RESUMO
In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Desenho de Fármacos , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Genes Reporter , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatócitos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Oligopeptídeos/química , Oxazóis/síntese química , Oxazóis/química , Replicon , Inibidores de Serina Proteinase/químicaRESUMO
Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC50s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.
Assuntos
HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Reversa , Alcinos , Benzoxazinas/metabolismo , Benzoxazinas/farmacologia , Bioensaio , Sistema Livre de Células , Ciclopropanos , Feminino , HIV-1/metabolismo , Humanos , Nevirapina/metabolismo , Nevirapina/farmacologia , Transcrição Reversa/efeitos dos fármacos , Sensibilidade e Especificidade , Virologia/métodosRESUMO
The unspliced human immunodeficiency virus type 1 (HIV-1) RNA is both the messenger for Gag and Gag-Pol and the viral genomic RNA (vRNA) that is packaged into the virion. Although Gag alone is sufficient for the incorporation of vRNA into virus particles, Gag-Pol molecules play an important role in vRNA dimerization and virion maturation. Here, a cis model for vRNA packaging was demonstrated, in which nascent Gag-Pol molecules were preferentially co-encapsulated with their cognate RNA used as the template. Genome-incorporation frequencies were evaluated for two distinct HIV-1 proviral clones differing in their ability to respond to nevirapine (NVP) treatment in one round of infection. It was shown that, under NVP selection, there was a twofold-higher incorporation of vRNAs and integration of provirus genome carrying NVP resistance when compared with the wild-type counterpart. Although cis incorporation has been already demonstrated for Gag, the novelty of these findings is that newly acquired resistant mutations in Gag-Pol will select their specific genomic RNA during virus replication, thus rapidly increasing the chance of the emergence of resistant viruses during the course of anti-retroviral treatment.
Assuntos
Genes gag , Genes pol , Transcriptase Reversa do HIV/genética , HIV-1/genética , Mutação , RNA Viral/metabolismo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , DNA Viral/genética , Farmacorresistência Viral/genética , Genoma Viral , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Modelos Biológicos , Nevirapina/farmacologia , Provírus/genética , Montagem de Vírus , Replicação ViralRESUMO
Human immunodeficiency virus type 1 subtype B and C proteases were manipulated to contain 90M, 88D, or 89L, and their in vitro biological properties were studied. We showed that D30N has significantly more impact in subtype C than in subtype B counterparts, accounting for the reported low prevalence of this mutation in patients failing nelfinavir-based regimens.