A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS.

Endogenous nucleosides and nucleotides in biosamples are frequently highlighted as the most differential metabolites in recent metabolomics studies. We developed a rapid, sensitive, high-throughput and reliable quantitative method to simultaneously profile 20 endogenous nucleosides and nucleotides in cancer cell lines based on ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC- MS/MS) by using a porous graphitic carbon column and basic mobile phase. The results indicated that high pH value of mobile phase containing 0.12% diethylamine (DEA) and 5mM NH4OAC (pH 11.5) was the critical factor to prevent the adsorption of multi-phosphorylated species, and significantly improved peak shape and sensitivity. The optimized method was successfully validated with satisfactory linearity, sensitivity, accuracy, precision, matrix effects, recovery and stability for all analytes. The limit of quantification (LOQ) was in the range of 0.6-6nM (6-60 fmol on column). The validated method was applied to the extract of three epithelial cancer cell lines, and the significant difference in the profiling of the nucleosides and nucleotides among the cancer cell lines enables discrimination of breast cancer cell line from the colon cancer cell line and the lung cancer cell line. This quantified analytical method of 20 endogenous nucleosides and nucleotides in cancer cell lines meets the requirement of quantification in specific expanded metabolomics studies, with good selectivity and sensitivity.

Inhibitory activities of three classes of acyclic nucleoside phosphonates against murine polyomavirus and primate simian virus 40 strains.

Murine polyomavirus and simian virus 40 were used to evaluate the potencies of the compounds of three classes of acyclic nucleoside phosphonates: (i) the original HPMP (3-hydroxy-2-phosphonomethoxypropyl) and PME (2-phosphonomethoxyethyl) derivatives, (ii) the 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (DAPy) derivatives, and (iii) a new class of HPMP derivatives containing a 5-azacytosine moiety. The last class showed the highest activities and selectivities against both polyomaviruses.

Recent advances in antiviral nucleoside and nucleotide therapeutics.

Recent developments in nucleoside/nucleotide therapeutics and antiviral drug targets are described covering progress in the development of nucleoside/nucleotide mimetics for the treatment of influenza virus, human immunodeficiency virus type 1, hepatitis B and C virus, herpes virus infections; including herpes simplex virus, cytomegalovirus and varicella zoster virus infections, and the highly pathogenic poxviruses (variola, vaccinia and monkey pox) and filoviruses (Ebola and Marburg).

MraY Inhibitors as Novel Antibacterial Agents.

MraY presents all necessary biological requirements to be considered as a target of interest for the discovery of novel antibacterials. Furthermore, several inhibitors aimed at this enzyme have been discovered. Amphomycin, which is currently used as a topical antibacterial in the veterinary industry is one of them, but the major source of future developments resides in the nucleoside based inhibitors group. This group has been subdivided into classes: Tunicamycins, Ribosamino-uridines, Uridylpeptides and Capuramycins. Analysis of pharmacological behaviours observed with several compounds within these classes, shows that broad-spectrum antibacterial activity, including relevant resistant strains and in vivo efficacy without toxicity are achievable. Among them, Caprazamycins, Muraymycins, Riburamycins and Capuramycins present the most promising profiles.

Post mortem degradation of nucleosides in the brain: comparison of human and rat brains for estimation of in vivo concentration of nucleosides.

There is an increasing attention paid for nucleoside metabolism and changes of nucleoside concentrations in human brain because of its pathological and physiological relevance. In order to determine the post mortem degradation of nucleosides and nucleoside metabolites, the concentrations of four nucleosides and three nucleobases were measured in rat and neurosurgical human cerebral cortical samples with 30s to 24h post mortem delay. Adenosine degradation coefficient (a multiplying factor for calculating concentrations of investigated substances for the living state) was 0.886 for human brain at 2 h post mortem time, while it was 1.976 for rats. Hypoxanthine, an adenosine degradation product had coefficients 0.564 for human brain and 0.812 for the rat brain. We provide data and degradation coefficients for the concentrations of adenosine, guanosine, inosine, uridine, uracil, hypoxanthine and xanthine with 2, 4, 6 and 24 h post mortem delay. We also report a method how to validate human neurosurgical brain samples in terms of sample preparation and statistical analysis.

Prolactin, cortisol, and insulin regulation of nucleoside uptake into mouse mammary gland explants.

Nucleosides are essential components of milk that are used for the nourishment of newborns. Effects of the three primary lactogenic hormones, including prolactin (PRL), insulin (I), and cortisol (H), on nucleoside uptake and incorporation into cultured mammary tissues taken from 12- to 14-day pregnant mice were determined; most experiments focused on the regulation of uridine uptake. Insulin alone, as well as PRL in the presence of insulin and cortisol, was shown to stimulate uridine uptake and incorporation into RNA in mammary explants taken from 12- to 14-day pregnant mice. The PRL effects were expressed at concentrations of 25 ng/ml and above, which are physiological plasma concentrations. In the absence of sodium, uridine uptake and incorporation were diminished, suggesting the presence of a sodium-dependent uridine transporter. In kinetic studies the apparent Km for uridine uptake was calculated to be 312 microM, and the Vmax 2.90 micromol/hr/L cell water; PRL had no effect on the Km but increased the Vmax to 5.88 micromol/hr/L cell water. When assessing uridine uptake in the presence of the other nucleosides at 0.1 mM, only cytidine competed with uridine uptake. The fact that distribution ratios of greater than 15:1 were achieved with uridine indicates that uridine uptake may be via an active transporter. These studies show that PRL enhances uridine update in mammary tissues by stimulating the activity, and probably synthesis, of a sodium-dependent, active uridine and cytosine transporter.

Application of multivariate data analysis methods to comparative molecular field analysis (CoMFA) data: proton affinities and pKa prediction for nucleic acids components.

Multivariate data analysis methods (Principal Component Analysis (PCA) and Partial Least Squares (PLS)) are applied to the analysis of the CoMFA (Comparative Molecular Field Analysis) data for several nucleic acids components. The data set includes nitrogenated bases, nucleosides, linear nucleotides, 3', 5'-cyclic nucleotides and oligonucleotides. PCA is applied to study the structure of the CoMFA data and to detect possible outliers in the data set. PLS is applied to correlate the CoMFA data with either calculated AM1 proton affinities or with experimental pKa values. The possibility of making a prediction of pKa values directly from 3D structures of the monomers for polynucleotides is also shown. The influence of the superposition criteria and of conformational changes along the glycosidic bond on the pKa prediction are studied as well.