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 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.
Zhang
X
X
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 376, Boyles Street, NCI-FCRDC, PO Box 13, Frederick, MD 21702-1201, USA.
Neamati
N
N
Lee
Y K
YK
Orr
A
A
Brown
R D
RD
Whitaker
N
N
Pommier
Y
Y
Burke
T R
TR
D019943:Amino Acid Substitution; D001483:Base Sequence; D002109:Caffeic Acids / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004952:Esters; D019427:HIV Integrase / Q000737:chemistry / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology*; D017879:Isothiocyanates / Q000737:chemistry*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009838:Oligodeoxyribonucleotides; D012694:Serine / Q000737:chemistry; D016339:Spectrometry, Mass, Fast Atom Bombardment
D002109:Caffeic Acids; D004952:Esters; D017879:Isothiocyanates; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D012694:Serine; D019427:HIV Integrase; D003545:Cysteine