The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

Evidence for an Alternative Glycolytic Pathway in Rapidly Proliferating Cells

Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells. We used mass spectrometry to show that the phosphate from PEP is transferred to the catalytic histidine (His11) on human PGAM1. This reaction occurred at physiological concentrations of PEP and produced pyruvate in the absence of PKM2 activity. The presence of histidine-phosphorylated PGAM1 correlated with the expression of PKM2 in cancer cell lines and tumor tissues. Thus, decreased pyruvate kinase activity in PKM2-expressing cells allows PEP-dependent histidine phosphorylation of PGAM1 and may provide an alternate glycolytic pathway that decouples adenosine triphosphate production from PEP-mediated phosphotransfer, allowing for the high rate of glycolysis to support the anabolic metabolism observed in many proliferating cells.Damon Runyon Cancer Research FoundationBurroughs Wellcome FundAmerican Cancer SocietyDana-Farber/Harvard Cancer CenterNational Institutes of Health (U.S.) (NIH 1K08CA136983)National Institutes of Health (U.S.) (NIH 5P30CA006516-43)National Institutes of Health (U.S.) (NIH 5 T32 CA009361-28)National Institutes of Health (U.S.) (NIH R21/R33 DK070299)National Institutes of Health (U.S.) (NIH P01GM047467)National Institutes of Health (U.S.) (NIH R01 AI078063)National Institutes of Health (U.S.) (NIH R21 CA12862)National Institutes of Health (U.S.) (NIH R01-GM56302)United States. Public Health Service (NIH P01CA089021)National Institutes of Health (U.S.) (NIH 1P01CA120964-01A

Chasing Phosphohistidine, an Elusive Sibling in the Phosphoamino Acid Fa mily

This year (2012) marks the 50th anniversary of the discovery of protein histidine phosphorylation. Phosphorylation of histidine (pHis) is now widely recognized as being critical to signaling processes in prokaryotes and lower eukaryotes. However, the modification is also becoming more widely reported in mammalian cellular processes and implicated in certain human disease states such as cancer and inflammation. Nonetheless, much remains to be understood about the role and extent of the modification in mammalian cell biology. Studying the functional role of pHis in signaling, either in vitro or in vivo, has proven devilishly hard, largely due to the chemical instability of the modification. As a consequence, we are currently handicapped by a chronic lack of chemical and biochemical tools with which to study histidine phosphorylation. Here, we discuss the challenges associated with studying the chemical biology of pHis and review recent., progress that offers some hope that long-awaited biochemical reagents for studying this elusive posttranslational modification (PTM) might soon be availableclose353