Background: The interactions between the viral envelope glycoprotein gp120, its cellular receptor CD4, and the specific co-receptor are part of the early events occurring during cell infection by HIV-1. Antagonists of HIV-1 replication at the entry level, via blockade of attachment, gp120/CD4/co-receptor interactions, or gp41 structural changes, represent a potential source for HIV-1 treatment. Synthetic combinatorial libraries (SCLs) made up of thousands to millions of compounds are a powerful approach for the development of such antagonists.

Methods: 2 recombinant vaccinia virus-based assays mimicking a T-cell line-tropism (IIIb-X4) and macrophage-tropism (JRFL-R5) have been developed to quantify the inhibition of the fusogenic activity of HIV-1 envelope glycoproteins. The expression of Lac Z from a reporter gene upon fusion was used to measure colorimetrically the fusion of target and effector cells. 2 peptide SCLs were screened in these assays to develop fusion inhibitors: a l-amino acid nonapeptide and a d-amino acid decapeptide SCLs. The inhibitory activities of the identified peptides were assayed in the vaccinia virus-based fusion and replication assays using different laboratory virus strains as well as clinical isolates.

Results:  Following the screening of the 2 SCLs in the X4 fusion system, 38 d-amino acid decapeptides and 44 l-amino acid nonapeptides were generated and their inhibitory activities determined. 5 nonapeptides exhibited inhibitory activity with IC₉₀ values lower than 10 µg/mL, while the most active decapeptides have IC₉₀ values around 25 µg/mL when tested in the X4 fusion system. Lower activity was observed in the R5 fusion system. These peptides have similar efficacy in the inhibition of virus replication (IC₉₀ values = 10 to 50 µg/ml in an assay using IIIb/X4 virus and between 50 and 80% inhibition at 25 µg/mL in assays using other X4, R5, and X4R5 virus strains). Studies toward the understanding the peptides mechanism and target of inhibition have been started.

Conclusions: These studies allowed the rapid identification of novel HIV fusion inhibitors that represent leads for new therapies targeting the entry of HIV-1, and useful tools to further study and enhance the understanding of the fusion process.