Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization,core and urea optimization and in vivo efficacy

Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.     

Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action

We systematically analyzed the relationships between gene fitness profiles (co-fitness) and drug inhibition profiles (co-inhibition) from several hundred chemogenomic screens in yeast. Co-fitness predicted gene functions distinct from those derived from other assays and identified conditionally dependent protein complexes. Co-inhibitory compounds were weakly correlated by structure and therapeutic class. We developed an algorithm predicting protein targets of chemical compounds and verified its accuracy with experimental testing. Fitness data provide a novel, systems-level perspective on the cell.     

Dietary Determinants of Changes in Waist Circumference Adjusted for Body Mass Index – a Proxy Measure of Visceral Adiposity

Background

Given the recognized health effects of visceral fat, the understanding of how diet can modulate changes in the phenotype “waist circumference for a given body mass index (WC_BMI)”, a proxy measure of visceral adiposity, is deemed necessary. Hence, the objective of the present study was to assess the association between dietary factors and prospective changes in visceral adiposity as measured by changes in the phenotype WC_BMI.

Methods and Findings

We analyzed data from 48,631 men and women from 5 countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Anthropometric measurements were obtained at baseline and after a median follow-up time of 5.5 years. WC_BMI was defined as the residuals of waist circumference regressed on body mass index, and annual change in WC_BMI (ΔWC_BMI, cm/y) was defined as the difference between residuals at follow-up and baseline, divided by follow-up time. The association between energy, energy density (ED), macronutrients, alcohol, glycemic index (GI), glycemic load (GL), fibre and ΔWC_BMI was modelled using centre-specific adjusted linear regression, and random-effects meta-analyses to obtain pooled estimates. Men and women with higher ED and GI diets showed significant increases in their WC_BMI, compared to those with lower ED and GI [1 kcal/g greater ED predicted a ΔWC_BMI of 0.09 cm (95% CI 0.05 to 0.13) in men and 0.15 cm (95% CI 0.09 to 0.21) in women; 10 units greater GI predicted a ΔWC_BMI of 0.07 cm (95% CI 0.03 to 0.12) in men and 0.06 cm (95% CI 0.03 to 0.10) in women]. Among women, lower fibre intake, higher GL, and higher alcohol consumption also predicted a higher ΔWC_BMI.

Conclusions

Results of this study suggest that a diet with low GI and ED may prevent visceral adiposity, defined as the prospective changes in WC_BMI. Additional effects may be obtained among women of low alcohol, low GL, and high fibre intake.     

EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.     

Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Autophagy is a well-conserved catabolic process, involving the degradation of a cell''s own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.     

The ARC1 E3 Ligase Promotes a Strong and Stable Self-Incompatibility Response in Arabidopsis Species: Response to the Nasrallah and Nasrallah Commentary

Following the identification of the male (S-locus Cysteine Rich/S-locus Protein 11) and female (S Receptor kinase [SRK]) factors controlling self-incompatibility in the Brassicaceae, research in this field has focused on understanding the nature of the cellular responses activated by these regulators. We previously identified the ARM Repeat Containing1 (ARC1) E3 ligase as a component of the SRK signaling pathway and demonstrated ARC1’s requirement in the stigma for self-incompatible pollen rejection in Brassica napus, Arabidopsis lyrata, and Arabidopsis thaliana. Here, we discuss our findings on the role of ARC1 in reconstructing a strong and stable A. thaliana self-incompatibility phenotype, in the context of the putative issues outlined in a commentary by Nasrallah and Nasrallah. Additionally, with their proposed standardized strategy for studying self-incompatibility in A. thaliana, we offer our perspective on what constitutes a strong and stable self-incompatibility phenotype in A. thaliana and how this should be investigated and reported to the greater community.With many angiosperms possessing hermaphroditic flowers, self-incompatibility (SI) systems have evolved to avoid the deleterious effects of inbreeding (Figures 1A and 1B). As defined by Charlesworth et al. (2005), “plant SI systems all prevent self-fertilization through recognition and rejection of pollen by pistils expressing ‘cognate’ allelic specificity.” In Brassicaceae species, the allele specificity is conferred by two well-characterized polymorphic genes encoding the female S Receptor kinase (SRK) and the male S-locus Protein 11/S-locus Cysteine Rich (SP11/SCR; hereby referred to as SCR) (reviewed in Iwano and Takayama, 2012). The major outstanding area in this field is identifying the signaling proteins activated by SRK, determining their function at the cellular level, and investigating whether these signaling proteins have conserved functions across the self-incompatible species in the Brassicaceae. Despite strong interest in finding these potential factors by us and other groups, only the Brassica rapa M Locus Protein Kinase (Murase et al., 2004; Kakita et al., 2007a, 2007b) and the ARM Repeat Containing1 (ARC1) E3 ligase have emerged as direct downstream signaling proteins. We demonstrated a conserved role for ARC1 in self-incompatible Brassica napus (Gu et al., 1998; Stone et al., 1999, 2003), self-incompatible Arabidopsis lyrata (Indriolo et al., 2012), and self-incompatible Arabidopsis thaliana expressing A. lyrata SCRb, SRKb, and ARC1 transgenes (Indriolo et al., 2014). The commentary by Nasrallah and Nasrallah (2014) focuses on our proposed role for ARC1 in reconstituting self-incompatibility in transgenic A. thaliana.Open in a separate windowFigure 1.Pathways for Compatible and Self-Incompatible Pollen Responses in A. thaliana.(A) Compatible (arrow) and self-incompatible (bar) pollen-pistil interactions.(B) Criteria for assessing compatible versus self-incompatible pollinations.(C) Model for the basal compatible pollen response. An unknown basal pollen response pathway is activated in the stigmatic papilla under the compatible pollen grain leading to the activation of vesicle secretion. Our research on Brassica and Arabidopsis Exo70A1 revealed a putative role for the exocyst complex in docking secretory vesicles at the stigmatic papillae plasma membrane (Samuel et al., 2009; Safavian and Goring, 2013; Safavian et al., 2014). Exo70A1 is proposed to assemble with the remaining subunits of the exocyst complex to dock secretory vesicles (reviewed in Zárský et al., 2013). SNARE proteins mediate vesicle fusion to the plasma membrane, and unknown cargo (ACA13 as one candidate; Iwano et al., 2014) are released to enable pollen hydration pollen tube entry through the stigmatic papillar cell wall (compatible pollen is accepted).(D) Model for the reconstituted self-incompatibility signaling pathway in the Sha ecotype. Following self-pollination in transgenic SCR-SRK+ARC1 Sha ecotype flowers, the pollen SCR ligand binds to SRK at the stigmatic papillar plasma membrane, resulting in the activation of the downstream signaling pathway. The ARC1 E3 ligase is recruited by SRK and targets Exo70A1 for ubiquitination. Even though the basal compatible pollen response pathway has been also activated, ubiquitinated Exo70A1 is somehow inhibited so that exocyst-mediated vesicle secretion to the self-incompatible pollen grain is blocked. In addition, secretory vesicles are degraded in the vacuole through autophagy. An unknown signaling protein (yellow) also has activity in the Sha ecotype in blocking exocytosis (see Samuel et al. [2009], Safavian and Goring [2013], and Indriolo et al. [2014] for further details and references therein).(E) Transmission electron microscopy image of a self-incompatible pollen-stigmatic papillar interaction at 10 min postpollination from the transgenic SCRb-SRKb+ARC1 Sha ecotype. Pseudocoloring has been added to distinguish the pollen (brown) from the stigmatic papilla (green). Autophagy is detected with the autophagic vacuole in the vacuole (see Rose et al. [2006] and Indriolo et al. [2014] for details). (Figures 1C to 1E adapted from Indriolo et al. [2014], Figures 9 and 10.)