[show abstract][hide abstract] ABSTRACT: Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a β,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfey's method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 μg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 μg/mL) and Trichophyton mentagrophytes (MIC 4 μg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.
Journal of Natural Products 03/2013; 76(3):334-45. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: In a whole-cell mechanism of action (MOA)-based screening strategy for discovery of antifungal agents, Candida albicans was used, followed by testing of active extracts in the C. albicans fitness test (CaFT), which provides insight into the mechanism of action. A fermentation extract of an undescribed species of Metulocladosporiella that inhibited proteasome activity in a C. albicans fitness test was identified. The chemical genomic profile of the extract contained hypersensitivity of heterozygous deletion strains (strains that had one of the genes of the diploid genes knocked down) of genes represented by multiple subunits of the 25S proteasome. Two structurally related peptide aldehydes, named fellutamides C and D, were isolated from the extract. Fellutamides were active against C. albicans and Aspergillus fumigatus with MICs ranging from 4 to 16 μg/mL and against fungal proteasome (IC₅₀ 0.2 μg/mL). Both compounds showed proteasome activity against human tumor cell lines, potently inhibiting the growth of PC-3 prostate carcinoma cells, but not A549 lung carcinoma cells. In PC-3 cells compound treatment produced a G2M cell cycle block and induced apoptosis. Preliminary SAR studies indicated that the aldehyde group is critical for the antifungal activity and that the two hydroxy groups are quantitatively important for potency.
Journal of Natural Products 07/2011; 74(8):1721-30. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several potent inhibitors of squalence synthetase have been discovered. Zaragozic acid A is produced by several fungi; zaragozic acid B is produced by several strains of Sporormiella intermedia; zaragozic acids C, E, and F are produced by Leptodontidium elatius; zaragozic acids D and D2 are produced by Amauroascus niger. L-731,120 and L-731,128 are minor components and coproduced with zaragozic acids A and B, respectively. Viridiofungins A, B, and C are produced by Trichoderma viride. Viridiofungin A is also produced by an unidentified sterile fungus. Several of the zaragozic acids are also potent inhibitors of farnesyl-protein transferase (FPTase). Inhibitors of FPTase may act as potential anticancer drugs. Chaetomellic acids A and B are produced by a fungus, Chaetomella acutiseta, while fusidienol is produced by Fusidium griseum. All three compounds are potent inhibitors of FPTase. Our experiences suggest that many novel inhibitors of both squalene synthase and FPTase are produced within a diverse phylogenetic array of filamentous fungi. Several of the zaragozic acids are potent inhibitors of both FPTase and squalene synthases. This is consistent with our observations that zaragozic acids and chaetomellic acids share some structural similarity. Key words: natural inhibitors, squalene synthase, farnesyl-protein transferase.
Canadian Journal of Botany 03/2011; 73:898-906. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Starting with the discovery of penicillin, the pharmaceutical industry has relied extensively on natural products (NPs) as an unparalleled source of bioactive small molecules suitable for antibiotic development. However, the discovery of structurally novel and chemically tractable NPs with suitable pharmacological properties as antibiotic leads has waned in recent decades. Today, the repetitive "rediscovery" of previously known NP classes with limited antibiotic lead potential dominates most industrial efforts. This limited productivity, exacerbated by the significant financial and resource requirements of such activities, has led to a broad de-emphasis of NP research by most pharmaceutical companies, including most recently Merck. Here we review our strategies--both technological and philosophical--in addressing current antifungal discovery bottlenecks in target identification and validation and how such efforts may improve NP-based antimicrobial discoveries when aligned with NP screening and dereplication.
[show abstract][hide abstract] ABSTRACT: Sordarins are a class of natural antifungal agents which act by specifically inhibiting fungal protein synthesis through their interaction with the elongation factor 2, EF2. A number of natural sordarins produced by diverse fungi of different classes have been reported in the literature. We have run an exhaustive search of sordarin-producing fungi using two different approaches consecutively, the first one being a differential sensitivity screen using a sordarin-resistant mutant yeast strain run in parallel with a wild type strain, and the second one an empiric screen against Candida albicans followed by early detection of sordarins by LC-MS analysis. Using these two strategies we have detected as many as 22 new strains producing a number of different sordarin analogues, either known (sordarin, xylarin, zofimarin) or novel (isozofimarin and 4'-O-demethyl sordarin). Sordarin and xylarin were the most frequently found compounds in the class. The producing strains were subjected to sequencing of the ITS region to determine their phylogenetic affinities. All the strains were shown to belong to the Xylariales, being distributed across three families in this order, the Xylariaceae, the Amphisphaeriaceae, and the Diatrypaceae. Despite being screened in large numbers, we did not find sordarin production in any other fungal group, including those orders where sordarin producing fungi are known to exist (i.e., Sordariales, Eurotiales, and Microascales), suggesting that the production of sordarin is a trait more frequently associated to members of the Xylariales than to any other fungal order.
Mycological Research 03/2009; 113(Pt 6-7):754-70. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Evaluation of fungal fermentation extracts with whole cell Candida albicans activity resulted in the identification of a novel class of isoxazolidinone-containing metabolites named parnafungins. Chemical-genetic profiling with the C. albicans fitness test identified the biochemical target as inhibition of polyadenosine polymerase, a component of the mRNA cleavage and polyadenylation complex. Parnafungins were discovered from fermentation extracts of fungi resembling F. larvarum isolated from plants, plant litter and lichens. Furthermore authentic strains of F. larvarum var. larvarum and F. larvarum var. rubrum could be induced to produce parnafungins and their degradation products in low titers. Relationships among strains of the F. larvarum complex (FLC), including parnafungin-producing strains, were examined by cladistic analyses of rDNA, mitochondrial rDNA, and two protein-coding genes, comparisons of antifungal activity and antifungal metabolite profiles, and morphological phenotypes. Integrated analyses of these data led to the conclusion that the diversity within the FLC exceeded the one-to-one correspondence between F. larvarum and its teleomorph Cosmospora aurantiicola. Based on multiple gene sequence analyses, strains of the FLC formed a monophyletic clade inclusive of the parnafungin-producing strains. The FLC, including newly discovered parnafungin-producing strains, could be resolved into at least six different lineages, possibly representing cryptic' species, of which one was not fully resolved from F. larvarum var. rubrum. Fusarium larvarum var. rubrum represents a species distinct from var. larvarum. Finally we report that two other species from the Hypocreales, Trichonectria rectipila and Cladobotryum pinarense, are able to produce parnafungins and their open-ring forms.
[show abstract][hide abstract] ABSTRACT: To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).
Journal of the American Chemical Society 01/2009; 130(49):16704-10. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Parnafungins, natural products containing an isoxazolidinone ring, have been isolated from Fusarium larvarum and have been shown to be potent inhibitors of the fungal polyadenosine polymerase. The extraction and analysis of fermentation broths of taxonomically related organisms identified as closely related Fusarium spp. produce not only parnafungin A and B, but also significant quantities of two related components. These members of the paranfungin family of natural products have been isolated and the structure of each has been elucidated. While structurally analogous to parnafungin A, parnafungin C is further elaborated by methylation of a phenolic hydroxyl group, and parnafungin D has both the methyl phenol ether as well as an epoxide in the xanthone ring system. Parnafungin C and D have potent, broad spectrum antifungal activity and also have been shown to target fungal mRNA cleavage and polyadenylation.
[show abstract][hide abstract] ABSTRACT: We isolated a cyclic lipodepsipeptide, phomafungin, from a Phoma sp. The distinct antifungal activity of phomafungin in the crude extract was initially discovered by mechanistic profiling in the Candida albicans fitness test. The purified compound contains a 28 member ring consisting of eight amino acids and a beta-hydroxy-gamma-methyl-hexadecanoic acid, and displays a broad spectrum of antifungal activity against Candida spp., Aspergillus fumigatus and Trichophyton mentagrophytes with MIC of 2-8 microg/ml, and toxicity to mice at 25 mg/kg. The linear peptide derived from opening of the lactone ring was devoid of antifungal activity as well as toxicity. Phomafungin has been identified in a number of Phoma spp. collected from Africa and the Indian and Pacific Ocean islands.
[show abstract][hide abstract] ABSTRACT: A glycosylated tetramic acid, virgineone (1), was isolated from saprotrophic Lachnum virgineum. The antifungal activity of the fermentation extract of L. virgineum was characterized in the Candida albicans fitness test as distinguishable from other natural products tested. Bioassay-guided fractionation yielded 1, a tyrosine-derived tetramic acid with a C-22 oxygenated chain and a beta-mannose. It displayed broad-spectrum antifungal activity against Candida spp. and Aspergillus fumigatus with a MIC of 4 and 16 microg/mL, respectively. Virgineone was also identified in a number of Lachnum strains collected from diverse geographies and habitats.
Journal of Natural Products 01/2009; 72(1):136-41. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Candida albicans Fitness Test, a whole-cell screening platform, was used to profile crude fermentation extracts for novel antifungal natural products with interesting mechanisms of action. An extract with intrinsic antifungal activity from the fungus Fusarium larvarum displayed a Fitness Test profile that strongly implicated mRNA processing as the molecular target responsible for inhibition of fungal growth. Isolation of the active components from this sample identified a novel class of isoxazolidinone-containing natural products, which we have named parnafungins. These natural products were isolated as an interconverting mixture of four structural- and stereoisomers. The isomerization of the parnafungins was due to a retro-Michael ring-opening and subsequent reformation of a xanthone ring system. This interconversion was blocked by methylation of an enol moiety. Structure elucidation of purified parnafungin derivatives was accomplished by X-ray crystallography and NMR analysis. The biochemical target of these natural products has been identified as the fungal polyadenosine polymerase. Parnafungins demonstrated broad spectrum antifungal activity with no observed activity against gram-positive or gram-negative bacteria. The intact isoxazolidinone ring was required for antifungal activity. In addition, the natural products were efficacious in a mouse model of disseminated candidiasis.
Journal of the American Chemical Society 07/2008; 130(22):7060-6. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.
[show abstract][hide abstract] ABSTRACT: A novel sordarin derivative, moriniafungin (1), containing a 2-hydroxysebacic acid residue linked to C-3' of the sordarose residue of sordarin through a 1,3-dioxolan-4-one ring was isolated from the fungus Morinia pestalozzioides. Isolation of moriniafungin employed a highly specific bioassay consisting of a panel of Saccharomyces cerevisiae strains containing chimeric eEF2 for Candida glabrata, Candida krusei, Candida lusitaniae, Crytpococcus neoformans, and Aspergillus fumigatus as well as wild type and human eEF2. Moriniafungin exhibited an MIC of 6 microg/mL versus Candida albicans and IC(50)'s ranging from 0.9 to 70 microg/mL against a panel of clinically relevant Candida strains. Moriniafungin was shown to inhibit in vitro translation in the chimeric S. cerevisae strains at levels consistent with the observed IC(50). Moriniafungin has the broadest antifungal spectrum and most potent activity of any natural sordarin analog identified to date.
[show abstract][hide abstract] ABSTRACT: In order to synthesize derivatives of galbonolide A and B with improved chemical stability and antifungal activity profiles, a panel of microorganisms consisting of various species of actinomycetes and fungi were screened. As a result, an organism, Streptomyces halstedii, was identified, which catalyzed the formation of two polar compounds, one from each of the galbonolides. The synthesis and the relative stability of these compounds were optimized by using washed cells, which had been prepared from the transforming organism, and reaction conditions, which included the usage of MES buffer with pH adjusted to 5.5 and incubation at 27°C. Under conditions thus established, two compounds were isolated and characterized by a combination of UV, mass, and NMR spectroscopic analysis. The data indicate the synthesis of 21-hydroxy derivatives of galbonolides A and B with reduced but still significant anti-fungal activity when compared to the parent galbonolides.
Journal of Molecular Catalysis B: Enzymatic. 01/2001;
[show abstract][hide abstract] ABSTRACT: The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-beta-D-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B(o), is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-beta-D-glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-beta-D-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1, the gene which encodes the large subunit of glucan synthase.
Antimicrobial Agents and Chemotherapy 03/2000; 44(2):368-77. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rustmicin is a 14-membered macrolide previously identified as an inhibitor of plant pathogenic fungi by a mechanism that was not defined. We discovered that rustmicin inhibits inositol phosphoceramide synthase, resulting in the accumulation of ceramide and the loss of all of the complex sphingolipids. Rustmicin has potent fungicidal activity against clinically important human pathogens that is correlated with its sphingolipid inhibition. It is especially potent against Cryptococcus neoformans, where it inhibits growth and sphingolipid synthesis at concentrations <1 ng/ml and inhibits the enzyme with an IC50 of 70 pM. This inhibition of the membrane-bound enzyme is reversible; moreover, rustmicin is nearly equipotent against the solubilized enzyme. Rustmicin was efficacious in a mouse model for cryptococcosis, but it was less active than predicted from its in vitro potency against this pathogen. Stability and drug efflux were identified as two factors limiting rustmicin's activity. In the presence of serum, rustmicin rapidly epimerizes at the C-2 position and is converted to a gamma-lactone, a product that is devoid of activity. Rustmicin was also found to be a remarkably good substrate for the Saccharomyces cerevisiae multidrug efflux pump encoded by PDR5.
Journal of Biological Chemistry 07/1998; 273(24):14942-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two new alkyl citrates, L-731,120 and L-731,128, with alkyl chains corresponding to those of zaragozic acids A and B, were isolated as minor components of large scale fungal fermentations producing zaragozic acid A and B. They are submicromolar inhibitors of squalene synthase in vitro.
[show abstract][hide abstract] ABSTRACT: The search for squalene synthase inhibitors of microbial origin has resulted in the discovery of a new class of fungal metabolites, the zaragozic acids (squalestatins). During our survey of representatives of most major groups of fungi and filamentous bacteria, the zaragozic acids were not found in prokaryotes Zygomycotina, or Basidiomycotina. All the fungal producers encountered to date are Ascomycotina, their related anamorphic states or sterile organisms with ascomycete affinities. Members of at least II different taxa of fungi are capable of making zaragozic acids. Zaragozic acid A (squalestatin 1) appears to be the most prevalent among the different fungal taxa. In several cases we have observed production in multiple strains of the same species; for example, nearly all strains of Sporormiella intermedia, that we have examined, produce zaragozic acid B. The discovery of the zaragozic acids illustrates how knowledge of fungal biology and biochemistry can enhance the search for new chemical entities. Simultaneous screening of fungi from diverse phylogenetic and ecological origins was emphasized to discover new zaragozic acids rather than simply relying on organisms from a single kind of substratum from geographically disparate sources.