4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV – by recruiting PI4KIIIα in the RNA replication complex – hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product.

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Background:The Snf1/AMPK and PKA pathways are crucial for nutrient sensing and utilization in yeast. Results: A novel cross-talk mechanism between Snf1/AMPK and PKA is proposed. Conclusion: Snf1 and Cyr1 interact in a nutrient-independent manner. Active Snf1 phosphorylates Cyr1 and negatively regulates cAMP content and PKA-dependent transcription. Significance: This is the first evidence of regulation of PKA pathway by Snf1/AMPK.

The metabolism of proliferating cells shows common features even in evolutionary distant organisms such as mammals and yeasts, for example the requirement for anabolic processes under tight control of signaling pathways. Analysis of the rewiring of metabolism, which occurs following the dysregulation of signaling pathways, provides new knowledge about the mechanisms underlying cell proliferation. The key energy regulator in yeast Snf1 and its mammalian ortholog AMPK have earlier been shown to have similar functions at glucose limited conditions and here we show that they also have analogies when grown with glucose excess. We show that loss of Snf1 in cells growing in 2% glucose induces an extensive transcriptional reprogramming, enhances glycolytic activity, fatty acid accumulation and reliance on amino acid utilization for growth. Strikingly, we demonstrate that Snf1/AMPK-deficient cells remodel their metabolism fueling mitochondria and show glucose and amino acids addiction, a typical hallmark of cancer cells.

bThe hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase III␣ (PI4KIII␣) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIII␣ complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIII␣-targeting inhibitor. In addition, both the NS5A and PI4KIII␣ classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIII␣. Because of the similarities between the effects induced by treatment with PI4KIII␣ or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIII␣ complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIII␣ complex.T he recent advent of direct-acting antivirals (DAAs) against hepatitis C virus (HCV) is radically transforming the treatment scenario for patients with chronic hepatitis C infection. These new drugs offer the promise of well-tolerated interferonfree oral regimens that are able to cure the majority of infected patients (1).Initially, the effort to identify DAAs focused primarily on inhibitors of two virally encoded enzymes: the nonstructural 3/4A (NS3/4A) protease and the NS5B polymerase. More recently, however, the clinical validation of NS5A inhibitors (2) has generated increasing interest in this target class. The first NS5A inhibitors were discovered by a phenotypic screen based on the genotype 1b replicon system (3, 4). The initial lead compounds had moderate potency and a narrow spectrum of anti-HCV activity, mainly on genotype 1b. Subsequent medicinal chemistry efforts (4) resulted in the design of picomolar inhibitors characterized by a peculiar and highly symmetrical dimeric structure (reviewed in reference 5). The most-studied agent of this "palindromic" NS5A inhibitor class is daclatasvir (DCV, formerly BMS-790052) (6), a highly optimized biphenyl derivative inhibitor for which regulatory approval is currently being sought.Different chemical isotypes were initially claimed to ...