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Antimicrobial Agents and Chemotherapy, June 2002, p. 1785-1792, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1785-1792.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases

Shabana I. Khan,¹ Alison C. Nimrod,^1, Mohammed Mehrpooya,² John L. Nitiss,² Larry A. Walker,^1,3 and Alice M. Clark^1,4*

National Center for Natural Products Research,¹ Department of Pharmacognosy,⁴ Department of Pharmacology, The University of Mississippi, University, Mississippi 38677,³ Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105²

Received 23 July 2001/ Returned for modification 18 September 2001/ Accepted 11 March 2002

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 µg/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.

Present address: Eli Lilly and Co., Indianapolis, IN 46285.