Identification of Fungus-Derived Natural Products as New Antigiardial Scaffolds

ABSTRACT There is an unmet need for effective therapies for treating diseases associated with the intestinal parasite Giardia lamblia. In this study, a library of chemically validated purified natural products and fungal extracts was screened for chemical scaffolds that can inhibit the growth of G. lamblia. The phenotypic screen led to the identification of several previously unreported classes of natural product inhibitors that block the growth of G. lamblia. Hits from phenotypic screens of these naturally derived compounds are likely to possess a variety of mechanisms of action not associated with clinically used nitroimidazole and thiazolide compounds. They may therefore be effective against current drug-resistant parasite strains. IMPORTANCE There is a direct link between widespread prevalence of clinical giardiasis and poverty. This may be one of the reasons why giardiasis is a significant contributor to diarrheal morbidity, stunting, and death of children in resource-limited communities around the world. FDA-approved treatments for giardiasis include metronidazole, related nitroimidazole drugs, and albendazole. However, a substantial number of clinical infections are resistant to these treatments. The depth of the challenge is partly exacerbated by a lack of investment in the discovery and development of novel agents for treatment of giardiasis. Applicable interventions must include new drug development strategies that will result in the identification of effective therapeutics, particularly those that are inexpensive and can be quickly advanced to clinical uses, such as products from nature. This study identified novel chemical scaffolds from fungi that can form the basis of future medicinal chemistry optimization of novel antigiardial agents.

IMPORTANCE There is a direct link between widespread prevalence of clinical giardiasis and poverty. This may be one of the reasons why giardiasis is a significant contributor to diarrheal morbidity, stunting, and death of children in resource-limited communities around the world. FDA-approved treatments for giardiasis include metronidazole, related nitroimidazole drugs, and albendazole. However, a substantial number of clinical infections are resistant to these treatments. The depth of the challenge is partly exacerbated by a lack of investment in the discovery and development of novel agents for treatment of giardiasis. Applicable interventions must include new drug development strategies that will result in the identification of effective therapeutics, particularly those that are inexpensive and can be quickly advanced to clinical uses, such as products from nature. This study identified novel chemical scaffolds from fungi that can form the basis of future medicinal chemistry optimization of novel antigiardial agents.
KEYWORDS Giardia lamblia, antigiardial, fungal extracts, pure natural compounds T he intestinal pathogen Giardia lamblia causes morbidity and mortality on a global scale (1). Unfortunately, higher prevalence of clinical giardiasis correlates with lower socioeconomic status, thus inflicting a disproportionate burden on the young, especially in resource-limited countries (1,2). Available treatments are limited, cause side effects, and have an increasing risk of drug resistance, which sets the stage for a greater public health concern (3-5). Furthermore, there are limited prospects for additional treatment options due to a lack of investment in the drug development pipelines. These facts emphasize the need for new drug development strategies to discover effective therapeutics, particularly those that can be quickly moved to clinical use. Bioactive compounds from nature have been optimized by evolution to perform specific biological functions without impairing the producing organism's physiological proficiencies (6,7). This extrapolative safety profile potentially reduces the cost for clinical development, especially for some antimicrobial agents. A significant number of clinically used antimicrobial agents are chemically optimized derivatives of natural products (8). Artemisinin and ivermectin are examples of antiparasitic agents derived from natural products (9,10). Naturally derived molecules are an important, yet relatively underexplored resource for new bioactive chemical scaffolds. The World Health Organization (WHO) has suggested the application of natural products for health care needs since they are widely used, inexpensive, and considered mostly safe (11,12). We report here a phenotypic screening of a natural product library with the aim of identifying compounds that can be optimized as potent antigiardial treatments.
For in vitro Giardia assays, we used a click beetle green (CBG99) luciferase-based reporter system that was developed for quantitative bioluminescence readouts of growth in trophozoites (13)(14)(15). The CBG99 reporter gene is under the control of the glutamate dehydrogenase promoter, which is highly expressed in the G. lamblia trophozoite stage, making it useful for monitoring growth even in mouse infection models (15). The compound library was obtained from the Natural Products Discovery Group (NPDG) at the University of Oklahoma (16). A combination of 755 purified natural products (pNPs) and 1,056 fungal extracts were screened against G. lamblia strain CBG99. Hits were defined as samples that caused $50% growth inhibition activity at 10 mM (pNPs) or 5 mg/mL (extracts). Analysis of the pNP inhibition data showed 38 compounds with $50% inhibition of G. lamblia growth and proliferation. Only 25 of the 38 compounds were further analyzed, since 13 showed signs of cytotoxicity in a counterscreen employing human ileocecal adenocarcinoma cells (HCT-8; ATCC, Manassas, VA) tested at 10 mM. Of the noncytotoxic compounds, 16 had $75% inhibition of G. lamblia trophozoite growth, with one showing $90% inhibition. A summary of the screening data is presented in Table 1. The concentration at which compounds inhibited 50% of G. lamblia trophozoite growth in vitro (EC 50 ) was determined for the 16 noncytotoxic hits with $75% inhibition of G. lamblia trophozoite growth in the primary screening. Only 2 were considered false positives (EC 50 . 10 mM) ( Table 2). The chemical structures of the 16 pNPs are shown in Fig. 1. Four compounds, fumagillin, cis-fumagillin, RES-1149-2, and NPDG 150137, had EC 50 values of ,200 nM against G. lamblia trophozoite and were .25 times more potent than metronidazole, which has an EC 50 of 5 mM (17). As noted, HCT-8 growth and proliferation were not inhibited at 10 mM by those compounds determined to be noncytotoxic, demonstrating that a good safety index could be achieved.
Testing of the extract library against G. lamblia CBG99 identified 87 extracts with $50% inhibition, with 8 of those samples also causing HCT-8 cytotoxicity. Further analysis generated a refined list of 39 noncytotoxic extracts exhibiting $75% inhibition of cell growth, among which 13 showed $90% inhibition, and 4 of these extracts had $95% inhibition. The most potent, noncytotoxic fungal extracts included samples prepared from Pochonia chlamydosporia (with 84% inhibition of Giardia growth), Cytospora sp. (95% inhibition), Scedosporium apiospermum (94%), Penicillium chlamydosporia (95%), Cladosporium cladosporioides (91%), Coniothyrium sp. (91%), Aspergillus fumigatus (95%), Penicillium janczewskii (85%), Penicillium oxalicum (95%), and Penicillium camemberti (92%). None of the listed crude fungal extracts inhibited HCT-8 cells at 40 mg/mL. Since the dearth of antigiardial drug development is associated with limited available resources (13), it is critical that extracts selected represent the best starting points for identifying additional natural product scaffolds with antiparasitic activity and reduced host toxicity, to provide the shortest path to clinical application. The mammalian cytotoxicity of crude extracts was therefore included as a selection criterion, in addition to EC 50 values against G. lamblia.  Extracts from cultures of Penicillium janczewskii were selected based on availability, ease of regrowth, and historical references (18,19). The observed EC 50 value of regrown P. janczewskii crude extract was 0.13 ng/mL, thereby validating its antigiardiasis activity. P. janczewskii is a common filamentous fungus found in nature. The P. janczewskii metabolite, griseofulvin, has been shown to be of particular therapeutic value in the treatment of human skin mycoses (19,20). Further characterization of the antigiardiasis activity and cytotoxicity of P. janczewskii crude extract (J145A) was carried out to find agents with no toxic signals (Fig. 2). J145A and its 3 antigiardial fractions did not inhibit the growth or proliferation of HepG2 and CRL-8155 cells at 40 mg/mL (21).
Highly potent inhibitors of G. lamblia can be obtained from natural products and fungal extracts, thereby expanding the diversity of sources and chemical spaces available for the discovery of new antigiardial therapeutic agents. Validation of the antigiardial effect of some of the active pNPs was provided by the observed inhibition of G. lamblia growth by other compounds with the same core chemical scaffolds ( Fig. 1 and  2). The new chemical scaffolds would be a basis for accelerated development of highly specific antigiardial drugs that utilize new mechanisms of action. Since the new scaffolds discovered in this study are different from the nitroimidazole and thiazolide classes of compounds, there is the possibility that the potential new drugs from the series will be effective against metronidazole-resistant strains. Many of the compounds in this library are not available from commercial sources and were identified as bioactive entities while conducting bioassay-driven discovery projects. Examples include NPDG-150029 and NPDG-150137 (Table 2). Inactive compounds in the extract help to define the SAR of scaffolds. For instance, dechlorogriseofulvin J16A, which is inactive at 15 mg/mL (Fig. 2), helps define likely SAR for griseofulvin analogs. This combination of factors makes the collection especially useful as a unique first-stage screening tool and a highly valuable resource for drug discovery.
Data availability. Data will be made available upon request for peer review.