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Role of Heme in the Antifungal Activity of the Azaoxoaporphine Alkaloid Sampangine ^,

Ameeta K. Agarwal,^1,* Tao Xu,^1, Melissa R. Jacob,¹ Qin Feng,¹ Michael C. Lorenz,² Larry A. Walker,^1,3 and Alice M. Clark^1,4

National Center for Natural Products Research,¹ Department of Pharmacology,³ Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, Mississippi 38677,⁴ Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, Texas 77030²

Received 30 August 2007/ Accepted 2 December 2007

Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling experiments coupled with analyses of mutants were performed in an effort to elucidate its mechanism of action. Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low-oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the hem1 mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analyses of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed with C. albicans to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles, suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.

Published ahead of print on 21 December 2007.

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A.K.A. and T.X. contributed equally to this work.