Identification of efficiently cleaved substrates for HIV-1 protease using a phage display library and use in inhibitor development

The recognition sequences for substrate cleavage by aspartic protease of HIV-1 are diverse and cleavage specificities are controlled by complex interactions between at least six amino acids around the cleavage site. We have identified 45 efficiently cleaved peptide substrates of HIV-1 protease (PR) using substrate phage display, an approach that can elucidate both context-dependent and context-independent preferences at individual subsites of a protease substrate. Many of the selected peptides were cleaved more efficiently and had lower K-m values than physiologically relevant substrates of HIV-1 PR. Therefore, mutations occurring in the cleavage sites of the Gag and Gag-pol polyproteins of HIV-1 could significantly lower the K-m values to better compete against drugs for protease binding while maintaining cleavage rates necessary for viral replication. The most efficiently cleaved peptide substrate derived from these phage, Ac-GSGIF*LETSL-NH2, was cleaved 60 times more efficiently and had a K-m approximately 260 times lower than a nine-amino-acid peptide based on the natural reverse transcriptase/integrase cleavage site when assayed at pH 5.6, 0.2 M NaCl. The peptide substrates selected served as frameworks for synthesis of tight binding reduced amide inhibitors of HIV-1 PR. The results show that the most efficiently cleaved substrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resistant proteases may aid in the development of more efficacious inhibitors. (C) 2000 Academic Press.