Paul L. Darke

HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins.

The mature proteins of retroviruses originate as a result of proteolytic cleavages of polyprotein precursors. Retroviruses encode proteases responsible for several of these processing events, making them potential antiviral drug targets. A 99-amino acid HIV-1 protease, produced by chemical synthesis or by expression in bacteria, is shown here to hydrolyze peptides corresponding to all of the known cleavage sites in the HIV-1 gag and pol polyproteins. It does not hydrolyze peptides corresponding to an env cleavage site or a distantly related retroviral gag cleavage site.

Affinity purification of the HIV-1 protease

An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column.

Stereoselective synthesis of the hydroxyethylene isostere of the HIV protease (TYR-PRO) cleavage site

Abstract The stereoselective, syntheses and IC 50 s of Tyr-HE-Pro based inhibitors are reported. The [2S,3S,4S,5S]-stereochemistry was preferred by HIV-1 protease.

HIV-1 protease inhibitory activity of L-694,746, a novel metabolite of L-689,502

L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 (Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.

Interaction of Mutant Forms of the HIV-1 Protease with Substrate and Inhibitors

The protease of HIV-1 is considered to be a prime target for the treatment of AIDS with small molecular weight inhibitors. 1,2,3 Many groups have now demonstrated that compounds designed as potent inhibitors of the enzyme in vitro are effective inhibitors of viral polyprotein processing and viral spread in cultured cells.4–7 Continued effort to discover new potent inhibitors with suitable properties for use in humans is thus justified.