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 HI V-1 are diverse and cleavage specificities are controlled by complex intera ctions between at least six amino acids around the cleavage site. We have i dentified 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 p rotease substrate. Many of the selected peptides were cleaved more efficien tly and had lower K-m values than physiologically relevant substrates of HI V-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 cleavag e rates necessary for viral replication. The most efficiently cleaved pepti de substrate derived from these phage, Ac-GSGIF*LETSL-NH2, was cleaved 60 t imes more efficiently and had a K-m approximately 260 times lower than a ni ne-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 se lected served as frameworks for synthesis of tight binding reduced amide in hibitors of HIV-1 PR. The results show that the most efficiently cleaved su bstrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resist ant proteases may aid in the development of more efficacious inhibitors. (C ) 2000 Academic Press.