Fungal Topoisomerases as Targets for Antifungal Drug Discovery

Shen, Linus

Abstract

DNA topoisomerases, a class of enzymes that change the topological structure of cccDNA through a concerted DNA breaking, passing and resealing process, have been shown to be the target of many therapeutic agents including antibacterial agents (quinolones) and anticancer agents (camptothecin, m-AMSA, ellipticines, adriamycin, etoposide etc.). These drugs inhibit the enzyme in a unique way that the enzyme and the drug are converted into a poison resulting in cell killing. The key event of such deleterious effect to the cell is the formation of a cleavable complex that triggers certain unknown cellular processes causing cell death. The strategy of discovering therapeutic agents of this class is plausible due to the fact that the enzyme need not be essential to cell viability. In principle, any cells having a high level of topoisomerase are vulnerable to the killing of such inhibitors. Our preliminary results using Candida albicans and Aspergillus niger show that these fungi have high levels of both type I and type II topoisomerases. Therefore, finding inhibitors to these enzymes will very possibly lead to the discovery of potent antifungal agents.
The specific aims of this project are: (1) to purify topoisomerases from pathogenic fungi, (2) to characterize their structural and catalytic properties, and to determine the differential response to various topoisomerase inhibitors between the fungal and the mammalian enzymes, (3) to develop screening methods for discovering new structural types of fungal topoisomerase inhibitors, and (4) to study the inhibition mechanism of active leads and to guide future synthetic efforts. The topoisomerase will be purified from fungi (Candida albicans, Aspergillus niger) using a combination of hydroxylapatite, ion-exchange, or hydrophobic chromatographic procedures. Purification to near homogeneity may be achieved using blue-agarose, DNA-cellulose, or novobiocin-Sepharose affinity chromatography. The activity of type I and II enzymes at each purification step will be monitored with specific assays already developed in our laboratory. The type of purified enzyme will be verified by the linking number change test. A microtiter SDS-KC1 precipitation method will be developed as primary screen for compounds that stabilize the enzyme-DNA complex. The potency of inducing enzyme-dependent DNA cleavage, a criterion that likely determines the therapeutic value of the inhibitor, will be first evaluated by a convenient unlabeled DNA method. The detailed mapping of the cleavage products of active compounds will then be examined by using the end-labeled DNA. The active leads will be studied for their interactions with the purified enzyme, DNA and enzyme-DNA complex. Results from such mechanistic studies will guide future synthetic efforts. These studies will allow us to discover potent fungal topoisomerase inhibitors that have potential to be effective antifungal agents.