Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity.

Activation of the inositol-requiring enzyme-1 alpha (IRE1α) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1α dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1α-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1α. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1α RNase activity appears to be caused by a conformational change, whereby the αC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1α, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1α RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.

Dysfunctional Dopaminergic Neurones in Mouse Models of Huntington's Disease: A Role for SK3 Channels.

BACKGROUND: Huntington's disease (HD) is a late-onset fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the gene coding for the protein huntingtin and is characterised by progressive motor, psychiatric and cognitive decline. We previously demonstrated that normal synaptic function in HD could be restored by application of dopamine receptor agonists, suggesting that changes in the release or bioavailability of dopamine may be a contributing factor to the disease process. OBJECTIVE: In the present study, we examined the properties of midbrain dopaminergic neurones and dopamine release in presymptomatic and symptomatic transgenic HD mice. METHODS AND RESULTS: Using intracellular sharp recordings and immunohistochemistry, we found that neuronal excitability was increased due to a loss of slow afterhyperpolarisation and that these changes were related to an apparent functional loss and abnormal distribution of SK3 channels (KCa2.3 encoded by the KCNN3 gene), a class of small-conductance calcium-activated potassium channels. Electrochemical detection of dopamine showed that this observation was associated with an enhanced dopamine release in presymptomatic transgenic mice and a drastic reduction in symptomatic animals. These changes occurred in the context of a progressive expansion in the CAG repeat number and nuclear localisation of mutant protein within the substantia nigra pars compacta. CONCLUSIONS: Dopaminergic neuronal dysfunction is a key early event in HD disease progression. The initial increase in dopamine release appears to be related to a loss of SK3 channel function, a protein containing a polyglutamine tract. Implications for polyglutamine-mediated sequestration of SK3 channels, dopamine-associated DNA damage and CAG expansion are discussed in the context of HD.

Assessing the knowledge of the potential harm to others caused by second-hand smoke and its impact on protective behaviours at home.

BACKGROUND: Smokers' knowledge of the risks of second-hand smoke (SHS) and the role this plays in implementing behaviours to reduce the SHS exposure of others have not been thoroughly explored. Mass media health promotion is used to promote behaviour change partly by providing information on the consequences of behaviour. In England, between 2003 and 2006, frequent mass media campaigns highlighted the toxicity of SHS. OBJECTIVES: To examine peoples' knowledge of SHS-related illnesses in England over time, identify the determinants of good knowledge and to assess its importance in predicting SHS-protective behaviours. METHODS: Statistical analysis of repeat cross-sectional data (1996-2008) from the Omnibus Survey to explore the trends and determinants of knowledge of SHS-related illnesses and the determinants of SHS-protective behaviours. RESULTS: Only 40% of smokers had 'good' knowledge of SHS-related illnesses compared with 65% of never smokers. Knowledge increased markedly when frequent SHS-related mass media campaigns (2003-06) ran, compared with earlier years (1996-2002). Smokers with better knowledge were more likely to have smoke-free homes [odds ratio (OR): 1.10, 1.04-1.16] and abstain from smoking in a room with children (OR: 1.11, 1.09-1.14). CONCLUSIONS: The low levels of knowledge of some SHS-related conditions, especially among smokers, and the relationship between knowledge and SHS-protective behaviours, suggest that greater efforts to educate smokers about the risks associated with SHS are worthwhile.

Phenoxyacetic acids as PPARδ partial agonists: synthesis, optimization, and in vivo efficacy.

A series of phenoxyacetic acids as subtype selective and potent hPPARδ partial agonists is described. Many analogues were readily accessible via a single solution-phase synthetic route which resulted in the rapid identification of key structure-activity relationships (SAR), and the discovery of two potent exemplars which were further evaluated in vivo. Details of the SAR, optimization, and in vivo efficacy of this series are presented herein.

3-Urea-1-(phenylmethyl)-pyridones as novel, potent, and selective EP3 receptor antagonists.

A series of 3-urea-1-(phenylmethyl)-pyridones was discovered as novel EP(3) antagonists via high-throughput screening and subsequent optimization. The synthesis, structure-activity relationships, and optimization of the initial hit that resulted in potent and selective EP(3) receptor antagonists such as 11g are described.

Synthesis and structure-activity relationships of a series of 3-aryl-4-isoxazolecarboxamides as a new class of TGR5 agonists.

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent agonists of the human TGR5 G-protein-coupled receptor is described. Many analogues were readily accessible via solution-phase synthesis which resulted in the rapid identification of key structure-activity relationships (SAR), and the discovery of potent exemplars (up to pEC50=9). Details of the SAR and optimization of this series are presented herein.

The prescribed duration algorithm: utilising 'free text' from multiple primary care electronic systems.

PURPOSE: To develop and test an algorithm that translates total dose and daily regimen, inputted as 'free text' on a prescription, into numerical values to calculate the prescribed treatment duration. METHOD: The algorithm was developed using antibiotic prescriptions (n = 711,714) from multiple primary care computer systems. For validation, the prescribed treatment duration of an independent sample of antibiotic scripts was calculated in two ways: (a) computer algorithm, (b) manually reviewed by a researcher blinded to the results of (a). The outputs of the two methods were compared and the level of agreement assessed, using confidence intervals for differences in proportions. This was repeated on sample of antidepressant scripts to test generalisability of the algorithm. RESULTS: For the antibiotic prescriptions, the algorithm processed 98.5% with an accuracy of 99.8% and the manual review processed 98.5% with 98.9% accuracy. The differences between these proportions are 0.0% (95%CI of -0.9, 0.9%) and 1.0% (95%CI of -0.1, 2.3%), respectively. For the antidepressant prescriptions, the algorithm processed 91.5% with an accuracy of 96.6% compared to the manual review with 96.4% processed and 99.8% accuracy; difference between these proportions is 4.9% (95%CI of 2.0, 8.0%) and 3.2% (95%CI of 1.6, 5.3%), respectively. CONCLUSION: The algorithm proved to be applicable and efficient for assessing prescribed duration, with sensitivity and specificity values close to the manual review, but with the added advantage that the computer can process large volume of scripts rapidly and automatically.

Amino acid anthranilamide derivatives as a new class of glycogen phosphorylase inhibitors.

A series of amino acid anthranilamide derivatives identified from a high-throughput screening campaign as novel, potent, and glucose-sensitive inhibitors of human liver glycogen phosphorylase a are described. A solid-phase synthesis using Wang resin was also developed which provided efficient access to a variety of analogues, and resulted in the identification of key structure-activity relationships, and the discovery of a potent exemplar (IC(50)=80 nM). The SAR scope, synthetic strategy, and in vitro results for this series are presented herein.

A highly efficient, asymmetric synthesis of benzothiadiazine-substituted tetramic acids: potent inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

[reaction: see text] An efficient two-pot, asymmetric synthesis of benzothiadiazine-substituted tetramic acids is reported. Starting from commercially available alpha-amino acids or esters, reductive amination followed by a novel one-pot amide bond formation/Dieckmann cyclization provided the desired products in high yield and optical purity. An analogous solid-phase approach to the same targets is also presented. These compounds were found to be potent inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

Novel synthesis of 5-amino-3-bromo-1-(tert-butyl)-1H-pyrazole-4-carbonitrile: a versatile intermediate for the preparation of 5-amino-3-aryl-1-(tert-butyl)-1H-pyrazole-4-carboxamides.

A simple, novel, and efficient route for the synthesis of 5-amino-3-aryl-1-(tert-butyl)-1H-pyrazole-4-carboxamides 1 has been devised. Preparation of pyrazole bromide 3 from potassium tricyanomethanide can be accomplished in only two steps in good yield and features a selective Sandmeyer reaction on the corresponding diaminopyrazole. This allows for a more versatile synthesis of 5-amino-3-aryl-1-(tert-butyl)-1H-pyrazole-4-carboxamides 1 than was previously possible.

Discovery of 3-aryl-4-isoxazolecarboxamides as TGR5 receptor agonists.

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.

Synthesis and structure-activity relationships of 3-phenyl-2-propenamides as inhibitors of glycogen phosphorylase a.

A series of 3-phenyl-2-propenamides discovered from a high-throughput screening campaign as novel, potent, glucose-sensitive inhibitors of human liver glycogen phosphorylase a is described. A solid-phase synthesis on DMHB resin was also developed which provided efficient access not only to certain analogues that could not be cleanly made using more traditional means, but also to a variety of additional analogues. The SAR scope and synthetic strategy are presented herein.

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids: potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids is reported. Starting from commercially available chiral Fmoc-protected alpha-amino acids loaded onto Wang resin, Fmoc removal, reductive amination followed by amide bond formation, and base-catalyzed cyclization with simultaneous cleavage from the resin provided the desired products. Compounds described are potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.