Pseudo-peptides derived from isomannide: inhibitors of serine proteases.

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.

Development of a new methodology for screening of human immunodeficiency virus type 1 microbicides based on real-time PCR quantification.

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.

Gag-Pol bearing a reverse transcriptase drug-resistant mutation influences viral genomic RNA incorporation into human immunodeficiency virus type 1 particles.

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.

[Pregnancy and delivery outcomes in women with previous cervical conization]. / Desfecho da gravidez e do parto em mulheres submetidas a conização cervical.

OBJECTIVE: The purpose of this study was to assess the effect of cervical conization on subsequent pregnancy and delivery outcomes. METHOD/STUDY DESIGN: We used a retrospective design that included the records of all women who had conization of the cervix in our department between March 1st 1993 and September 1st 2001. Loop electrosurgical excision procedure (LEEP) was performed in every woman. From the totality of records we selected the ones who subsequently got pregnant (20 patients) and determined the pregnancy and delivery events. RESULTS: 113 women in the reproductive years underwent cervical conization during this period; 45 (39.8%) were lost to follow up, 48 (42.5%) didn't get pregnant and 20 (11.7%) women achieved 21 pregnancies. Among these 21 pregnancies there were 2 (9.5%) spontaneous abortions, 2 (9.5%) voluntary terminations of pregnancies, 1 (4.8%) elective termination of pregnancy, 1 (4.8%) ectopic pregnancy, 1 (4.8%) pregnancy evolving in the first trimester at the time of the study and 14 (66.7%) term deliveries. This last group was studied with more detail. Half of the women were nulliparous and half were multiparous. There were 2 cases of threatened preterm labour, 2 gestation diabetes, 1 pregnancy induced hypertension, 1 intrauterine growth restriction, 1 macrosomic foetus, 1 premature rupture of membranes and in three cases the labour was induced. The mean time between conization and delivery was 35.75 months. All deliveries occurred between 37 and 40 weeks of gestation; 11 (78.6%) women delivered vaginally and 3 (21.4%) had caesarean section. Only one newborn weighted less than 2500 g. The mean duration of labour (active phase of the first stage and 2nd stage) was 130.92 minutes (95 minutes for multiparous and 157.5 minutes for nulliparous women). CONCLUSION: Despite the small number of cases, pregnant patients who previously underwent LEEP don't seem to be at increased risk of adverse pregnancy outcome, preterm delivery, caesarean delivery or low birth weight. There was, however, a tendency to short duration of labour.

Impact of nelfinavir resistance mutations on in vitro phenotype, fitness, and replication capacity of human immunodeficiency virus type 1 with subtype B and C proteases.

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.