Evaluation of the anti-hepatitis C virus effect of novel potent, selective, and orally bioavailable JNK and VEGFR kinase inhibitors

Screening of a focused library of TGF beta kinase inhibitors in the cellular HCV replicon model with luciferase read out yielded a number of low micromolar HCV inhibitors. Medicinal chemistry driven optimization resulted in the discovery of 4-[2-(5-bromo-2-fluoro-phenyl)pteridin-4-ylamino]-N-[3-(2- oxopyrrolidin-1-yl)propyl]nicotinamide 36 with a replicon EC(50) of 64nM, associated with a selective kinase inhibitory profile for human JNK kinases 2 and 3 as well as VEGFR-1, 2, and 3 kinases. Moreover, 36 showed an advantageous PK profile in mice. Experiments performed using different replicon constructs suggest that this series of kinase inhibitors might mediate their effect through the HCV non-structural protein 5A (NS5A).     

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.     

Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region

Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21-26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca(2+) influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca(2+) influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr(26); this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp(25), a part of the consensus sulfation site. The expressed CCR2B was found to be N:-glycosylated, as N:-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.     

Comprehensive gene expression profiling of rat lung reveals distinct acute and chronic responses to cigarette smoke inhalation

Chronic obstructive pulmonary disease (COPD) is a smoking-related disease that lacks effective therapies due partly to the poor understanding of disease pathogenesis. The aim of this study was to identify molecular pathways that could be responsible for the damaging consequences of smoking. To do this, we employed Gene Set Enrichment Analysis to analyze differences in global gene expression, which we then related to the pathological changes induced by cigarette smoke (CS). Sprague-Dawley rats were exposed to whole body CS for 1 day and for various periods up to 8 mo. Gene Set Enrichment Analysis of microarray data identified that metabolic processes were most significantly increased early in the response to CS. Gene sets involved in stress response and inflammation were also upregulated. CS exposure increased neutrophil chemokines, cytokines, and proteases (MMP-12) linked to the pathogenesis of COPD. After a transient acute response, the CS-exposed rats developed a distinct molecular signature after 2 wk, which was followed by the chronic phase of the response. During this phase, gene sets related to immunity and defense progressively increased and predominated at the later time points in smoke-exposed rats. Chronic CS inhalation recapitulated many of the phenotypic changes observed in COPD patients including oxidative damage to macrophages, a slowly resolving inflammation, epithelial damage, mucus hypersecretion, airway fibrosis, and emphysema. As such, it appears that metabolic pathways are central to dealing with the stress of CS exposure; however, over time, inflammation and stress response gene sets become the most significantly affected in the chronic response to CS.     

Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1’s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.     

CD40L protects against mouse hepatitis virus-induced neuroinflammatory demyelination

Neurotropic mouse hepatitis virus (MHV-A59/RSA59) infection in mice induces acute neuroinflammation due to direct neural cell dystrophy, which proceeds with demyelination with or without axonal loss, the pathological hallmarks of human neurological disease, Multiple sclerosis (MS). Recent studies in the RSA59-induced neuroinflammation model of MS showed a protective role of CNS-infiltrating CD4⁺ T cells compared to their pathogenic role in the autoimmune model. The current study further investigated the molecular nexus between CD4⁺ T cell-expressed CD40Ligand and microglia/macrophage-expressed CD40 using CD40L^-/- mice. Results demonstrate CD40L expression in the CNS is modulated upon RSA59 infection. We show evidence that CD40L^-/- mice are more susceptible to RSA59 induced disease due to reduced microglia/macrophage activation and significantly dampened effector CD4⁺ T recruitment to the CNS on day 10 p.i. Additionally, CD40L^-/- mice exhibited severe demyelination mediated by phagocytic microglia/macrophages, axonal loss, and persistent poliomyelitis during chronic infection, indicating CD40-CD40L as host-protective against RSA59-induced demyelination. This suggests a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to immunosuppression which holds only for autoimmune mechanisms of inflammatory demyelination.