Arylisothiocyanate-containing esters of caffeic acid designed as affinity ligands for HIV-1 integrase

Integrase is an enzyme found in human immunodeficiency virus, which is requ ired for the viral life cycle, yet has no human cellular homologue. For thi s reason, HIV integrase (IN) has become an important target for the develop ment 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 ag ainst IN, we appended isothiocyanate functionality onto caffeic acid phenet hyl eater (CAPE), a known HIV integrase inhibitor. The choice of isothiocya nate as the reactive functionality, was based on its demonstrated utility i n the preparation of affinity ligands directed against a number of other pr otein targets. Several isomeric CAFE isothiocyanates were prepared to explo re the enzyme topography for reactive nitrogen and sulfur nucleophiles vici nal to the enzyme-bound CAFE. The preparation of these CAFE isothiocyanates . required development of new synthetic methodology which employed phenyl t hiocarbamates as latent isothiocyanates which could be unmasked near the en d of the synthetic sequence. When it was observed that beta -mercaptoethano l (beta -ME), which is required to maintain the catalytic activity of solub le IN (a F185KC280S mutant), reacted with CAFE isothiocyanate functionality to form the corresponding hydroxyethylthiocarbamate, a variety of mutant I N were examined which did not require the presence of beta -ME for catalyti c activity. Although in these latter enzymes, CAFE isothiocyanate functiona lity was presumed to be present and available for acylation by IN nucleophi les, they were equally effective against Cys to Ser mutants. One conclusion of these studies. is that upon binding of CAFE 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 rea ctive functionality, such as isothiocyanate groups. The fact that introduct ion of the isothiocyanate group onto various positions of the phenethyl rin g or replacement of the phenyl ring with naphthyl rings, failed to signific antly affect inhibitory potency, indicates a degree of insensitivity of thi s 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. (C) 2001 Published by Elsevier Science Ltd.