DNA topoisomerases from pathogenic fungi: targets for the discovery of antifungal drugs

Abstract

(11,19,20,36,60DNA break to form a gate allowing subsequent DNA strand passing. Effective topoisomerase inhibitors stabilize the intermediate, termed the cleavage complex, and this stabilization in the cell triggers an unknown process causing cell death (7,30). The stabilization of the cleavage complex by topoisomerase inhibitors thus converts these enzymes into cellular poisons (28). The strategy of discovering therapeutic agents of this class is, therefore, plausible and easily justifiable because of the fact that the enzyme need not be proven to be essential to the viability of that particular cell. In principle, any cell which contains high levels of a topoisomerase is vulnerable to killing by topoisomerase inhibitors capable of stabilizing the complex.Candida albicans andAspeigilus niger are two important, life-threatening systemic fungal pathogens in individuals with impaired immunity, such as transplant recipients, patients with AIDS, and patients with cancer undergoing chemotherapy. More effective and safe antifungal agents are urgently needed for the treatment of these increasingly important opportunistic infections. Our results showed that both types of topoisomerases exist in abundance in these pathogenic fungi, indicating that the enzymes are potential targets for the discovery of fungicidal agents. By using isolated C. albicans DNA topoisomerase II and DNA breakage assays, we have further found that the responses of the fungal and the mammalian topoisomerase II to topoisomerase poisons differ in magnitude, suggesting that the discovery of specific inhibitors safe for the host is feasible. MATERIAL1 AND METHODSChemicals and reaents. Agarose (type II), Tris, EDTA, dimethyl sulfoxide, and amiloride were from Sigma. EcoRI

Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast

“…These results are in agreement with previous studies using a partially purified topoisomerase II preparation from C. albicans and calf thymus topoisomerase II as a representative mammalian enzyme [26]. Differential DNA cleavage was reported for four compounds, and similar or lower levels of DNA breakage were observed for the fungal enzyme in most cases, e.g.…”

“…Purification of C. albicans topoisomerase I and its inhibition by aminocatechols have been reported and the corresponding TOP1 gene has been characterized [23][24][25]. In the case of C. albicans topoisomerase II, only one paper has appeared that describes a partial purification procedure [26]. By using this preparation in parallel with mammalian topoisomerase II, the authors identified one compound, a quinolone derivative, that preferentially inhibited the fungal enzyme.…”

Relationship of the Glyoxylate Pathway to the Pathogenesis of Cryptococcus neoformans

“…Fifth, there are already a series of genes in C. neoformans associated with infection and their quantitative and qualitative impact on virulence has been described through gene disruption experiments. These genes include those for the following: capsule formation (8)(9)(10)(11)27), melanin production (20,31,49), high-temperature growth (36,48), purine metabolism (47), myristylation (32), topoisomerase (54), signal transduction (2,3,12,18,41,55), alpha-mating type locus (30), phospholipase (13), urease (14), and mannosylation (57). In addition to a single gene disruption strategy, a more global approach has used signature-tagged mutagenesis to identify virulence genes (40).…”

In vitro Synergistic Effects of Anthracycline Antitumor Agents and Fluconazole Against Azole-Resistant Candida albicans Clinical Isolates

“…Several studies have suggested that DNA topoisomerase is a potential target of antifungal agents [12][13][14][15]16] found that the DNA topoisomerase inhibitor aclarubicin at 0.8-7.3 µg/mL inhibited the growth of C. albicans in vitro, whereas other inhibitors, including daunorubicin, doxorubicin, idarubicin, beta-lapachone, camptothecin, irinotecan, topotecan, etoposide, and mitoxantrone, did not inhibit the growth. Nevertheless, the first four of these compounds affected the morphology of C. albicans.…”

“…Topoisomerase is an enzyme that plays roles in the processes of DNA cleavage and recombination and is divided into two classes, I and II, based on the mode of DNA cleavage. Topoisomerase I cleaves one strand of a duplex DNA molecule, whereas topoisomerase II cleaves both strands of the DNA molecule [12]. Topoisomerase inhibitors are further classified into four classes chemically: camptothecins, anthracyclines, epipodophyllotoxins, and quinolones.…”