Interaction of the antileukemic alkaloid, 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[ c]phenanthridine (fagaronine), with nucleic acids

Abstract

The novel alkaloid 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[ c]phenanthridine was isolated from the roots of Fagara zanthoxyloides, an indigenous plant of Ghana, structurally characterized and assigned the trivial name fagaronine. Fagaronine is a potent antileukemic agent. We presently report a detailed isolation procedure and the results of studies designed to examine its mechanism of action. Admixture of calf thymus DNA produced a hypsochromic shift in the absorption spectra of fagaronine, with a remarkable increase in intensity in the range of 400 nm. The extent of the spectral alterations were dependent on the concentration of DNA which was added, and clear isosbestic points, indicative of a single mode of interaction, were generated. As determined by gel filtration column chromatography, the DNA-fagaronine complex formed with an association constant of 4.7 × 10 4 M −1 and the maximum extent of binding was 1.0 fagaronine molecule per 4.5 base pairs. Contrary to previous literature reports, the binding of fagaronine was not limited to regions of DNA containing adenine and thymine. Isosbestic points were observed following admixture with poly-(dA-dT), as well as poly(dG) · poly(dC). The most likely mode of interaction is intercalation. As judged by spectral titrations, fagaronine could also interact with ribonucleic acids. As compared with single-stranded homopolymers [e.g., poly(A), poly(U)], the interaction with double-stranded species [e.g., poly(A) · poly(U), poly(I) · poly(C)] was more extensive. Although the spectra differed from those obtained with deoxyribonucleic acids, single isosbestic points could be generated. Additionally, utilizing Salmonella typhimurium strain TM677, fagaronine was found to be bactericidal but not mutagenic. The bactericidal activity was enhanced about 1000-fold in the presence of a 9000 × g supernatant fraction derived from rat liver and the activating component was further characterized as being heat labile and localized in the microsomal subfraction. Preliminary investigations did not support the notion that fagaronine was metabolized to an active species responsible for this effect. When KB cells were treated with fagaronine, it was also found to be cytotoxic. In contrast, N-demethylfagaronine was not bactericidal or cytotoxic, nor could it effectively interact with DNA. Thus, the cationic (quaternary) nitrogen atom of fagaronine is of requisite importance, and a key mechanism of cytotoxic activity may be interaction with cellular nucleic acids.