C-Methylated Spermidine Derivatives: Convenient Syntheses and Antizyme-Related Effects.

The biogenic polyamines, spermidine (Spd) and spermine (Spm), are present at millimolar concentrations in all eukaryotic cells, where they participate in the regulation of vitally important cellular functions. Polyamine analogs and derivatives are a traditional and important instrument for the investigation of the cellular functions of polyamines, enzymes of their metabolism, and the regulation of the biosynthesis of antizyme-a key downregulator of polyamine homeostasis. Here, we describe convenient gram-scale syntheses of a set of C-methylated analogs of Spd. The biochemical properties of these compounds and the possibility for the regulation of their activity by moving a methyl group along the polyamine backbone and by changing the stereochemistry of the chiral center(s) are discussed.

Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions.

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)2-Polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone-2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)2-Polyamine complex formation. Thus, the (OAZ1)2-Polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds-2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.

Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation.

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus.

Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.

Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication.

Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N(3)-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N(1) position.

Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells.

Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N(1)-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-κB transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT - acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress.

Inhibition of the helicase activity of the HCV NS3 protein by symmetrical dimeric bis-benzimidazoles.

Dimeric bis-benzimidazoles (DBn) are the compounds specifically binding to A-T enriched sequences in the DNA minor groove. Due to this property they can inhibit DNA-dependent enzymes. We show that inhibition of the helicase activity of HCV NS3 protein by DBn was due to a novel mechanism, which involved direct binding of the ligands to the enzyme. The binding potency and inhibition efficacy depended on the length of the linker between the benzimidazole fragments. The most effective inhibitor DB11 partially prevented activation of NTPase activity of NS3 by poly(U) and increased affinity of the enzyme to the helicase substrate DNA.

Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system.

Hepatitis C virus (HCV) NS5A phosphoprotein is a component of virus replicase. Here we demonstrate that in vitro unphosphorylated NS5A protein inhibits HCV RNA-dependent RNA polymerase (RdRp) activity in polyA-oligoU system but has little effect on synthesis of viral RNA. The phosphorylated casein kinase (CK) II NS5A protein causes the opposite effect on RdRp in each of these systems. The phosphorylation of NS5A protein with CKII does not affect its affinity to the HCV RdRp and RNA. The NS5A phosphorylation with CKI does not change the RdRp activity. Herein we report evidence that the NS5A prevents template binding to the RdRp.

Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins.

The plasmid pET-21d-2c-5BDelta55 effectively expressing a C-terminally truncated form (NS5BDelta55) of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed. It was derived from pET-21d-5BDelta55 plasmid and contained six mutations in the ATG-start codon region and an additional cistron upstream the target gene. The C-terminally His-tagged NS5BDelta55 protein was expressed in Rosetta(DE3) Escherichia coli strain bearing an additional pRARE plasmid encoding extra copies of rare tRNAs. The yield of the target enzyme exceeded by a factor of 29 the yield of NS5BDelta55 protein expressed from the parental pET-21d-5BDelta55 plasmid (5 mg/L). The increase in the protein yield could be explained by facilitated protein translation initiation, resulted from disruption of the stable secondary mRNA structure. The pET-21d-2c-5BDelta55 plasmid yielded one third amount of the protein when expressed in BL-21(DE3) strain, indicating that the pRARE plasmid is required for a high-level expression of NS5BDelta55 protein. The 29-fold enhancement of the protein yield was accompanied by only a 2.5-fold increase of the corresponding mRNA level. The expression of another HCV NS5A protein His-tagged at the C-terminus in the developed system yielded a similar amount of the protein (4 mg/L), whereas its N-terminally His-tagged counterpart was obtained in a 30 mg/L yield. The NS5A protein purified under denaturing conditions and renatured in solution inhibited the HCV RdRp and was a substrate for human casein kinase II.