Search

Curcumin (diferuloylmethane) is the yellow pigment in turmeric (Curcuma longa L.) that is widely used as a spice, food coloring (curry) and preservative. Curcumin exhibits a variety of pharmacological effects including antitumor, anti-inflammatory, and anti-infectious activities and is currently in clinical trials for AIDS patients. The effects of curcumin have been determined on purified human immunodeficiency virus type 1 (HIV-1) integrase. Curcumin has an inhibitory concentration50 (IC50) for strand transfer of 40 microM. Inhibition of an integrase deletion mutant containing only amino acids 50-212 suggests that curcumin interacts with the integrase catalytic core. Two structural analogs, methyl cinnamate and chlorogenic acid, were inactive. Energy minimization studies suggest that the anti-integrase activity of curcumin could be due to an intramolecular stacking of two phenyl rings that brings the hydroxyl groups into close proximity. The present data suggest that HIV-1 integrase inhibition may contribute to the antiviral activity of curcumin. These observations suggest new strategies for antiviral drug development that could be based upon curcumin as a lead compound for the development of inhibitors of HIV-1 integrase.

The inhibition of HIV-1 integrase by flavones and related compounds was investigated biochemically and by means of structure-activity relationships. Purified enzyme and synthetic oligonucleotides were used to assay for three reactions catalysed by integrase: (1) processing of 3' termini by cleavage of the terminal dinucleotide; (2) strand transfer, which models the integration step; and (3) "disintegration," which models the reversal of the strand transfer reaction. Inhibitions of all three reactions by flavones generally occurred in parallel, but caffeic acid phenethyl ester (CAPE) appeared to inhibit reaction 2 selectively. CAPE, however, inhibited reactions 1 and 3 effectively when preincubated with the enzyme, suggesting that this compound differs from the flavones primarily in requiring more time to block the enzyme. The core integrase fragment consisting of amino acids 50-212 retained the ability to catalyse reaction 3, and flavones and CAPE retained the ability to inhibit. Hence, the putative zinc-finger region that is deleted in this fragment is probably not the target of inhibition. Inhibition by flavones usually required the presence of at least one ortho pair of phenolic hydroxyl groups and at least one or two additional hydroxyl groups. Potency was enhanced by the presence of additional hydroxyl groups, especially when present in ortho pairs or in adjacent groups of three. Inhibitory activity was reduced or eliminated by methoxy or glycosidic substitutions or by saturation of the 2,3 double bond. These structure-activity findings for flavones were generally concordant with those previously reported for reverse transcriptase and topoisomerase II. These findings are discussed in the context of a review of the effects of flavones on various enzymes, the possible mechanisms of inhibition, and the potential for building upon a general pharmacophore to generate target specificity.

The anticancer drug 4′-(9-acridinylamino)-methanesulfon- m -anisidide (m-AMSA) is known to bind to DNA by intercalation and to produce protein-associated DNA strand breaks in cells. Previous work [Zwelling et al., Biochemistry 20 , 6553 (1981)] had shown that m-AMSA is in rapid equilibrium between extracellular and intracellular compartments, and that the DNA strand breaks exist in a steady state of rapid formation and resealing. The current work reports an unusual uptake phenomenon of m-AMSA by mouse leukemia L1210 cells that occurs at higher drug concentrations than previously studied. The new uptake phenomenon was characterized by cooperativity, hysteresis, irreversibility, saturability, slowness and temperature dependence. It is concluded that m-AMSA concentrations above a critical value can initiate the irreversible sequestration of m-AMSA into a new phase, probably in an extranuclear compartment of the cell, from which the drug has no access to the nuclear DNA and probably does not contribute to cytotoxicity.

An attempt was made to analyze the mechanism by which 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits mammalian DNA topoisomerase II. The effects of various 9-aminoacridine derivatives differing by their DNA affinities and DNA sequence selectivity of binding were compared in the presence of purified mouse leukemia L1210 DNA topoisomerase II. No correlation was found between DNA unwinding and topoisomerase II inhibition. 9-Aminoacridine was inactive as a topoisomerase II inhibitor and o-AMSA was only weakly active. The location of L1210 topoisomerase II mediated DNA breaks produced in the absence or presence of 9-aminoacridines were studied in [32P]-end-labeled pBR 322 DNA. All 9-aminoacridines, even those differing by their DNA sequence selectivity of binding, produced similar DNA cleavage patterns. Most drug-induced topoisomerase II mediated DNA breaks appeared at sites that were already cleaved by the enzyme in the absence of drug. The present results suggest that 9-aminoacridines inhibit L1210 DNA topoisomerase II by interacting at or near enzyme-DNA complexes by some unknown DNA effect or by direct protein interaction.

DNA intercalating agents, including ellipticine, had been found previously to produce protein-associated DNA single-strand breaks and double-strand breaks in mammalian cells. The relationship between these effects on DNA and cytotoxicity could not be determined reliably for ellipticine, because of the poor solubility characteristics of this compound. Studies were therefore carried out using the cationic derivative, 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+), which has adequate water solubility and retains antitumor activity. DNA single-strand breaks (SSB) and DNA-protein crosslinks (DPC) were measured using the alkaline elution (pH 12) technique, and double-strand breaks (DSB) were measured by the neutral elution (pH 10) method. The effects of ellipticine and 2-Me-9-OH-E+ were compared in mouse leukemia L1210 cells. Like ellipticine, moderate concentrations of 2-Me-9-OH-E+ produced protein-associated SSB, indicated by the appearance of SSB and DPC at approximately equal frequencies and localized with respect to each other. Below 20 μM (1-hr treatments), the effects of the two drugs were comparable in magnitude. At higher concentrations, ellipticine produced extensive DNA breakage not associated with protein; this is perhaps secondary to an action on membranes or other non-DNA targets. However, 2-Me-9-OH-E+ produced only protein-associated strand breaks, even at 4-fold higher concentrations. The two compounds produced similar and relatively large extents of double-strand scission. The measured DSB/SSB ratio was higher than that produced by X-ray or certain other intercalators that have been similarly studied. The DNA effects of 2-Me-9-OH-E+, unlike those of ellipticine, were readily reversible and, therefore, permitted a meaningful correspondence between the magnitudes of the DNA effects and the inhibition of colony-forming ability. Comparison with two other types of intercalating agents indicated that neither the SSB nor the DSB predicts the magnitude of cell killing.