echinocandin-b and cilofungin

The echinocandins are large lipopeptide molecules that are inhibitors of beta-(1,3)-glucan synthesis, an action that damages fungal cell walls. In vitro and in vivo, the echinocandins are rapidly fungicidal against most Candida spp and fungistatic against Aspergillus spp. They are not active at clinically relevant concentrations against Zygomycetes, Cryptococcus neoformans, or Fusarium spp. No drug target is present in mammalian cells. The first of the class to be licensed was caspofungin, for refractory invasive aspergillosis (about 40% response rate) and the second was micafungin. Adverse events are generally mild, including (for caspofungin) local phlebitis, fever, abnormal liver function tests, and mild haemolysis. Poor absorption after oral administration limits use to the intravenous route. Dosing is once daily and drug interactions are few. The echinocandins are widely distributed in the body, and are metabolised by the liver. Results of studies of caspofungin in candidaemia and invasive candidiasis suggest equivalent efficacy to amphotericin B, with substantially fewer toxic effects. Absence of antagonism in combination with other antifungal drugs suggests that combination antifungal therapy could become a general feature of the echinocandins, particularly for invasive aspergillosis.

Topics: Amphotericin B; Antifungal Agents; Aspergillosis; Candidiasis; Caspofungin; Clinical Trials as Topic; Echinocandins; Fungal Proteins; Humans; Lipopeptides; Lipoproteins; Micafungin; Peptides; Peptides, Cyclic; Treatment Outcome

The echinocandins are large lipopeptide molecules that are inhibitors of beta-(1,3)-glucan synthesis, an action that damages fungal cell walls. In vitro and in vivo, the echinocandins are rapidly fungicidal against most Candida spp and fungistatic against Aspergillus spp. They are not active at clinically relevant concentrations against Zygomycetes, Cryptococcus neoformans, or Fusarium spp. No drug target is present in mammalian cells. The first of the class to be licensed was caspofungin, for refractory invasive aspergillosis (about 40% response rate) and the second was micafungin. Adverse events are generally mild, including (for caspofungin) local phlebitis, fever, abnormal liver function tests, and mild haemolysis. Poor absorption after oral administration limits use to the intravenous route. Dosing is once daily and drug interactions are few. The echinocandins are widely distributed in the body, and are metabolised by the liver. Results of studies of caspofungin in candidaemia and invasive candidiasis suggest equivalent efficacy to amphotericin B, with substantially fewer toxic effects. Absence of antagonism in combination with other antifungal drugs suggests that combination antifungal therapy could become a general feature of the echinocandins, particularly for invasive aspergillosis.

Topics: Amphotericin B; Antifungal Agents; Aspergillosis; Candidiasis; Caspofungin; Clinical Trials as Topic; Echinocandins; Fungal Proteins; Humans; Lipopeptides; Lipoproteins; Micafungin; Peptides; Peptides, Cyclic; Treatment Outcome

A whole-cell C. albicans screen was designed to identify novel inhibitors interacting with the synthesis, assembly and regulation of the fungal cell wall. C. albicans was grown in a paired broth assay in 96-well plates with natural product extracts or pure chemical compounds in the presence and absence of the osmotic stabilizer, sorbitol. Growth was visually examined over a 7-day period and scored into different growth categories. Positives from the sorbitol rescue were then examined under the microscope for morphological alterations and grouped into several morphological classes. Sorbitol protection and cell morphology were indicators of novel antifungal agents from natural product extracts and pure compounds.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Benzenesulfonates; beta-Glucans; Candida albicans; Cell Wall; Chitin; Echinocandins; Fungal Proteins; Glucans; Peptides; Peptides, Cyclic; Protein Kinase C; Sorbitol

When grown on a range of surfaces in conditions favouring hyphal growth, hyphae of Candida albicans grew in a right-handed helical fashion. This phenomenon was observed with eight strains and with two nutrient media. It is suggested that this is a result of rotation of the hyphal apex as it extends, which on some surfaces results in a helical hyphal wall, but which in a liquid results in a straight hypha. The consequence is that on a surface, a helically growing hypha will be exposed to a more diverse environment than a straight hypha. This phenomenon may have significance in the colonization of tissue by C. albicans.

Topics: Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Cell Division; Echinocandins; Fungal Proteins; Peptides; Peptides, Cyclic

A systematic evaluation of the in vitro (1,3)-beta-glucan synthase assay parameters was performed using microsomes prepared from Candida albicans from either yeast or mycelial phase cells. Enzyme activities of both yeast and mycelial phase microsomes depended on the presence of guanosine-5'-O-(3-thiophosphate) and either bovine serum albumin or a detergent [W-1 (polyoxyethylene ether detergent) or Brij-35 (polyoxyethylene ether, 23 lauryl ether)]. Brij-35 was included in standard assays as it was compatible with the permeabilized whole-cell assay. Microsomes derived from both the yeast and mycelial phases generally yielded similar glucan synthase activities under a range of different assay conditions. Brij-35 significantly stabilized the enzyme, yielding a half-life of 5.6 d at 4 degrees C, compared with 0.9 d without detergent. The addition of detergent during mechanical breakage of yeast cells dramatically improved glucan synthase stability and activity. Enzyme catalysis was linear for at least 75 min with 100 micrograms protein from microsomes of yeast cells grown to mid-exponential phase, with an apparent Km for UDP-glucose of 1.1 mM. The pH and temperature optima were 7.75 and 30 degrees C, respectively. Glucan synthase activity was highest in cells derived from early mid-exponential phase and declined to a basal level by stationary phase. A permeabilization-based in situ assay for glucan synthase was developed. Cells were permeabilized with 2% (v/v) solution of toluene/methanol (1:1) and assayed for glucan synthase activity using standard reaction mixtures. Reactions were linear for 30 min and were inhibited by known inhibitors of glucan synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Cell Membrane Permeability; Detergents; Echinocandins; Enzyme Stability; Fungal Proteins; Glucosyltransferases; Guanosine Triphosphate; Kinetics; Membrane Proteins; Microsomes; Peptides; Peptides, Cyclic; Polidocanol; Polyethylene Glycols; Schizosaccharomyces pombe Proteins

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Centrifugation, Density Gradient; Echinocandins; Fungal Proteins; Glucosyltransferases; Kinetics; Membrane Proteins; Neurospora crassa; Peptides; Peptides, Cyclic; Protoplasts; Schizosaccharomyces pombe Proteins; Sorbose; Uridine Diphosphate

(1,3)-beta-D-Glucan synthase of Candida albicans was rendered soluble by treatment of membrane preparations with the polyoxyethylene ether detergent W-1. Extraction with 0.025% W-1 at 4 degrees C for 24 h effectively solubilized and activated the enzyme. Under these conditions, greater than 85% of the protein in membrane preparations was released, and about 64% of the glucan synthase activity could be recovered in the soluble form. Soluble enzyme activity was stable for more than 12 days at 4 degrees C. Also, glucan synthase activity in the extracted membrane preparations could be activated to achieve more than twice the enzyme activity in the original, unextracted membrane preparations. The soluble glucan synthase had characteristics similar to those of the membrane-bound enzyme. Soluble glucan synthase had an apparent Km of 2.0 mM, and particulate glucan synthase had an apparent Km of 2.5 mM. Kinetics of cilofungin inhibition for both enzyme preparations were noncompetitive, with an apparent Ki of 2.5 microM; both preparations could be inhibited by cilofungin but not by its peptide nucleus or side chain, either alone or in combination. The reaction products from both forms of the enzyme were sensitive to (1,3)-beta-D-glucanase degradation but not to alpha-amylase, alpha-glucosidase, or proteinase K degradation and thus were shown to be beta(1----3) glucan.

Topics: Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Detergents; Echinocandins; Enzyme Activation; Fungal Proteins; Glucosyltransferases; Kinetics; Membrane Proteins; Membranes; Peptides; Peptides, Cyclic; Polyethylene Glycols; Schizosaccharomyces pombe Proteins; Solubility

Topics: Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Echinocandins; Fungal Proteins; Glucosyltransferases; Membrane Proteins; Molecular Structure; Neurospora; Neurospora crassa; Peptides; Peptides, Cyclic; Schizosaccharomyces pombe Proteins; Structure-Activity Relationship

The antifungal antibiotic, echinocandin B (ECB), was modified by a sequential procedure in which the initial step involved enzymatic removal of the native N-linoleoyl group from the N-terminus using an Actinoplanes utahensis culture. The resulting product, ECB nucleus, was reacylated using active esters or acid halides of various substituted acids to give a series of ECB analogs. These analogs possessed anti-Candida activity both in vitro and in vivo (mice). Other studies have shown that one of these, cilofungin, the 4-n-octyloxybenzoyl-ECB analog (LY121019), has excellent anti-Candida activity, low toxicity and is superior to other available antifungal antibiotics.

Topics: Acylation; Anti-Bacterial Agents; Antifungal Agents; Echinocandins; Fungal Proteins; Peptides; Peptides, Cyclic; Structure-Activity Relationship

Topics: Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Drug Design; Echinocandins; Fungal Proteins; Microbial Sensitivity Tests; Molecular Structure; Peptides; Peptides, Cyclic; Structure-Activity Relationship