In natural products drug discovery, the challenge of identifying novel chemotypes is met through developing novel and unique sourcing strategies coupled with success in isolation and structural characterization of novel compounds from these sources. However, the chal- lenge of obtaining a truly interesting biologically active compound, especially one that is likely to be nontoxic, begins with the biological assay design and is improved upon with lead selection and optimization studies. The fundamental problem in antifungal drug dis- covery, as noted earlier, is the similarity in the cell systems, leading to problems in obtaining
©2000 by CRC Press LLC
selective and nontoxic compounds. Furthermore, this disease state is further complicated by the fact that it occurs only in the immunocompromised host; thus patients cannot rely on their immune systems to assist in clearing the invading organisms from the body (as is the case in the immunocompetent host). Thus, the two major problems to overcome are (1) both cell types (host and pathogen) are eukaryotic, leading to greater likelihood of tox- icity to the host, and (2) it is unlikely that a microbiological cure will be achieved in an immunocompromised host.
In considering how to meet these challenges, general approaches to controlling infectious diseases were considered, and several points of prevention or intervention could be identified:
• Preventative
– Personal preventative good health (e.g., good immune status) – Public health (public hygiene, immunizations)
– Prophylactic drug treatment • Curative
– Restoration of good health (eliminate the cause of the immunosuppression) – Therapeutic drug treatment
As noted earlier, significant difficulties and challenges are encountered when consider- ing these approaches as applied to the circumstance of an immunocompromised host. For example, personal preventative good health is not an option for patients with AIDS or can- cer, where a severe, incurable disease is the underlying cause of the immunosuppression. Likewise, public health measures are not relevant, since there are no immunizing biologics or public hygiene measures that could control disseminated fungal infections. The use of a therapeutic agent for prophylaxis of infection, as noted earlier, may select for resistant strains or nonsusceptible minor species. To affect a cure, the cause of the immunosuppres- sion must be eliminated (restoration of the good health of the patient), or there must be a therapeutic agent that can affect at least a clinical, if not microbiological, cure. It is not cur- rently possible to remove the source of immunosuppression in patients with AIDS, and while this may be possible with patients with cancer (i.e., cease anticancer drug treatment), it leaves the patient at risk for worsening of the underlying condition, which itself may also be a cause of immunosuppression. The shortcomings of the various therapeutic drug treat- ments have already been cited and provide strong justification for an innovative approach to the discovery of new drugs to control disseminated fungal infections. Toward that goal, we began to question how one might alternatively approach the discovery of new antifun- gal agents that could be useful in preventing the pathogenecity of the fungal pathogen. In other words, if the good health or immune status of the patient cannot be restored, can the pathogenicity of the microorganism be destroyed?
There are a growing number of reports regarding the role of various virulence factors in the pathogenesis of a variety of infectious diseases, including the opportunistic fungal pathogens. A virulence factor is a substance produced by the pathogen to enhance its abil- ity to establish infection, spread, and/or evade host defenses. Since the major fungal OI pathogens are ubiquitous in the environment, exposure of immunocompromised patients to these organisms cannot reasonably be prevented. However, the prevention of the devel- opment of invasive systemic mycoses is a major therapeutic strategy in such patients,27 and
with the development of resistant strains the search for new prophylactic therapies is gain- ing increasing importance. In the search for prophylactic agents, a reasonable approach is to identify known virulence factors that contribute to invasiveness of the organism and
©2000 by CRC Press LLC
search for agents that counter the effects of such factors. The rationale of this approach is that interference with the production or function of a virulence factor should mitigate dis- ease progression and could, therefore, be the functional equivalent of restoring the immune status of the patient. Virulence factors produced by the opportunistic fungal pathogens include the secreted aspartic proteases of Candida species,28-32 phenoloxidase produced by Cryptococcus neoformans,33,34 the polysaccharide capsule of Cryptococcus,34 and the iron-scav-
enging siderophores produced by Candida, Cryptococcus, Histoplasma, and Aspergillus.35-37
As part of a National Cooperative Drug Discovery Group for Opportunistic Infections (NIH-UO1-35203), we undertook a program to identify novel natural product-derived inhibitors of several important fungal virulence factors and to validate the utility of these agents or their derivatives as prophylactic drugs to control the progression of disease in immunocompromised hosts.
7.4.1 Secreted Acid Proteases of Candida albicans
The secreted acid proteases of C. albicans appeared to us to be an important target for the discovery of novel antifungal compounds. Although its specific function in pathogenesis has not yet been established, this enzyme is believed to be an important virulence factor.28-32
It has been reported that the protease from Candida destroys certain tissues and antibodies and that pepstatin-A, a protease inhibitor, alters the adherence and invasiveness of
C. albicans in the epithelium.41 Moreover, the administration of pepstatin prior to infection
of mice with C. albicans reduced the mortality rate and a protease-deficient mutant of
C. albicans had lower virulence for mice and was more readily phagocytized by polymor-
phonuculear leukocytes than the protease-positive strain.41 Thus, control of the acid pro-
tease of Candida could provide a means to control the colonization of Candida and therefore limit the spread of the organism; i.e., protection against invasive systemic candidiasis seems possible with a selective protease inhibitor. Furthermore, it is speculated that the inhibition of growth of Candida is not an absolute requirement for therapeutic potential (although it would obviously be an added benefit). Even in the absence of intrinsic activity, such an agent may find use in combination with other inhibitory compounds such as a polyene antibiotic or an azole.
To determine if higher plants produce nonpeptide inhibitors of Candida-secreted aspartic proteases (SAPS), it was necessary to develop a sensitive and specific primary bioassay suit- able for screening large numbers of plant extracts, to have access to a unique and diverse plant collection to evaluate for the presence of inhibitors, and to have available purified recombinant Saps for use in a secondary assay to corroborate any observed activity.
A simple and rapid fluorometric assay based on the method reported by Capobianco et al.38 was established as a primary screen for crude plant extracts that inhibit protease.
This assay uses a fluorogenic decapeptide substrate initially designed for use with the human aspartic protease renin, but suitable for detecting the rapid appearance of SAP activity in cultures of C. albicans induced to secrete the proteases by providing protein (bovine serum albumin) under limited-nitrogen conditions. The assay is sensitive, amena- ble to high-throughput applications and the substrate is commercially available (Molecular Probes, Inc., Eugene, OR); therefore, it is useful as a primary assay for screening plant extracts.
The assay was used to screen over 215 randomly selected extracts of plants from the National Center for the Development of Natural Products repository of plant extracts (half from South America and the other half from the U.S.). Plant extracts that inhibited the increase in fluorescence by Š50% were considered positive. Seven extracts (3% total)
©2000 by CRC Press LLC
showed moderate to excellent inhibition in this primary assay (>50% inhibition of SAP activity). A secondary assay to determine the effects of extracts and compounds on specific recombinant Saps was developed by our collaborators on this project, Dr. Jordan Tang, Dr. Xinli Lin, and Dr. Gerald Koelsch (Oklahoma Medical Research Foundation, University of Oklahoma). The advantage of screening inhibitory activity against individual Saps is that the differences in specificity may be exploited. There was good correlation of activity between the primary and secondary assays, and bioassay-directed fractionation of several active plants is in progress (results of these studies will be reported elsewhere).
7.4.2 Phenoloxidase Inhibitors
Phenoloxidase, an enzyme produced by Cryptococcus neoformans that catalyzes the conver- sion of phenolic compounds to the pigment melanin, is considered to be an important vir- ulence factor for this pathogen.33 Interest in phenoloxidase activity in Cryptococcus has
previously been associated mostly with its application as a diagnostic and identification tool, since neoformans is unique among the Cryptococcus species in its melanin-pigment pro- duction.34 Growth of C. neoformans on phenol-containing agar results in the development
of dark brown colonies when phenoloxidase polymerizes the phenols to melanin. Kwon- Chung and Rhodes34 studied the relationship of phenoloxidase activity and encapsulation
and their effects on virulence. The ability to produce melanin (as a result of phenoloxidase activity) and the polysaccharide capsule were both demonstrated to be critical virulence factors. With this in mind, we undertook a study to determine if extracts of higher plants contain inhibitors of melanin production. As was the case with the Candida proteases, a simple in vitro assay to detect potential inhibitors was developed following methods pre- viously published.39
In this case, the assay relies on visual assessment of reduced melanin production vs. direct antifungal effect when C. neoformans is grown on phenol-containing agar medium in the presence of extracts or compounds. Inhibitors of melanin production will cause the col- onies surrounding the sample to remain white, creating an “albino” zone of growth around the sample. A total of 216 randomly selected samples (66 plant extracts; 150 pure com- pounds) from the NCDNP Repository were evaluated. Of the total 216 samples evaluated, 13 showed activity (4 plant extracts, 6%; 9 pure compounds, 6%). Further studies are needed to establish if the observed inhibition of melanin production is due to direct inhibi- tion of phenoloxidase activity or to other effects (e.g., interference with transport or chem- ical interactions), and efforts are underway to purify cryptococcal phenoloxidase for this purpose (results to be published elsewhere).