorate immunosenescence in these women

Structural and functional insight into human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value

The ProPrems trial: investigating the effects of probiotics on late onset sepsis in very preterm infants

BACKGROUND: Late onset sepsis is a frequent complication of prematurity associated with increased mortality and morbidity. The commensal bacteria of the gastrointestinal tract play a key role in the development of healthy immune responses. Healthy term infants acquire these commensal organisms rapidly after birth. However, colonisation in preterm infants is adversely affected by delivery mode, antibiotic treatment and the intensive care environment. Altered microbiota composition may lead to increased colonisation with pathogenic bacteria, poor immune development and susceptibility to sepsis in the preterm infant.Probiotics are live microorganisms, which when administered in adequate amounts confer health benefits on the host. Amongst numerous bacteriocidal and nutritional roles, they may also favourably modulate host immune responses in local and remote tissues. Meta-analyses of probiotic supplementation in preterm infants report a reduction in mortality and necrotising enterocolitis. Studies with sepsis as an outcome have reported mixed results to date.Allergic diseases are increasing in incidence in "westernised" countries. There is evidence that probiotics may reduce the incidence of these diseases by altering the intestinal microbiota to influence immune function. METHODS/DESIGN: This is a multi-centre, randomised, double blinded, placebo controlled trial investigating supplementing preterm infants born at < 32 weeks' gestation weighing < 1500 g, with a probiotic combination (Bifidobacterium infantis, Streptococcus thermophilus and Bifidobacterium lactis). A total of 1,100 subjects are being recruited in Australia and New Zealand. Infants commence the allocated intervention from soon after the start of feeds until discharge home or term corrected age. The primary outcome is the incidence of at least one episode of definite (blood culture positive) late onset sepsis before 40 weeks corrected age or discharge home. Secondary outcomes include: Necrotising enterocolitis, mortality, antibiotic usage, time to establish full enteral feeds, duration of hospital stay, growth measurements at 6 and 12 months' corrected age and evidence of atopic conditions at 12 months' corrected age. DISCUSSION: Results from previous studies on the use of probiotics to prevent diseases in preterm infants are promising. However, a large clinical trial is required to address outstanding issues regarding safety and efficacy in this vulnerable population. This study will address these important issues. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN012607000144415The product "ABC Dophilus Probiotic Powder for Infants®", Solgar, USA has its 3 probiotics strains registered with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ--German Collection of Microorganisms and Cell Cultures) as BB-12 15954, B-02 96579, Th-4 15957