RESUMO
OBJECTIVES: A dynamic G-quadruplex region has been previously shown to form in the long terminal repeat (LTR) promoter of the HIV-1 integrated DNA genome. Inhibition of promoter activity and antiviral effects have been observed when this region was stabilized by BRACO-19, a trisubstituted acridine derivative that binds G-quadruplexes. Here, we aimed at characterizing the antiviral mechanism of action of BRACO-19 by analysing its activity towards a broad range of HIV-1 strains, host cells and infection modes. METHODS: The antiviral activity of BRACO-19 in cell lines and primary cells infected or persistently infected by HIV-1 strains was evaluated at different times post-infection. Virucidal, viral binding, time-dependent and drug-dependent assays were performed to identify the viral target step. Circular dichroism, UV spectroscopy and a reverse transcriptase (RT) stop assay were used to assess RNA G-quadruplex folding and inhibition of RT processing. RESULTS: Thorough virological assays demonstrated that BRACO-19 acts both at the reverse transcription and the post-integration level during the virus life cycle. This behaviour was rationalized by the observation that a G-quadruplex-forming sequence identical to that of the LTR DNA is present at the 3'-end of the virus RNA genome. Biophysics and biomolecular testing showed that this region has the ability to fold into very stable G-quadruplex structures that are even more stabilized by BRACO-19, therefore inhibiting the reverse transcription process at the template level. CONCLUSIONS: Our findings strongly support the activity of BRACO-19 at the viral G-quadruplex level and therefore strengthen the use of viral G-quadruplexes as new anti-HIV-1 targets.
Assuntos
Acridinas/farmacologia , Fármacos Anti-HIV/farmacologia , Quadruplex G/efeitos dos fármacos , HIV-1/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , HIV-1/fisiologia , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Provírus/efeitos dos fármacos , Transcrição Reversa/efeitos dos fármacosRESUMO
Influenza viruses have developed resistance to the current classes of drugs, which means they could eventually become more virulent and cause more mortality and hospitalization. Our study aims to investigate the antiviral activity of Rhazya stricta Decne leaves extract in vitro and search for new promising drugs from R. stricta identified compounds in silico. The study was performed in vitro by utilizing Madin-Darby Canine Kidney cell line (MDCK) as a substrate for the influenza virus and estimating the inhibition performance of the plant leaves extract. Additionally, in silico screening was conducted to explore the antiviral activity of R. stricta phytochemicals. We investigated the cytotoxicity of R. stricta leaves extract and its antiviral activity against influenza virus (A/Puerto Rico/8/34 (H1N1)) using the MTT assay. The mode of action of the plant leaves extract during the viral life cycle was tested using time-of-addition assay. In silico analyses were performed, including molecular docking, drug-likeness analysis, and toxicity risk assessment, to state the leading compounds to be developed into an anti-influenza virus drug. The 50% cytotoxicity concentration of the leaves extract was CC50: 184.6 µg/mL, and the 50% inhibition concentration was CI50: 19.71 µg\mL. The time of addition assay revealed that R. stricta leaves extract exerted its activity in the late step of the influenza virus replication cycle. In comparison to Oseltamivir, the leading compounds showed better binding affinity and can be developed into oral drugs with low toxicity risk. Isolation and purification of the leading compounds and testing their antiviral activity in vitro and in vivo are required.
RESUMO
In the prevention of epidemic and pandemic emerging and neglected viral infections, natural products are an important source of lead compounds. Hornstedtia bella Skornickis is a rhizomatous herb growing in the forest of central Vietnam. Hornstedtia bella essential oil (Hb EO) was recently characterised by our group as endowed of antimicrobial activity against Staphylococcus aureus Methicillin-Resistant strains. Here, we describe for the first time the evaluation of Hb EO against a spectrum of viruses responsible for important human diseases. Hb EO resulted active against Vaccinia virus (VV) (EC50 values 80 µg/mL), closely related to variola virus, causative agent of smallpox. Hb EO was able to strongly reduce the viral VV titer in cell-based assay at not cytotoxic concentration and its potential mode of action was characterised by virucidal activity evaluation followed by time-of-addition assay. Furthermore, Hb EO antiviral activity was implemented in a combination study with the mycophenolic acid.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Óleos Voláteis , Zingiberaceae , Antivirais/farmacologia , Humanos , Óleos Voláteis/farmacologia , Vaccinia virusRESUMO
Viruses infect their host cells to produce progeny virus particles through the sequential steps of the viral life cycle, such as viral attachment, entry, penetration and post-entry events. This protocol describes time-of-addition and temperature-shift assays that are employed to explore which step(s) in the viral life cycle is blocked by an antiviral substance(s).