Repurposing of approved antivirals against dengue virus serotypes: an in silico and in vitro mechanistic study.

Dengue is an emerging, mosquito-borne viral disease of international public health concern. Dengue is endemic in more than 100 countries across the world. However, there are no clinically approved antivirals for its cure. Drug repurposing proves to be an efficient alternative to conventional drug discovery approaches in this regard, as approved drugs with an established safety profile are tested for new indications, which circumvents several time-consuming experiments. In the present study, eight approved RNA-dependent RNA polymerase inhibitors of Hepatitis C virus were virtually screened against the Dengue virus polymerase protein, and their antiviral activity was assessed in vitro. Schrödinger software was used for in silico screening, where the compounds were passed through several hierarchical filters. Among the eight compounds, dasabuvir was finally selected for in vitro cytotoxicity and antiviral screening. Cytotoxicity profiling of dasabuvir in Vero cells revealed changes in cellular morphology, cell aggregation, and detachment at 50 µM. Based on these results, four noncytotoxic concentrations of dasabuvir (0.1, 0.25, 0.5, and 1 µM) were selected for antiviral screening against DENV-2 under three experimental conditions: pre-infection, co-infection, and post-infection treatment, by plaque reduction assay. Viral plaques were reduced significantly (p < 0.05) in the co-infection and post-infection treatment regimens; however, no reduction was observed in the pretreatment group. This indicated a possible interference of dasabuvir with NS5 RdRp, as seen from in silico interaction studies, translating into a reduction in virus plaques. Such studies reiterate the usefulness of drug repurposing as a viable strategy in antiviral drug discovery. In this drug repurposing study, dasabuvir, a known anti-hepatitis C drug, was selected through virtual screening and assessed for its anti-dengue activity.

Quantifying the significance of phage attack on starter cultures: a mechanistic model for population dynamics of phage and their hosts isolated from fermenting sauerkraut.

We investigated the possibility of using starter cultures in sauerkraut fermentation and thereby reducing the quantity of salt used in the process. This, in turn, would reduce the amount of waste salt that would enter in our water resources. Phage, naturally present in sauerkraut fermentation, could potentially affect the starter cultures introduced. Thus, a mechanistic mathematical model was developed to quantify the growth kinetics of the phage and starter cultures. The model was validated by independent experiments with two Leuconostoc mesenteroides strains isolated from sauerkraut and their corresponding phage. Model simulations and experimental evidence showed the presence of phage-resistant cell populations in starter cultures which replaced phage-sensitive cells, even when the initial phage density (P(0)) and multiplicity of infection (MOI) were low (P(0) < 1 x 10(3) PFU/ml; MOI < 10(-4)) in the MRS media. Based on the results of model simulation and parameter optimization, it was suggested that the kinetic parameters of phage-host interaction, especially the adsorption rate, vary with the initial phage and host densities and with time. The model was validated in MRS broth. Therefore, the effects of heterogeneity and other environmental factors, such as temperature and pH, should be considered to make the model applicable to commercial fermentations.

Casein kinase II activity of buffalo sperm chromatin.

Two cyclic AMP-independent protein kinase activities have been found associated with buffalo sperm chromatin: a histone kinase highly specific for arginine-rich histone was reported recently (Mudgal et al., 1997: Arch Andrology 38:191-199) and a casein kinase II is described here. Casein kinase activity was solubilized with 0.35 M NaCl, which extracted 90% of the initial enzyme activity associated with buffalo sperm chromatin. Of the two acidic proteins tested, casein was preferred substrate over phosvitin. Among the casein fractions, the order of preference for casein kinase was beta-casein > alpha-casein > casein. Cyclic AMP at concentrations up to 50 microM had no effect on the phosphorylation of casein. Phosphoamino acid analysis using casein as the substrate showed threonine to be the acceptor amino acid for phosphoester link. Phosphorylation specificity was determined by phosphorylating buffalo beta-casein followed by the preparation of tryptic peptides and identification of amino acid residue phosphorylated. Threonine residue at position 41 having clusters of acidic amino acid residues (Thr. Glu. Asp. Glu) C-terminal to it was phosphorylated, a phosphorylation specificity akin to CKII. It is thought that phosphorylation of histones decreases their association with DNA and probably makes the DNA more available for replication, while phosphorylation of nonhistone proteins modifies their interaction with histones, allowing control of template activity. Two protein kinases found in buffalo sperm chromatin may perform a similar function.

Histone kinase activity of buffalo sperm chromatin.

Phosphorylation of buffalo sperm chromatin proteins under optimum conditions (8 mM Mg2+, pH 8.0, and at 30 degrees C) using [gamma-32P]ATP and endogenous protein kinase activity was linear for 15 min incubation time and up to 330 micrograms protein. The 32P transferred from [gamma-32P]ATP was located in protein as a phosphoester bond. Fractionation with 1.2 M NaCl-4 M urea-0.2 M 2-mercaptoethanol-1 mM PMSF followed by acid treatment solubilized 87% of the total chromatin proteins termed "sperm histones." The remaining 21% nonhistone protein was tightly bound to DNA. Follow-up of the label showed 91% of the 32P in sperm histone and 9% with DNA-associated proteins. Histone kinase activity was solubilized with 0.35 M NaCl, which extracted 70% of the initial enzyme activity associated with chromatin. Of the different histones tested as substrates, histone kinase phosphorylated only histone H3 and, therefore, is highly specific for arginine rich histone. It also phosphorylates the acidic protein, casein. Cyclic AMP at concentrations up to 50 microM had no effect on the phosphorylation of histone H3. Phosphoamino acid analysis using histone H3 as the substrate showed serine to be the acceptor amino acid for phosphoester link.