Synthesis and SAR of novel 1,1-dialkyl-2(1H)-naphthalenones as potent HCV polymerase inhibitors

A series of gem-dialkyl naphthalenone derivatives with varied alkyl substitutions were synthesized and evaluated according to their structure-activity relationship. This investigation led to the discovery of potent inhibitors of the hepatitis C virus at low nanomolar concentrations in both enzymatic and cell-based HCV genotype 1a assays.     

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.     

Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development

The heart is the first organ required to function during embryonic development and is absolutely necessary for embryo survival. Cardiac activity is dependent on both the sinoatrial node (SAN), which is the pacemaker of heart''s electrical activity, and the cardiac conduction system which transduces the electrical signal though the heart tissue, leading to heart muscle contractions. Defects in the development of cardiac electrical function may lead to severe heart disorders. The Erbb2 (Epidermal Growth Factor Receptor 2) gene encodes a member of the EGF receptor family of receptor tyrosine kinases. The Erbb2 receptor lacks ligand-binding activity but forms heterodimers with other EGF receptors, stabilising their ligand binding and enhancing kinase-mediated activation of downstream signalling pathways. Erbb2 is absolutely necessary in normal embryonic development and homozygous mouse knock-out Erbb2 embryos die at embryonic day (E)10.5 due to severe cardiac defects. We have isolated a mouse line, l11Jus8, from a random chemical mutagenesis screen, which carries a hypomorphic missense mutation in the Erbb2 gene. Homozygous mutant embryos exhibit embryonic lethality by E12.5-13. The l11Jus8 mutants display cardiac haemorrhage and a failure of atrial function due to defects in atrial electrical signal propagation, leading to an atrial-specific conduction block, which does not affect ventricular conduction. The l11Jus8 mutant phenotype is distinct from those reported for Erbb2 knockout mouse mutants. Thus, the l11Jus8 mouse reveals a novel function of Erbb2 during atrial conduction system development, which when disrupted causes death at mid-gestation.     

Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K_ATP) channels. K_ATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K_ATP channel subunit were compared with littermate controls. Evaluation of K_ATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80–85% reduction in cardiac K_ATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K_ATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.     

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study

BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.ConclusionsThis study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.     

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.     

Polyinosinic-polycytidylic acid treatment of Friend retrovirus-infected mice improves functional properties of virus-specific T cells and prevents virus-induced disease

The induction of type I IFN is the most immediate host response to viral infections. Type I IFN has a direct antiviral activity mediated by antiviral enzymes, but it also modulates the function of cells of the adaptive immune system. Many viruses can suppress type I IFN production, and in retroviral infections, the initial type I IFN is weak. Thus, one strategy of immunotherapy in viral infection is the exogenous induction of type I IFN during acute viral infection by TLR ligands. Along these lines, the TLR3/MDA5 ligand polyinosinic-polycytidylic acid [poly(I:C)] has already been used to treat viral infections. However, the immunological mechanisms underlying this successful therapy have not been defined until now. In this study, the Friend retrovirus (FV) mouse model was used to investigate the mode of action of poly(I:C) in antiretroviral immunotherapy. Postexposure, poly(I:C) treatment of FV-infected mice resulted in a significant reduction in viral loads and protection from virus-induced leukemia. This effect was IFN dependent because type I IFN receptor-deficient mice could not be protected by poly(I:C). The poly(I:C)-induced IFN response resulted in the expression of antiviral enzymes, which suppressed FV replication. Also, the virus-specific T cell response was augmented. Interestingly, it did not enhance the number of virus-specific CD4(+) and CD8(+) T cells, but rather the functional properties of these cells, such as cytokine production and cytotoxic activity. The results demonstrate a direct antiviral and immunomodulatory effect of poly(I:C) and, therefore, suggests its potential for clinical treatment of retroviral infections.     

Identification of a novel NF-kappaB-binding site with regulation of the murine alpha2(I) collagen promoter

Hepatic fibrosis is due to the increased synthesis and deposition of type I collagen. Acetaldehyde activates type I collagen promoters. Nuclear factor kappaB (NF-kappaB) was previously shown to inhibit expression of murine alpha(1)(I) and human alpha(2)(I) collagen promoters. The present study identifies binding of NF-kappaB, present in nuclear extracts of stellate cells, to a region between -553 and -537 of the murine alpha(2)(I) collagen promoter. The NF-kappaB (p65) expression vector inhibited promoter activity. Mutation of the promoter at the NF-kappaB-binding site increased basal promoter activity and abrogated the activating and inhibitory effects of transforming growth factor beta and tumor necrosis factor alpha, respectively, on promoter activity. Acetaldehyde increased IkappaB-alpha kinase activity and phosphorylated IkappaB-alpha, NF-kappaB nuclear protein, and its binding to the promoter. However, the activating effect of acetaldehyde was not affected by the mutation of the promoter. In conclusion, although acetaldehyde increases the binding of NF-kappaB to the murine alpha(2)(I) collagen promoter, this binding does not mediate the activating effect of acetaldehyde on promoter activity. The effects of acetaldehyde in increasing the translocation of NF-kappaB to the nucleus with increased DNA binding activity may be important in mediating the effects of acetaldehyde on other genes.     

Retinyl esters are the substrate for isomerohydrolase

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.