Synthesis and Antifungal Activity of Novel Cationic Pneumocandin Bo Derivatives

Bouffard, F. Aileen; Zambias, Robert A.; Dropinski, James F.; Balkovec, James M.; Hammond, Milton L.; Abruzzo, George K.; Bartizal, Ken; Marrinan, Jean A.; Kurtz, Myra B.

doi:10.1021/jm00028a003

Cited by 104 publications

(58 citation statements)

References 2 publications

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“…A. nidulans wild-type strain FGSC 4 was compared with A. fumigatus MF5668, a strain used for animal models of disseminated aspergillosis, for sensitivity to pneumocandins in vitro. The minimum effective concentrations that altered growth and morphology (38) were the same for both strains: 4.0 g/ml (3,800 nM) with the natural product, L-688,786 (55), and 0.002 g/ml (1.7 nM) with the semisynthetic analog L-733,560 (12). Identical morphological effects were also seen for both strains; i.e., the hyphae grew abnormally, producing compact clumps instead of the highly branched filaments that were observed at concentrations of inhibitor below the minimal effective concentration.…”

Section: Resultsmentioning

confidence: 97%

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

et al. 1996

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Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-␤-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of ϳ200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched ϳ300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.

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Section: Resultsmentioning

confidence: 97%

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

et al. 1996

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“…Once cell wall proteins are released into the cell wall space by secretory vesicles, they become covalently crosslinked into the cell wall glucan-chitin matrix (5,8). This process is expected to take some time, and cell wall proteins that have been secreted but not yet cross-linked into the wall matrix could be extracted from the cell wall.…”

Section: Resultsmentioning

confidence: 99%

“…The wall is known to contain ␤-1,3-glucan, ␣-1,3-glucan, chitin, and a number of identified cell wall glycoproteins (5,7,26). In the ER, the cell wall glycoproteins are modified by N-linked glycosylation, and many cell wall proteins receive a GPI anchor.…”

Section: Discussionmentioning

confidence: 99%

α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

Maddi

Free

2010

Eukaryot Cell

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The enzyme ␣-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.The fungal cell wall is an important organelle that protects the cell from various environmental stresses. It is a dynamic structure that interacts with the environment and is modified to accommodate growth, cell division, and development. Fungal cell walls have been shown to contain ␤-1,3-glucan, ␣-1,3-glucan, ␤-1,6-glucan, mixed ␤-1,3/␤-1,4-glucans, chitin, and mannan/galactomannan (6, 19). These polysaccharide polymers constitute 80 to 85% of the cell wall mass, while glycoproteins constitute the remaining 15 to 20% (6). The cell wall glycoproteins are required for vital functions, like structural support, signal transduction, biofilm formation, and cell wall biosynthesis. In the case of pathogenic fungi, the cell wall is critical for the invasion of host tissues (8). Because of their accessibility and the crucial functions they perform, cell wall proteins could be important targets for the development of antifungal therapeutics.The glucan and chitin cell wall polymers are synthesized by enzyme complexes (glucan synthases and chitin synthases) that are associated with the plasma membrane. Glucan and chitin are vectorially passed into the cell wall space during synthesis and cross-linked together in the cell wall space. The mannan and galactomannan present in the cell wall are found as glycoconjugates on cell wall proteins. Mannosylation of cell wall proteins occurs in the endoplasmic reticulum (ER) and Golgi apparatus at O-linked and N-linked glycosylation sites. In Saccharomyces cerevisiae, mannosylation of N-linked glycosylation is initiated by the addition of an ␣-1,6-linked mannose ...

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“…Clinical studies have shown that caspofungin is as active as amphotericin B and fluconazole in the treatment of invasive candidal disease (4,23,33). Caspofungin exerts its fungicidal activity by inhibiting the synthesis of (1,3)-␤-glucan, which leads to cell wall damage (9,19). The pharmacodynamic parameter linked with the efficacy of caspofungin has not been adequately defined.…”

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confidence: 99%

Pharmacodynamics of Caspofungin in a Murine Model of Systemic Candidiasis: Importance of Persistence of Caspofungin in Tissues to Understanding Drug Activity

Louie

Deziel

Liu

et al. 2005

Antimicrob Agents Chemother

152

141

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Pharmacokinetic and pharmacodynamic studies were conducted in a murine model of systemic candidiasis to determine the pharmacodynamic parameter linked with caspofungin efficacy. Additional studies defined the importance of persistent tissue drug concentrations to treatment outcome. The pharmacokinetics of caspofungin were determined in the serum and kidneys of infected mice over 96 h. Population pharmacokinetic analysis demonstrated a serum terminal half-life (t 1/2 ) for caspofungin of 20.2 h when only serum concentrations were considered, but the terminal t 1/2 increased to 59.2 h when serum and kidney concentration-time data were comodeled. In dose-range studies, the dose-response effect was well described by an inhibitory sigmoid curve for the exposure-effect killing caused by the drug (r 2 > 0.96; P Ͻ Ͻ 0.001). In dose-fractionation studies, fungal counts in kidneys were not statistically different for total doses given as one, two, or four equally divided doses over 96 h, indicating that the area under the concentration-time curve/MIC is the pharmacodynamic parameter that predicts caspofungin efficacy in our infection model. In a separate study, mice infected with Candida albicans 24 h after serum concentrations of caspofungin fell below the MIC for the fungal isolate had significant reductions in fungal densities in their kidneys compared with the growth of fungi in the kidneys of untreated controls (P ‫؍‬ 0.005). This in vivo biological assay demonstrates that therapeutic concentrations of caspofungin persist at the site of infection in kidney tissue well after serum concentrations fall below the MIC, underscoring the primacy of caspofungin levels in tissues on determining treatment outcome.

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Synthesis and Antifungal Activity of Novel Cationic Pneumocandin Bo Derivatives

Cited by 104 publications

References 2 publications

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

Pharmacodynamics of Caspofungin in a Murine Model of Systemic Candidiasis: Importance of Persistence of Caspofungin in Tissues to Understanding Drug Activity

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