Characterization of DNA topoisomerase I from Candida albicans as a target for drug discovery

Abstract

Candida albicans is an opportunistic pathogen responsible for life-threatening infections in persons with impaired immune systems. Topoisomerase I is a potential target for novel antifungal agents; however, in order for this enzyme to be a therapeutically useful target, it needs to be demonstrated that the fungal and human topoisomerases differ sufficiently as to allow the fungal topoisomerase to be selectively targeted. To address this question, we isolated the topoisomerase I from C. albicans and compared its biochemical properties with those of the mammalian enzyme. Similar to other eukaryotic type I topoisomerases, the C. albicans type I topoisomerase has an apparent molecular mass of 102 kDa and covalently links to the 3' end of DNA, as shown after the reaction is interrupted by sodium dodecyl sulfate. Topoisomerase poisons such as camptothecin act by stabilizing the cleavage complex formed by the topoisomerase I and DNA. We observed that the C. albicans and mammalian type I topoisomerases differ in that the C. albicans cleavage complex is approximately 10-fold less sensitive to camptothecin than the mammalian cleavage conmplex is. In addition, we found that the antifungal agent eupolauridine can stabilize the cleavage complex formed by both the C. albicans and human topoisomerases and that the response of the C. albicans topoisomerase I to this drug is greater than that of the human enzyme. Thus, the topoisomerase I from C. albicans is sufficiently distinct from the human enzyme as to allow differential chemical targeting and will therefore make a good target for antifungal drug discovery.Topoisomerases are enzymes which modulate the topological structure of DNA (for recent reviews, see references 7, 20, and 30). Topoisomerases are able to relax supercoiled DNA and unlink catenated DNA circles, among other functions. These enzymes perform these reactions by making a transient break in the DNA backbone, passing another segment of DNA through this break, and then resealing the break. Type I topoisomerases break only one DNA strand, while type II topoisomerases can introduce a double-strand break into DNA. In the eukaryotic cell, type I topoisomerases are associated with elongating transcription and replication forks, while type II topoisomerases are required for segregation of daughter chromosomes after cell division (1,12,13,18,34,37).Topoisomerases form a complex with DNA in which the transiently broken ends of the DNA are constrained. When a protein denaturant is added, a DNA break is revealed, and the denatured topoisomerase is found to be covalently attached to one end of the break (5,11,16,33,35). This complex between topoisomerase and DNA has been termed the cleavage complex (26; reviewed in reference 23). Certain drugs stabilize the cleavage complex, such as the anticancer drug camptothecin, which targets the eukaryotic topoisomerase I (19). A structure-activity relationship for camptothecin derivatives has been established, and a correlation is seen between the cytotoxicity of the compound in replic...

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.…”

“…Of these, camptothecin targets topoisomerase I, whereas the others target topoisomerase II. Several studies suggested that topoisomerase might be a target for antifungal drugs [13][14][15]. As an example, aclarubicin, an anthrax cycline antitumor agent, inhibited the growth of C. albicans at low concentrations [16].…”

Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases

“…Fungal topoisomerase I was purified from C. albicans as described previously (11). The specific activity of the enzyme preparation was 32,000 U/mg.…”

“…Eupolauridine was reported by Fostel et al to stabilize the cleavage complex with selectivity for Candida topoisomerase I, while camptothecin showed selectivity for human topoisomerase I (11). Fostel et al did not determine the effect of eupolauridine on catalytic activity of the enzyme (11).…”

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

“…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].…”