epiglucan and cilofungin

Analysis of the in vitro sensitivity of clinical isolates of different yeast species to amphotericine B and two inhibitors of cell wall beta-glucane synthesis: the lipopeptide cilofungin and the liposaccharide papulacandin B, as well as the possible interactions among these antifungal types.. The in vitro sensibility of C. albicans strains and other yeast species of clinical origin was studied, using the macrodilution method in RPMI broth as recommended by NCCLS. In six C. albicans and two T. glabrata strains the interaction between amphotericine B and one of the beta-glucan inhibitors was also assessed, by combining one antifungal agent in a two-fold dilution series with subinhibitory concentrations (1/4MIC) of the second antifungal.. Of all three antifungals, amphothericine B proved to be the more active one, with a MIC90 of 1.25 micrograms/ml for C. albicans. MIC90 of cilofungin and papulacandine B is 5 micrograms/ml for both antifungals. The spectrum of action for the different yeast species is broader for papulacandin B than for cilofungin. On the other hand, no synergism has been observed between the two glucane inhibitors and amphotericine B.

Topics: Aminoglycosides; Amphotericin B; Anti-Bacterial Agents; Antifungal Agents; Ascomycota; beta-Glucans; Drug Synergism; Echinocandins; Glucans; Microbial Sensitivity Tests; Peptides, Cyclic; Yeasts

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

The effect of the beta 1-3 glucan synthase inhibitor, cilofungin, on the incorporation of 35S-methionine-labelled glucan associated proteins (GAP) in the cell wall of Candida albicans was investigated in a susceptible strain C. albicans 3153 and resistant strain C. albicans CA-2. Cilofungin exerted a marked effect on the GAP composition of the cell wall at 0.25 mg/L, a concentration which reduced beta 1-3 glucan synthesis by approximately 50% and also inhibited the growth of the susceptible strain C. albicans 3153. A 46 kDa protein was present in large amounts in C. albicans 3153 but not in strain CA-2. This protein was probably not mannosylated and its incorporation was greatly reduced by cilofungin. In addition, a well defined 34 kDa protein was identified together with a distinct band of high molecular mass polydisperse material of between 65 and 96 kDa and another of > 200 kDa. These proteins were strongly reactive to concanavalin A indicating that they were mannosylated, and treatment with cilofungin caused an increase in their production which was also confirmed by immunoblotting with rabbit anti-Candida serum. In contrast, exposure of the drug-resistant strain CA-2 to cilofungin did not result in changes in the composition of the GAP constituents. Only the mannosylated proteins of 34 kDa and the high molecular mass polydisperse material 65-96 kDa were present in the cell wall. The pulse-chase labelling experiments showed that the 46 kDa protein was the first of the GAPs to be incorporated into the cell wall, and that this was suppressed in the presence of cilofungin whereas there was a concomitant increase in the incorporation of the 34 kDa and the high-molecular weight polydisperse material. Thus, cilofungin causes a profound imbalance in GAP incorporation into the growing cell wall which is possibly related to changes in the amount and type of glucan being synthesized at sub-inhibitory concentrations of the antimycotic.

Topics: Antifungal Agents; beta-Glucans; Candida albicans; Cell Wall; Echinocandins; Fungal Proteins; Glucans; Kinetics; Peptides, Cyclic

The in vivo anti-Candida activities of 1,3-beta-D-glucan synthesis inhibitors L-671,329, L-646,991 (cilofungin), L-687,901 (tetrahydroechinocandin B), and L-687,781 (a papulacandin analog) were evaluated by utilizing a murine model of disseminated candidiasis that has enhanced susceptibility to Candida albicans but increased sensitivity for discriminating antifungal efficacy. DBA/2 mice were challenged intravenously with 1 x 10(4) to 5 x 10(4) CFU of C. albicans MY1055 per mouse. Compounds were administered intraperitoneally at concentrations ranging from 1.25 to 10 mg/kg of body weight twice daily for 4 days. At 6 h and 1, 2, 3, 4, 7, and 9 days after challenge, five mice per group were sacrificed and their kidneys were homogenized and plated for enumeration of Candida organisms (CFU per gram). Progressiveness of response trends and no-statistical-significance-of-trend doses were derived to rank compound efficacy. 1,3-beta-D-Glucan synthesis 50% inhibitory concentrations were determined by using a C. albicans (MY1208) membrane glucan assay. Candida and Cryptococcus neoformans MICs and minimal fungicidal concentrations were determined by broth microdilution. L-671,329, L-646,991, L-687,901, and L-687,781 showed similar 1,3-beta-D-glucan activities, with 50% inhibitory concentrations of 0.64, 1.30, 0.85, and 0.16 micrograms/ml, respectively. Data from in vitro antifungal susceptibility studies showed that L-671,329, L-646,991, and L-687,901 had similar MICs ranging from 0.5 to 1.0 micrograms/ml, while L-687,781 showed slightly higher MICs of 1.0 to 2.0 micrograms/ml for C. albicans MY1055. Lipopeptide compounds were ineffective against C. neoformans strains. Results from in vivo experiments comparing significant trend and progressiveness in response analyses indicated that L-671,329 and L-646,991 were equipotent but slightly less active than L-687-901, while L-687,781 was ineffective at 10 mg/kg. Fungicidal activities of L-671,329, L-646,991, and L-687,901 were observed in vivo, with significant reduction in Candida CFU per gram of kidneys compared with those in sham-treated mice at doses of > or = 2.5 mg/kg evident as early as 1 day after challenge.

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Azoles; beta-Glucans; Candida albicans; Candidiasis; Echinocandins; Glucans; Kidney; Lethal Dose 50; Mice; Mice, Inbred DBA; Microbial Sensitivity Tests; Peptides; Peptides, Cyclic; Pyrans

The lipopeptide antimycotic agent, cilofungin, at a dose of 20 micrograms ml-1, inhibited beta 1-3 glucan synthesis in a drug-susceptible strain (3153; minimum inhibitory concentration (MIC) < 1 microgram ml-1) as well as in a drug-resistant strain of Candida albicans (CA-2, derived from 3153 by nitrosoguanidine mutagenesis; MIC > 50 micrograms ml-1). This was demonstrated for both whole cells under growing and non-growing conditions, and during protoplast regeneration. However, time-effect experiments, during growth of a CA-2 culture initially exposed to an inhibitory dose of cilofungin, showed that this strain was able to progressively regain both glucan synthesis and a growth rate comparable to that of cultures that had not been treated with the drug. This recovery was not attributable to cilofungin instability or degradation within the CA-2 culture. Our study suggests the existence of an as yet unknown drug-related and/or cell-related factor(s) modulating the inhibition of glucan synthesis, and then contributing to the actual inhibitory effects of cilofungin in C. albicans.

Topics: Antifungal Agents; beta-Glucans; Candida albicans; Drug Resistance, Microbial; Echinocandins; Glucans; Peptides, Cyclic; Protoplasts

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.

Topics: Amino Acid Sequence; Antifungal Agents; beta-Glucans; Candida; Echinocandins; Glucans; Molecular Sequence Data; Peptides; Peptides, Cyclic; Structure-Activity Relationship