Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases

Abstract

The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 g/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets.Candida albicans and Cryptococcus neoformans are common causes of life-threatening fungal infections in immunocompromised patients. Increased incidence of aspergillosis also contributes to prevalent mycosis in neutropenic patients. Both drug resistance and toxicity are associated with existing antifungal therapies, and there is a need for new broad-spectrum antifungal drugs to more efficiently manage systemic fungal infections. Several potential new antifungal targets are being investigated in a search for novel drugs with reduced toxicity and less likelihood of resistance.DNA topoisomerases are the targets of a number of antibacterial and anticancer chemotherapy agents, such as fluoroquinolones, pentamidines, acridines, camptothecins, and epipodophyllotoxins (5, 9, 16). Topoisomerases are ubiquitous enzymes that have a pivotal role in the processes of DNA replication, transcription, and recombination. The topological state of DNA is regulated by topoisomerases through the action of breaking and resealing DNA strands (23, 34). These enzymes have been classified into two major classes, based on their mode of cleaving DNA. Topoisomerase I acts by making a transient nick on one strand of duplex DNA molecule and changing the linking numbers in steps of 1. Topoisomerase II acts by transiently nicking both strands of DNA, passing another double-stranded DNA segment through the gap, and changing the linking number in steps of 2. Topoisomerase II can decatenate or catenate duplex DNA and is involved in the separation and resolution of daughter molecule...

Selective inhibition of human leukemia cell growth and induction of cell cycle arrest and apoptosis by pseudolaric acid B

“…Assay for topoisomerase activity Inhibition of topoisomerase I (topo I) and topoisomerase II (topo II) enzyme activities was measured by DNA relaxation assay using topo I and II drug screening kit (Topogen, Columbus, OH) as described earlier (Ma et al 2005;Khan et al 2002). Measurement of the catalytic activity of topo I or II was based on the conversion of supercoiled DNA to relaxed DNA.…”

DNA Topoisomerase I Inhibitory Alkaloids from Corydalis saxicola

“…In this complex, the religation step of the topoisomerase reaction is blocked by the inhibitor. The presence of the stabilized cleavage complex interferes with DNA metabolism and ultimately leads to irreversible DNA damage [33]. There are two general mechanisms of inhibitors of DNA topoisomerase I: direct interference of the inhibitor with the enzyme -DNA complex (stabilization of topoisomerase -DNA cleavable complexes), and non-specific interference resulting from the intercalation of the drug into DNA, leading to hindrance of the relaxation of plasmid DNA by the enzyme.…”

A Route to Azafluoranthene Natural Products Through Direct Arylation

“…Nevertheless, members of the azafluoranthene class of natural products have shown promising biological activity as anti-HIV [6] , antifungal and cytotoxic [5, 16] agents. Recently, the cytotoxic activity of the azafluoranthene eupolauridine was attributed to targeting of DNA topoisomerase II [17] In addition to the biological activities mentioned, the extensive conjugation in molecules containing the azafluoranthene core endow them with interesting spectral properties. For this reason, they have been studied as potentially useful agents in luminescent and electroluminescent applications and devices.…”