Integrase is an
enzyme found in human immunodeficiency virus, which is required for the viral life cycle, yet has no human cellular homologue. For this reason,
HIV integrase (IN) has become an important target for the development of new
AIDS therapeutics. Irreversible affinity
ligands have proven to be valuable tools for studying a number of
enzyme and
protein systems, yet to date there have been no reports of such affinity
ligands for the study of IN. As an initial approach toward irreversible
ligand design directed against IN, we appended
isothiocyanate functionality onto
caffeic acid phenethyl ester (CAPE), a known
HIV integrase inhibitor. The choice of
isothiocyanate as the reactive functionality, was based on its demonstrated utility in the preparation of affinity
ligands directed against a number of other
protein targets. Several isomeric CAPE
isothiocyanates were prepared to explore the
enzyme topography for reactive
nitrogen and
sulfur nucleophiles vicinal to the
enzyme-bound CAPE. The preparation of these CAPE
isothiocyanates, required development of new synthetic methodology which employed phenyl
thiocarbamates as latent
isothiocyanates which could be unmasked near the end of the synthetic sequence. When it was observed that beta-
mercaptoethanol (beta-ME), which is required to maintain the catalytic activity of soluble IN (a F185KC280S mutant), reacted with CAPE
isothiocyanate functionality to form the corresponding hydroxyethylthiocarbamate, a variety of mutant IN were examined which did not require the presence of beta-ME for catalytic activity. Although in these latter
enzymes, CAPE
isothiocyanate functionality was presumed to be present and available for acylation by IN nucleophiles, they were equally effective against Cys to Ser mutants. One conclusion of these studies, is that upon binding of CAPE to the
integrase,
nitrogen or
sulfur nucleophiles may not be properly situated in the vicinity of the phenethyl aryl ring to allow reaction with and covalent modification of reactive functionality, such as
isothiocyanate groups. The fact that introduction of the
isothiocyanate group onto various positions of the phenethyl ring or replacement of the phenyl ring with naphthyl rings, failed to significantly affect inhibitory potency, indicates a degree of insensitivity of this region of the molecule toward structural modification. These findings may be useful in future studies concerned with the development and use of
HIV-1 integrase affinity
ligands.