Direct Binding of a Hepatitis C Virus Inhibitor to the Viral Capsid Protein

Over 130 million people are infected chronically with hepatitis C virus (HCV), which, together with HBV, is the leading cause of liver disease. Novel small molecule inhibitors of Hepatitis C virus (HCV) are needed to complement or replace current treatments based on pegylated interferon and ribavirin, which are only partially successful and plagued with side-effects. Assembly of the virion is initiated by the oligomerization of core, the capsid protein, followed by the interaction with NS5A and other HCV proteins. By screening for inhibitors of core dimerization, we previously discovered peptides and drug-like compounds that disrupt interactions between core and other HCV proteins, NS3 and NS5A, and block HCV production. Here we report that a biotinylated derivative of SL209, a prototype small molecule inhibitor of core dimerization (IC50 of 2.80 µM) that inhibits HCV production with an EC50 of 3.20 µM, is capable of penetrating HCV-infected cells and tracking with core. Interaction between the inhibitors, core and other viral proteins was demonstrated by SL209–mediated affinity-isolation of HCV proteins from lysates of infected cells, or of the corresponding recombinant HCV proteins. SL209-like inhibitors of HCV core may form the basis of novel treatments of Hepatitis C in combination with other target-specific HCV drugs such as inhibitors of the NS3 protease, the NS5B polymerase, or the NS5A regulatory protein. More generally, our work supports the hypothesis that inhibitors of viral capsid formation might constitute a new class of potent antiviral agents, as was recently also shown for HIV capsid inhibitors

ENU Mutagenesis Identifies Mice with Morbid Obesity and Severe Hyperinsulinemia Caused by a Novel Mutation in Leptin

BACKGROUND: Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals. METHODOLOGY/PRINCIPAL FINDINGS: To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin(145E/145E) mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin(145E/145E) mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin(145E/145E) mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region. CONCLUSIONS/SIGNIFICANCE: Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome

Noradrenergic Control of Gene Expression and Long-Term Neuronal Adaptation Evoked by Learned Vocalizations in Songbirds

Norepinephrine (NE) is thought to play important roles in the consolidation and retrieval of long-term memories, but its role in the processing and memorization of complex acoustic signals used for vocal communication has yet to be determined. We have used a combination of gene expression analysis, electrophysiological recordings and pharmacological manipulations in zebra finches to examine the role of noradrenergic transmission in the brain’s response to birdsong, a learned vocal behavior that shares important features with human speech. We show that noradrenergic transmission is required for both the expression of activity-dependent genes and the long-term maintenance of stimulus-specific electrophysiological adaptation that are induced in central auditory neurons by stimulation with birdsong. Specifically, we show that the caudomedial nidopallium (NCM), an area directly involved in the auditory processing and memorization of birdsong, receives strong noradrenergic innervation. Song-responsive neurons in this area express α-adrenergic receptors and are in close proximity to noradrenergic terminals. We further show that local α-adrenergic antagonism interferes with song-induced gene expression, without affecting spontaneous or evoked electrophysiological activity, thus dissociating the molecular and electrophysiological responses to song. Moreover, α-adrenergic antagonism disrupts the maintenance but not the acquisition of the adapted physiological state. We suggest that the noradrenergic system regulates long-term changes in song-responsive neurons by modulating the gene expression response that is associated with the electrophysiological activation triggered by song. We also suggest that this mechanism may be an important contributor to long-term auditory memories of learned vocalizations

COLOCALIZATION OF MUSCARINIC ACETYLCHOLINE-RECEPTORS AND PROTEIN KINASE-C-GAMMA IN RAT PARIETAL CORTEX

The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the gamma isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present in layers 2, 3 and 5, whereas most PKCgamma-positive cells were found in layers 2, 5 and 6. Under normal, non-stimulated conditions, approximately 58% of all muscarinic cholinoceptive neurons were immunoreactive for PKCgamma. Conversely, nearly all PKCgamma-positive neurons were M35-immunoreactive. Although both pyramidal and nonpyramidal neurons express the two types of protein, the pyramidal cell type represents the vast majority. Of all cortical neurons, the large (15-25 mu m in diameter) muscarinic cholinoceptive pyramidal neurons in layer 5 express the gamma isoform of PKC most abundantly and most frequently. Approximately 96% of these cells are immunoreactive for PKCgamma. Stimulation of mAChRs by the cholinergic agonist carbachol resulted in a pronounced increase in the intensity of 36G9 immunoreactivity, which may suggest that the mAChRs are functionally linked to the colocalized PKCgamma. No change was found in the number of 36G9-immunoreactive neurons. In contrast, the number of immunocytochemically detectable muscarinic cholinoceptive neurons increased by approximately 38% after carbachol stimulation. The high degree of codistribution in cortical neurone of both transduction proteins suggests a considerable cholinergic impact upon the regulation of PKCgamma, a candidate key enzyme in cortical learning and memory mechanisms