An RNA Hybridization Assay for Screening Influenza A Virus Polymerase Inhibitors Using the Entire Ribonucleoprotein Complex.

Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.

Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon.

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent. At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromatographic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoroacetic acid. Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid. Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such as ATP, deoxyguanosine diphosphate (dGTP), and phospho-adenosine-5'-phosphosulfate or 3',5'-cyclic adenosine 5'-monophosphate (AMP) and its much more abundant isomer 2',3'-cyclic AMP. A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short-lived analytes such as cytidine 5'-monophosphate (CMP)-2-keto-deoxy-octulosonic acid. In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as uridine 5'-diphosphate (UDP)-l-arabinopyranose, UDP-L-arabinofuranose, guanosine 5'-diphosphate (GDP)-L-galactofuranose, UDP-L-rhamnose, and adenosine diphosphate (ADP)-sugars. Surprisingly, UDP-arabinopyranose was also found in Chinese hamster ovary (CHO) cells. Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches.

Improvement of the energy metabolism of recombinant CHO cells by cell sorting for reduced mitochondrial membrane potential.

One of the major problems in process performance of mammalian cell cultures is the production of lactic acid. Cell specific glucose uptake rates usually correlate to glucose concentration and approximately 80% of the metabolised glucose is converted into lactic acid. As the mitochondrial membrane potential was shown to correlate to cell specific glucose uptake rates, we used Rhodamine 123, a lipophilic cationic dye for cell sorting to improve the energy metabolism of existing production cell lines. Two recombinant CHO cell lines with known differences in lactic acid production rate were used to evaluate Rhodamine 123 staining as a descriptor for glucose uptake rates and to determine whether it is possible to isolate subclones with altered metabolic properties. Such subclones would exhibit an improved process performance, and in addition could be used as models for genomic and metabolic studies. From the cell line with high lactate production, a subclone sorted for reduced mitochondrial membrane potential was found to have a lower specific lactate formation rate compared to the parental cell line in batch cultures. In addition, the glucose consumption rate was also reduced, while both the growth rate and the final cell concentration were increased. A subclone sorted for high mitochondrial membrane potential, on the other hand, had a higher glucose consumption rate, a higher lactate production rate and reduced growth. The potential of using flow cytometric cell sorting methods based on physiological activity for cell line optimisation is discussed.

The Drosophila melanogaster homologue of the human histo-blood group Pk gene encodes a glycolipid-modifying alpha1,4-N-acetylgalactosaminyltransferase.

Insects express arthro-series glycosphingolipids, which contain an alpha1,4-linked GalNAc residue. To determine the genetic basis for this linkage, we cloned a cDNA (CG17223) from Drosophila melanogaster encoding a protein with homology to mammalian alpha1,4-glycosyltransferases and expressed it in the yeast Pichia pastoris. Culture supernatants from the transformed yeast were found to display a novel UDP-GalNAc:GalNAcbeta1,4GlcNAcbeta1-R alpha-N-acetylgalactosaminyltransferase activity when using either a glycolipid, p-nitrophenylglycoside or an N-glycan carrying one or two terminal beta-N-acetylgalactosamine residues. NMR and MS in combination with glycosidase digestion and methylation analysis indicate that the cloned cDNA encodes an alpha1,4-N-acetylgalactosaminyltransferase. We hypothesize that this enzyme and its orthologues in other insects are required for the biosynthesis of the N5a and subsequent members of the arthro-series of glycolipids as well as of N-glycan receptors for Bacillus thuringiensis crystal toxin Cry1Ac.