Robert A. Giacobbe

Discovery of Novel Antifungal (1,3)-β-d-Glucan Synthase Inhibitors

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)-β-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 Bo, 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)-β-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)-β-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.

Interlaboratory Comparison of Results of Susceptibility Testing with Caspofungin against Candida and Aspergillus Species

ABSTRACT Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual “prominent growth reduction” (MIC2) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ± one dilution range across all 17 laboratories. MIC2 at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.

The Discovery of Enfumafungin, a Novel Antifungal Compound Produced by an Endophytic Hormonema Species Biological Activity and Taxonomy of the Producing Organisms

In a screening of natural products with antifungal activity derived from endophytic fungi, we detected a potent activity in a culture belonging to the form-genus Hormonema, isolated from leaves of Juniperus communis. The compound is a new triterpene glycoside, showing an antifungal activity highly potent in vitro against Candida and Aspergillus and with moderate efficacy in an in vivo mouse model of disseminated candidiasis. The agent is especially interesting since its antifungal spectrum and its effect on morphology of Aspergillus fumigatus is comparable to that of the glucan synthase inhibitor pneumocandin B,,, the natural precursor of the clinical candidate MK-0991 (caspofungin acetate). An additional search for other Hormonema isolates producing improved titers or derivatives resulted in the isolation of two more strains recovered from the same plant host showing identical activity. The producing isolates were compared with other non-producing Hormonema strains by DNA fingerprinting and sequencing of the rDNA internal transcribed spacers. Comparison of rDNA sequences with other fungal species suggests that the producing fungus could be an undetermined Kabatina species. Kabatina is a coelomycetous genus whose members are known to produce Hormonema-like states in culture.

PAP Inhibitor with In Vivo Efficacy Identified by Candida albicans Genetic Profiling of Natural Products

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.

Caspofungin Susceptibility in Aspergillus and Non-Aspergillus Molds: Inhibition of Glucan Synthase and Reduction of β-d-1,3 Glucan Levels in Culture

ABSTRACT Caspofungin inhibits synthesis of β-d-1,3 glucan, essential to cell walls in Candida and Aspergillus spp., but activity against less common molds is largely uncharacterized. We demonstrate that caspofungin inhibits β-d-1,3 glucan synthesis and reduces in vitro growth of clinical isolates from the genera Alternaria, Curvularia, Scedosporium, Acremonium, Bipolaris, and Trichoderma.

Pramanicin, a novel antimicrobial agent from a fungal fermentation

Abstract The antimicrobial agent pramanicin ( 1 ), and a related fatty acid ( 6 ), were isolated from a corn-based solid or a lactose-containing liquid fermentation of a sterile fungus found growing in grass. The structures of these compounds were determined by a variety of spectral means including UV, IR, and NMR spectroscopy, as well as mass spectrometry. A number of chemical derivatives are also presented here. Pramanicin represents a new class of antimicrobial agents containing a highly functionalized head group and functionalized fatty side chain

Isolation, structure elucidation, and biological activity of virgineone from Lachnum virgineum using the genome-wide Candida albicans fitness test

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.

Tremorgenic mycotoxins, paspalitrem A and C, from a tropical Phomopsis

An endophytic Phomopsis sp. from living bark of Cavendishia pubescens in Colombia produced paspalitrem A and paspalitrem C in batch fermentations. These compounds previously were known only from sclerotia of Claviceps paspali as tremorgenic mycotoxins causing neurological disorders of livestock. A potential ecological role of these metabolites in regard to endophytism of the woody host is considered.

Antimicrobial activity of ergokonin A from Trichoderma longibrachiatum.

Aims: Natural fungal products were screened for antifungal compounds. The mode of action of one of the hits found and the taxonomy of the producing organism were analysed.

Improvement in the titer of echinocandin-type antibiotics: a magnesium-limited medium supporting the biphasic production of pneumocandins A0 and B0.

SummaryWe have developed a liquid fermentation medium for the submerged culture of the fungus,Zalerion arboricola, which supports the rapid production of an echinocandin-type antibiotic, pneumocandin A0 (formerly L-671, 329), in yields increased at least 4-fold over those reported previously. The improvements were achieved through medium simplification, substitution of high levels of mannitol for glycerol as the major source of carbon, and restriction of available magnesium. Antibiotic formation in batch cultures with this mannitol-based medium is not confined to the idiophase; rather production appears to be biphasic, with synthesis beginning during growth (i.e., at day 3) and increasing in rate at day 11, well after rapid growth has ended. Accumulation of antibiotic continues beyond 14 days, and by 21 days titers greater than 500 μg/ml are attained. For the synthesis of a related compound, pneumocandin B0, by a mutant strain ofZ. arboricola, the medium gives similar production kinetics and a titer of 800 μg/ml. Although supplementation of the medium with magnesium ions stimulates growth, it decreases titer by preferentially affecting the second phase of antibiotic synthesis. This decline in synthesis in the magnesium-supplemented medium is explained by the depletion of mannitol before the second phase of synthesis can begin. In contrast, mannitol in the magnesium-limited medium is used more slowly with approximately half still available at day 11 to support continued antibiotic formation.