The inhibition of Pseudomonas aeruginosa biofilm formation by micafungin and the enhancement of antimicrobial agent effectiveness in BALB/c mice.

Micafungin inhibits biofilm formation by impeding 1,3-ß-D-glucan synthesis in Candida albicans. Since Pseudomonas aeruginosa also has 1,3-ß-D-glucan in its cell wall, this study assessed the effects of antibacterial agents in vitro and in vivo on micafungin-treated biofilm-forming P. aeruginosa isolates. After treatment with micafungin as well as with a panel of four antibacterial agents, biofilm production was significantly reduced as measured by spectrophotometry. The relative mRNA transcription levels for the genes encoding pellicles (pelC) and cell wall 1,3-ß-D-glucan (ndvB), which were measured by quantitative reverse transcription PCR (qRT-PCR), significantly decreased with micafungin treatment. In vivo, the survival rates of P. aeruginosa-infected BALB/c mice significantly increased after combined treatment with micafungin and each of the antibacterial agents. Of these treatments, the combination of micafungin with levofloxacin had the highest survival rate; this combination was the most effective treatment against P. aeruginosa-induced infection.

Enfumafungin derivative MK-3118 shows increased in vitro potency against clinical echinocandin-resistant Candida Species and Aspergillus species isolates.

MK-3118 is as an orally active new antifungal in the early stage of clinical development that inhibits the biosynthesis of ß-(1,3)-glucan. We evaluated the in vitro activity of this compound against wild-type and echinocandin-resistant (ER) isolates containing mutations in the FKS gene(s) of Candida spp. and Aspergillus spp. MK-3118 demonstrated enhanced efficacy for most C. albicans and C. glabrata ER isolates relative to caspofungin, with decreased MICs and half-maximal inhibitory concentrations (IC50s).

New ß-glucan inhibitors as antifungal drugs.

INTRODUCTION: New classes of synthetic and semi-synthetic ß-glucan inhibitors have recently emerged, providing analogs that, in some cases, have been proven to have a high degree of activity against fungi, offering the prospect of alternatives to the commercially available lipopeptide/echinocandin agents caspofungin, micafungin and anidulafungin. AREA COVERED: This review covers applications disclosing compound classes that include synthetic pyridazinone analogs, bicyclic heteroaryl ring compounds, aniline derivates, and semi-synthetic echinocandin and enfumafungin derivatives. MK-3118 is an analog of the natural product enfumafungin that, in particular, shows promise as it has a spectrum of activity comparable with caspofungin but has the advantageous property of oral bioavailability. EXPERT OPINION: The diversity of chemical classes in the present review, which have demonstrable activity against ß-glucan and the prospect of oral bioavailability, offers hope that safe and effective antifungal drugs will emerge and be commercialized. Of particular note, the Merck compound MK-3118, with solid evidence of efficacy based on preclinical data, has moved into clinical trials.

Pga26 mediates filamentation and biofilm formation and is required for virulence in Candida albicans.

The Candida albicans gene PGA26 encodes a small cell wall protein and is upregulated during de novo wall synthesis in protoplasts. Disruption of PGA26 caused hypersensitivity to cell wall-perturbing compounds (Calcofluor white and Congo red) and to zymolyase, which degrades the cell wall ß-1,3-glucan network. However, susceptibility to caspofungin, an inhibitor of ß-1,3-glucan synthesis, was decreased. In addition, pga26Δ mutants show increased susceptibility to antifungals (fluconazol, posaconazol or amphotericin B) that target the plasma membrane and have altered sensitivities to environmental (heat, osmotic and oxidative) stresses. Except for a threefold increase in ß-1,6-glucan and a slightly widened outer mannoprotein layer, the cell wall composition and structure was largely unaltered. Therefore, Pga26 is important for proper cell wall integrity, but does not seem to be directly involved in the synthesis of cell wall components. Deletion of PGA26 further leads to hyperfilamentation, increased biofilm formation and reduced virulence in a mouse model of disseminated candidiasis. We propose that deletion of PGA26 may cause an imbalance in the morphological switching ability of Candida, leading to attenuated dissemination and infection.

1,3-Benzoxazole-4-carbonitrile as a novel antifungal scaffold of ß-1,6-glucan synthesis inhibitors.

Synthesis and in vitro antifungal evaluations of 1,3-benzoxazole-7-carbonitrile 3, 1,3-benzoxazole-4-carbonitrile 4, benzofuran 5, benzoxazine 7, and benzimidazole 8 were reported. Among them, 1,3-benzoxazole-4-carbonitrile was found to be a superior scaffold structure with moderate growth inhibition against Candida species. 1,3-Benzoxazole-4-carbonitrile 6 showed potent activity against Candida species compared to 5-desmethyl compound 4 and triazolopyridine 2. Compound 6 was efficiently prepared from versatile intermediate 24, which possessed six different substituents on the benzene ring. Conversion of benzene 24 into various 1,3-benzoxazole derivatives such as 2-aliphatic 34, 2-amino 35, and lactone 38 was demonstrated.

Evidence for DNA synthesis in Pneumocystis carinii trophozoites treated with the beta-1,3-glucan synthesis inhibitor pneumocandin L-693,989.

Pneumocandins inhibit beta-1,3-glucan synthesis preventing the development of Pneumocystis cysts that are absent from the lungs of treated rats. To determine whether treated trophozoites are capable of DNA replication, cytochemical analyses were performed on 4',6-diamidino-2-phenylindole (DAPI)- and DB181-stained Pneumocystis carinii isolated from pneumocandin L-693-989-treated rats. Fluorescence intensities of trophozoite nuclei from drug-treated rats were greater than those of untreated controls, suggesting that DNA replication was not inhibited but that cytokinesis and perhaps karyokinesis were blocked.

Novel antifungal agents: triazolopyridines as inhibitors of beta-1,6-glucan synthesis.

Preparations and in vitro antifungal activities of triazolopyridines, imidazopyridines, and a pyrazolopyridine were reported. Among those scaffolds, triazolopyridine was found to be the specific inhibitor of the synthesis of beta-1,6-glucan, an essential component of the fungal cell wall, and to show potent antifungal activities against several Candida species.

Novel pyridobenzimidazole derivatives exhibiting antifungal activity by the inhibition of beta-1,6-glucan synthesis.

Based on the HTS hit compound 1a, an inhibitor of beta-1,6-glucan synthesis, we synthesized novel pyridobenzimidazole derivatives and evaluated their antifungal activity. Among the compounds synthesized, we identified the potent compound 15e, which exhibits excellent activity superior to fluconazole against both Candida glabrata and Candida krusei. From the SAR study, we revealed essential moieties for antifungal activity.

Chitin synthesis and fungal pathogenesis.

Chitin is an essential part of the carbohydrate skeleton of the fungal cell wall and is a molecule that is not represented in humans and other vertebrates. Complex regulatory mechanisms enable chitin to be positioned at specific sites throughout the cell cycle to maintain the overall strength of the wall and enable rapid, life-saving modifications to be made under cell wall stress conditions. Chitin has also recently emerged as a significant player in the activation and attenuation of immune responses to fungi and other chitin-containing parasites. This review summarises latest advances in the analysis of chitin synthesis regulation in the context of fungal pathogenesis.

In vitro antifungal activities of D11-2040, a beta-1,6-glucan inhibitor, with or without currently available antifungal drugs.

We recently reported our discovery of small molecule beta-1,6-glucan inhibitor named D75-4590 and the anti-Candida activity of its derivatives D11-2040 and D21-6076. In this study, we further evaluated the antifungal profile of D11-2040. It alone strongly inhibited the vegetative growth and/or hyphal development of various Candida species, but no significant activity was observed against Cryptococcus neoformans or any of the filamentous fungi tested. Synergism was detected for C. albicans in the interaction of D11-2040 and caspofungin by the chequerboard method and in that of D11-2040 and fluconazole by the time-kill method. Slight but positive interactions were observed in several combinations for C. neoformans and Aspergillus fumigatus as well. These results suggested that beta-1,6-glucan inhibitors have promising potential as single drugs as well as concomitants.

Discovery and characterization of ß-1,6-glucan inhibitors.

IMPORTANCE OF THE FIELD: The increasing number of patient populations at high risk of opportunistic infections has highlighted the need for the improvement in antifungal treatments. Due to the limited number of currently available antifungal drugs and the concerns for possible prevalence of resistant strains, drugs with a new mechanism of action are most desirable. AREAS COVERED IN THIS REVIEW: Although the cell wall is considered to be one of the ideal targets for antifungal drugs, insufficient information on the enzymes involved in its construction has restricted the discovery of new inhibitors. This review introduces the recent discovery of the inhibitors of ß-1,6-glucan, one of the essential components of the yeast cell wall. WHAT THE READER WILL GAIN: The readers will gain the strategy to obtain the ß-1,6-glucan synthesis inhibitors, their mechanisms of actions, and antifungal activities in vitro as well as in vivo. TAKE HOME MESSAGE: The ß-1,6-glucan inhibitors are considered to be promising candidates for new antifungal drugs which could give valuable options in a clinical setting, although their usage may be limited because of their fungistatic action and limited spectrum. Additionally, they can be useful tools in the study on ß-1,6-glucan synthesis and the virulence of Candida species.

Effect of beta-1,6-glucan inhibitors on the invasion process of Candida albicans: potential mechanism of their in vivo efficacy.

Beta-1,6-glucan is a fungus-specific cell wall component that is essential for the retention of many cell wall proteins. We recently reported the discovery of a small molecule inhibitor of beta-1,6-glucan biosynthesis in yeasts. In the course of our study of its derivatives, we found a unique feature in their antifungal profile. D21-6076, one of these compounds, exhibited potent in vitro and in vivo antifungal activities against Candida glabrata. Interestingly, although it only weakly reduced the growth of Candida albicans in conventional media, it significantly prolonged the survival of mice infected by the pathogen. Biochemical evaluation of D21-6076 indicated that it inhibited beta-1,6-glucan synthesis of C. albicans, leading the cell wall proteins, which play a critical role in its virulence, to be released from the cell. Correspondingly, adhesion of C. albicans cells to mammalian cells and their hyphal elongation were strongly reduced by the drug treatment. The results of the experiment using an in vitro model of vaginal candidiasis showed that D21-6076 strongly inhibited the invasion process of C. albicans without a significant reduction in its growth in the medium. These evidences suggested that D21-6076 probably exhibited in vivo efficacy against C. albicans by inhibiting its invasion process.

[The mechanisms of resistance to echinocandin class of antifungal drugs].

The echinocandin (candin) class of antifungal drugs inhibit beta-1,3-glucan synthase and block synthesis of beta-1,3-glucan , an important polysaccharide in fungal cell walls. Candins are used widely for treatment of systemic infections caused by Candida and Aspergillus because of their high potency and low toxicity to humans. The incidence of candin resistance has been rare compared to that of azole resistance, although candin-resistant clinical isolates of C. albicans, C. glabrata, C. krusei and C. tropicalis have been reported in the USA and Europe in recent years. These isolates possess hundred-fold higher MIC values for candins than sensitive strains, as well as candin-resistant beta-1,3-glucan synthase activities. Their candin resistance is associated with amino acid substitutions in the echinocandin resistant region (Ech) of the FKS gene that encodes a catalytic subunit of the beta-1,3-glucan synthase. However, the effect of these amino acid substitutions on the drug-protein interaction and the molecular basis for the resistance is unknown. The exposure of fungi to candin drugs induces stress responses that activate networks involving transcriptional regulators and components controlling signal transduction of the pathways responsible for maintenance of fungal cell wall integrity. The fungal cell wall is still an attractive drug target and further investigation into the mechanisms of candin resistance and structural analysis of the beta-1,3-glucan synthase protein complex will facilitate the development of broad spectrum inhibitors of fungal cell wall synthesis.

Protection by anti-beta-glucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence.

Anti-beta-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective beta-glucan epitope(s) and protection mechanisms, we studied two anti-beta-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the beta1,3-beta1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to beta1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to beta1,6- and beta1,4-linked glucose sequences in addition to beta1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-beta-glucan antibodies may affect details of the beta-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize beta-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

Discovery of a small-molecule inhibitor of {beta}-1,6-glucan synthesis.

It is possible that antifungal drugs with novel modes of action will provide favorable options to treat fungal infections. In the course of our screening for antifungal compounds acting on the cell wall, a pyridobenzimidazole derivative with unique activities, named D75-4590, was discovered. During treatment of Saccharomyces cerevisiae with D75-4590, (i) incorporation of [(14)C]glucose into the beta-1,6-glucan component was selectively reduced, (ii) proteins released from the cell had lost the beta-1,6-glucan moiety, and (iii) cells tended to clump, resulting in impaired cell growth. Genetic analysis of a D75-4590-resistant mutant of S. cerevisiae indicated that its primary target was Kre6p, which is considered to be one of the beta-1,6-glucan synthases. These results strongly suggest that D75-4590 is a specific inhibitor of beta-1,6-glucan synthesis. D75-4590 showed potent activities against various Candida species. It inhibited hyphal elongation of C. albicans as well. KRE6 is conserved in various fungi, but no homologue has been found in mammalian cells. These lines of evidence indicate that D75-4590 is a promising lead compound for novel antifungal drugs. To our knowledge, this is the first report of a beta-1,6-glucan inhibitor.

Differential effects of inhibiting chitin and 1,3-{beta}-D-glucan synthesis in ras and calcineurin mutants of Aspergillus fumigatus.

Aspergillus fumigatus must be able to properly form hyphae and maintain cell wall integrity in order to establish invasive disease. Ras proteins and calcineurin each have been implicated as having roles in these processes. Here, we further delineate the roles of calcineurin and Ras activity in cell wall biosynthesis and hyphal morphology using genetic and pharmacologic tools. Strains deleted for three genes encoding proteins of these pathways, rasA (the Ras protein), cnaA (calcineurin), or crzA (the zinc finger transcription factor downstream of calcineurin), all displayed decreased cell wall 1,3-beta-d-glucan content. Echinocandin treatment further decreased the levels of 1,3-beta-d-glucan for all strains tested yet also partially corrected the hyphal growth defect of the DeltarasA strain. The inhibition of glucan synthesis caused an increase in chitin content for wild-type, dominant-active rasA, and DeltarasA strains. However, this important compensatory response was diminished in the calcineurin pathway mutants (DeltacnaA and DeltacrzA). Taken together, our data suggest that the Ras and calcineurin pathways act in parallel to regulate cell wall formation and hyphal growth. Additionally, the calcineurin pathway elements cnaA and crzA play a major role in proper chitin and glucan incorporation into the A. fumigatus cell wall.

Anti-beta-glucan-like immunoprotective candidacidal antiidiotypic antibodies.

Mycoses, candidiasis in particular, are relatively common opportunistic infections still characterized by an unacceptable high mortality rate. Furthermore, they are often complicated by resistance or refractoriness to the existing antimicrobial agents. In recent years new effective therapeutic and large-scale preventative strategies have been proposed by exploiting the identification of fungal beta-glucans as target of antifungal agents such as echinocandins, yeast killer toxins and protective antibodies. Anti-beta-glucan antibodies are detectable in animal and human sera. When elicited by glucan-based vaccines they can exert a fungicidal protective activity. Beta-glucan cell wall killer toxin receptors can elicit fungicidal protective antibodies following natural and experimental infections. When used as an immunogen a killer toxin-neutralizing monoclonal antibody (beta-glucan-like) is able to elicit a significant anticandidal protection mediated by anti-idiotypic anti-beta-glucan-like candidacidal antibodies. Polyclonal, monoclonal and recombinant anti-beta-glucan-like antibodies and peptide mimotopes are able to exert an in vitro and/or in vivo microbicidal activity against eukaryotic and prokaryotic killer toxin receptor-bearing pathogenic microorganisms. Implications and perspectives for transphyletic anti-infectious control strategies, as immunoprevention and immunotherapy, are discussed.

Synthesis of UDP-glucose derivatives modified at the 3-OH as potential chain terminators of beta-glucan biosynthesis.

A series of UDP-D-glucose derivatives and precursors that have been modified at C-3 were synthesised from D-glucose as potential chain terminators of beta-glucan biosynthesis. None of the UDP-derivatives or the precursors tested displayed significant anti-fungal activity in a series of germination assays on the dermatophyte Trichophyton rubrum.

Immunological actions of Sophy beta-glucan (beta-1,3-1,6 glucan), currently available commercially as a health food supplement.

We examined the immunological actions of Sophy beta-glucan(Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004-329077), a type of beta-1,3-1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO-202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy beta-glucan significantly (P<0.01) stimulated the (3)H-thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme-linked immunoassays (EIAs) revealed that Sophy beta-glucan stimulated the production of interleukin-8 (IL-8) or soluble Fas (sFas), but not that of IL-1beta, IL-2, IL-6, IL-12 (p70+40), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte-like cell line, U937. The induction by Sophy beta-glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen-class II (HLA-class II), Toll-like receptor-2 (TLR-2), and Toll-like receptor-4 (TLR-4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR-2 and TLR-4 did not block the Sophy beta-glucan-induced production of IL-8 in the U937 cells. Furthermore, a mAb to the beta-glucan receptor, Dectin-1, significantly (P<0.05) blocked the Sophy beta-glucan induced DNA synthesis in the PBMCs, and Sophy beta-glucan-induced production of IL-8 in the U937 cells. The Sophy beta-glucan-induced production of IL-8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Sophy beta-glucan stimulated the expression of IL-8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy beta-glucan also blocked the stimulator cell induction of DNA synthesis and IFN-gamma production in the responder cells in a one-way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy beta-glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy beta-glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries.

Blocking effect of anti-Dectin-1 antibodies on the anti-tumor activity of 1,3-beta-glucan and the binding of Dectin-1 to 1,3-beta-glucan.

Schizophyllan (SPG) is used to treat cervical cancer in combination with irradiation to enhance the immunological surveillance system. Dectin-1 is a cell surface receptor for 1,3-beta-glucan. In this study, we prepared two anti-Dectin-1 monoclonal antibodies, 4B2 and SC30 having a K(D) of 7.04 x 10(-8) M and 1.55 x 10(-7) M, respectively, and evaluated the role of Dectin-1 in SPG-induced anti-tumor activity in mice. Expression of Dectin-1 on peritoneal macrophages and binding of SPG to the cells were decreased by administration of 4B2 and SC30. SPG-mediated anti-tumor activity was inhibited by 4B2 and SC30. 4B2 and SC30 inhibited the binding of SPG to splenocytes from mice. The binding of SPG-biotin to Dectin-1-transfected HEK293 cells was inhibited by 4B2, but not SC30. 4B2 and SC30 differ in their influence on Dectin-1 between primary cells and transduced cells, and Dectin-1 effects 1,3-beta-glucan-mediated anti-tumor activity in mice by binding to SPG.