RESUMO
New RNA-binding small-molecule scaffolds are needed to unleash the pharmacological potential of RNA targets. Here we have applied a pharmacophore-based virtual screening approach, seldom used in the RNA recognition field, to identify novel conformational inhibitors of the hepatitis C virus internal ribosome entry site. The conformational effect of the screening hits was assessed with a fluorescence resonance energy transfer assay, and the affinity, specificity, and binding site of the ligands were determined using a combination of fluorescence intensity and NMR spectroscopy experiments. The results indicate that this strategy can be successfully applied to discover RNA conformational inhibitors bearing substantially less positive charge than the reference ligands. This methodology can potentially be accommodated to other RNA motifs of pharmacological interest, facilitating the discovery of novel RNA-targeted molecules.
RESUMO
Vector design and its characterization is an area of great interest in current vaccine research. In this article, we have formulated and characterized a multicompartmental lipopolyplex, which associates multiple liposomes and polyplexes in the same complex. These particles allow the simultaneous delivery of lipid or water-soluble antigens associated with genes to the same cell, in much higher amounts than conventional lipopolyplexes. The vector characterization and optimization were carried out using liposomes with entrapped carboxyfluorescein and adapted electrophoretic assays. Two types of lipopolyplexes (containing hydrophilic or lipophilic antigens) were employed to evaluate their interest in vaccination. The lipopolyplex loaded with an extract of water-soluble melanoma proteins proved to efficiently induce humoral response in murine melanoma model, increasing the levels of IgM and IgG. The specificity of the immune response induced by the lipopolyplex was demonstrated in mice with the lipopolyplex containing the GD3 ganglioside lipid antigen, abundant in melanoma cells. The levels of anti-GD3 IgG increased markedly without modifying the expression of humoral antibodies against other gangliosides.
RESUMO
The 3'X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3'X mutants that favor one of the two previously proposed conformations comprising either two or three stem-loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3'X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3'X domain does not occur as a result of a transition between two- and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3'X homodimers.
Assuntos
Regiões 3' não Traduzidas/genética , Hepacivirus/genética , Hepatite C/virologia , Conformação de Ácido Nucleico , RNA Viral/genética , Proteínas não Estruturais Virais/genética , Pareamento de Bases , Dimerização , Hepacivirus/fisiologia , Humanos , Sequências Repetidas Invertidas , Modelos Moleculares , Mutação , Nucleotídeos , Dobramento de RNA , RNA Viral/química , Replicação Viral/genéticaRESUMO
Therapeutic vaccination of mice bearing melanoma tumors with our genetically modified tumor cells, via DOTAP/GM-CSF lipoplexes, results in >85% tumor growth inhibition. These fresh transfected cells (irradiated, frozen and thawed) are able to produce high amounts of GM-CSF transgene (>200 ng/10(6) cells/24 h). After vaccination, significant increases (>eight-fold) in specific antitumor membrane protein IgG1 and IgG2a are obtained only in groups vaccinated with GM-CSF-producing cells, where also the highest rates of tumor inhibition, and significantly delayed mice death (P<0.05), are observed. The antitumor response obtained is long-lasting in survivors (GM-CSF-group) from 6 months after the first tumor challenge, and a full 100% of mice survived to a second tumor challenge. All these results suggest that antitumor cell vaccines engineered by nonviral procedures are suitable for use as therapeutic vaccines with potential clinical applications.
