Iron is required for growth by almost all microorganisms, including the pathogenic fungi. In the human body, iron is abundant but unavailable; i.e., it is located intracellularly as haem or ferritin or, when extracellular, is bound tightly to transferrin and lactoferrin. Therefore, pathogenic microorganisms growing in the human host must possess a means of scavenging iron. One such means is the synthesis of compounds known as sidero- phores.35,36 These are typically low-molecular-weight compounds that are released extra-
cellularly by the microbe, complex with extracellular iron(III), and reenter the cell to release
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the iron. Two major chemical types of siderophores have been identified: phenolates and hydroxamic acids.36 Recent studies have shown that all of the major AIDS-related opportu-
nistic fungal pathogens secrete siderophores, mostly of the hydroxamate type35; however,
some isolates appear to be capable of simultaneous production of both phenolate and hydroxamate siderophores.36 The production of a phytosiderophore has also been noted,41
leading us to speculate that other siderophore-type compounds (and/or their precursors) are present in higher plants. Therefore, inhibitors of siderophore synthesis or function might reasonably be expected to be present in higher plants. Miller and colleagues40 have
reported a simple assay to test for siderophore production in yeasts such as C. albicans, which fails to grow on deferrated agar medium. However, when a paper disk saturated with a hydroxamate siderophore is placed on the surface of the seeded agar plate, a zone of growth is observed around the disk. Such an assay can be used to identify plant extracts containing inhibitors of siderophore production or function.
7.6
Summary
Natural product drug discovery is a multistep, integrated process, requiring many key decisions, including selection of targets relevant for the therapeutic end point, choice of compounds for testing, the challenges of purification and structure elucidation, pharmaco- logical and toxicological characterization, formulation and other preclinical work required to move a lead compound to drug-candidate status. These efforts are aimed ideally at iden- tification of novel prototype compounds for new drug classes, preferably with new mech- anisms of action. Because of continual emergence of resistant organisms, these considerations are especially critical to the realization of major advances in the therapies available for the treatment of infectious diseases.
Chemotype novelty is best achieved through innovative sourcing strategies, coupled with effective dereplication. With the explosive growth in our understanding of microbio- logical (e.g., molecular biology, pathogenic mechanisms, or ultrastructural organiza- tion/function), as well as host systems, the opportunity for selection of novel targets for drug discovery is rapidly expanding. Critical to this is validation of the relevance of the tar- gets selected and development/adaptation of an efficient biological screen.
Among such novel targets are microbial virulence factors. An abundant literature sup- ports the concept that recently identified virulence factors can serve as potential drug tar- gets. For example, the SAPS of Candida species appear to be important for tissue invasion and dissemination of the organism within the mammalian host. In the case of Cryptococcus, the enzyme phenoloxidase appears to play an important role in pathogenicity, perhaps by protecting the organism against host chemical defense mechanisms. And for both of these fungi and others, extracellular release of siderophores allows assimilation of iron, an essen- tial nutrient, from host tissues.
The capacity of higher plants to produce inhibitors of fungal virulence factors has been reported, but not widely exploited as sources of new drugs. Presumably these inhibitors function to protect the plants from fungal pathogens encountered in the environment, and thus would be expected to have potential utility as antifungal drugs. Future studies will explore these sources for discovery of new inhibitors for the mentioned targets, as well as others that might be identified. Furthermore, in vivo evaluation of specific inhibitors already identified will be aimed at characterizing their effects on disease progression in animal models.
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ACKNOWLEDGMENTS: This work was supported by a grant from the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Division of AIDS, NIH UO1-AI- 32485, “Novel Approaches to Therapies for AIDS-Related OI.”
The authors would also like to acknowledge the contributions of Drs. Jordan Tang, Xingli Lin, and Gerald Koelsch of the Oklahoma Medical Research Foundation, and Drs. Hala ElSohly, David Pasco, Alison Nimrod, Xing-Cong Li, Deanna Hatch, and Leslie Rutherford of the National Center for the Development of Natural Products, all investigators in research projects alluded to in this chapter. Also gratefully acknowledged is the help of Meridith Wulff and Dr. Troy Smillie in preparation of this manuscript.
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