Echinocandins - structure, mechanism of action and use in antifungal therapy.

With increasing number of immunocompromised patients as well as drug resistance in fungi, the risk of fatal fungal infections in humans increases as well. The action of echinocandins is based on the inhibition of ß-(1,3)-d-glucan synthesis that builds the fungal cell wall. Caspofungin, micafungin, anidulafungin and rezafungin are semi-synthetic cyclic lipopeptides. Their specific chemical structure possess a potential to obtain novel derivatives with better pharmacological properties resulting in more effective treatment, especially in infections caused by Candida and Aspergillus species. In this review we summarise information about echinocandins with closer look on their chemical structure, mechanism of action, drug resistance and usage in clinical practice. We also introduce actual trends in modification of this antifungals as well as new methods of their administration, and additional use in viral and bacterial infections.

Feedback control of calcium driven alternans in cardiac myocytes.

Cardiac alternans is a beat-to-beat alternation of the action potential duration (APD), which has been implicated as a possible cause of ventricular fibrillation. Previous studies have shown that alternans can originate via a period doubling bifurcation caused by the nonlinear dependence of the APD on the previous diastolic interval. In this case, it has been demonstrated that alternans can be eliminated by applying feedback control on the pacing cycle length. However, studies have shown that alternans can also originate due to unstable calcium (Ca) cycling in cardiac myocytes. In this study, we explore the effectiveness of APD feedback control to suppress alternans when the underlying instability is due to unstable Ca cycling. In particular, we explore the role of the bi-directional coupling between Ca and voltage and determine the effectiveness of feedback control under a wide range of conditions. We also analyze the applicability of feedback control on a coupled two cell system and show that APD control induces spatially out-of-phase alternans. We analyze the onset and the necessary conditions for the emergence of these out-of-phase patterns and assess the effectiveness of feedback control to suppress Ca driven alternans in a multi-cellular system.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Preclinical Pharmacokinetics and Pharmacodynamic Target of SCY-078, a First-in-Class Orally Active Antifungal Glucan Synthesis Inhibitor, in Murine Models of Disseminated Candidiasis.

SCY-078 (MK-3118) is a novel, semisynthetic derivative of enfumafungin and represents the first compound of the triterpene class of antifungals. SCY-078 exhibits potent inhibition of ß-(1,3)-d-glucan synthesis, an essential cell wall component of many pathogenic fungi, including Candida spp. and Aspergillus spp. SCY-078 is currently in phase 2 clinical development for the treatment of invasive fungal diseases. In vitro disposition studies to assess solubility, intestinal permeability, and metabolic stability were predictive of good oral bioavailability. Preclinical pharmacokinetic studies were consistent with once-daily administration to humans. After intravenous delivery, plasma clearance in rodents and dogs was low, representing <15% and <25% of hepatic blood flow, respectively. The terminal elimination-phase half-life was 5.5 to 8.7 h in rodents, and it was ∼9.3 h in dogs. The volume of distribution at steady-state was high (4.7 to 5.3 liters/kg), a finding suggestive of extensive tissue distribution. Exposure of SCY-078 in kidney tissue, a target organ for invasive fungal disease such as candidiasis, exceeded plasma by 20- to 25-fold for the area under the concentration-time curve from 0 h to infinity (AUC0-∞) and Cmax SCY-078 achieved efficacy endpoints following oral delivery across multiple murine models of disseminated candidiasis. The pharmacokinetic/pharmacodynamic indices Cmax/MIC and AUC/MIC correlated with outcome. Target therapeutic exposure, expressed as the plasma AUC0-24, was comparable across models, with an upper value of 11.2 µg·h/ml (15.4 µM·h); the corresponding mean value for free drug AUC/MIC was ∼0.75. Overall, these results demonstrate that SCY-078 has the oral and intravenous (i.v.) pharmacokinetic properties and potency in murine infection models of disseminated candidiasis to support further investigation as a novel i.v. and oral treatment for invasive fungal diseases.

Real-time microscopic observation of Candida biofilm development and effects due to micafungin and fluconazole.

To understand the process of Candida biofilm development and the effects of antifungal agents on biofilms, we analyzed real-time data comprising time-lapse images taken at times separated by brief intervals. The growth rate was calculated by measuring the change of biofilm thickness every hour. For the antifungal study, 5-h-old biofilms of Candida albicans were treated with either micafungin (MCFG) or fluconazole (FLCZ). MCFG began to suppress biofilm growth a few minutes after the initiation of the treatment, and this effect was maintained over the course of the observation period. In contrast, the suppressive effects of FLCZ on biofilm growth took longer to manifest: biofilms grew in the first 5 h after treatment, and then their growth was suppressed over the next 10 h, finally producing results similar to those observed with MCFG. MCFG was also involved in the disruption of cells in the biofilms, releasing string-like structures (undefined extracellular component) from the burst hyphae. Thus, MCFG inhibited the detachment of yeast cell clusters from the tips of hyphae. In contrast, FLCZ did not disrupt biofilm cells. MCFG also showed fast antifungal activity against Candida parapsilosis biofilms. In conclusion, our results show that inhibition of glucan synthesis due to MCFG contributed not only to fungicidal activity but also to the immediate suppression of biofilm growth, while FLCZ suppressed growth by inhibiting ergosterol synthesis. Therefore, those characteristic differences should be considered when treating clinical biofilm infections.

Prevention of artificial dental plaque formation in vitro by plant extracts.

AIMS: A number of previous studies have shown that plant extracts can inhibit formation of dental plaque. The ability of extracts of Rosmarinus officianalis L., Salvia officianalis L., unfermented cocoa, red grape seed and green tea to inhibit plaque bacteria, glucosyltransferase activity, glucan and plaque formation in an in vitro model using bovine teeth was examined. METHODS AND RESULTS: The antimicrobial activity of the plant extracts against oral bacteria was determined using a standard susceptibility agar dilution technique. Inhibition of growth and acid production from glucose and sucrose by Streptococcus mutans in liquid culture was investigated. Prevention of plaque formation on bovine teeth initiated by Strep. mutans was studied using an artificial mouth. The plant extracts inhibited the growth of oral bacteria and prevented acid production by Strep. mutans. Extracts inhibited glucosyltransferase activity and glucan production and inhibited adhesion to glass. Extracts of R. officianalis L. and S. officianalis L. at 0·25 mg ml(-1) reduced plaque growth by >80%. Green tea extract completely inhibited plaque formation but resulted in a greenish discolouration of the teeth which could not be removed by scrubbing. CONCLUSIONS: The plant extracts, particularly those from R. officianalis L. and S. officianalis L., inhibited glucosyltranferase activity, glucan production and plaque formation in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the extracts of R. officianalis L. and S. officianalis L. may be useful as antiplaque agents in foods and dental preparations. Bovine teeth can be used as an alternative to hydroxyapatite for studies of plaque formation, but they need to be carefully sterilized before use.

The inhibition of eugenol on glucan is essential for the biofilm eradication effect on caries-related biofilm in an artificial mouth model.

This study aimed to evaluate the inhibitory effects of herbal extracts on caries-related bacteria, glucan and biofilm in vitro. Sensitive tests of bacteria were carried out by broth dilution method on a 96-microwell plate. Glucan inhibition tests were carried out using the phenol sulphate method. A minimum biofilm inhibitory concentration (MBIC) test was performed in an artificial mouth model. The results of the MBIC of agents were 8, 16 and 32 mg mL(-1) for eugenol, tannic acid and magnolol, respectively. For the results of glucan inhibition tests, over 63%, 28% and 27% inhibition occurred on insoluble glucan syntheses of Streptococcus sobrinus for eugenol, magnolol and tannic acid, respectively. Over 46%, 16% and 13% inhibition on soluble glucan syntheses for eugenol, magnolol and tannic acid, respectively, were also observed. In conclusion, the inhibition of eugenol on glucan is essential for the biofilm inhibition effect on caries-related biofilm in an artificial mouth model in vitro.

Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris.

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.

Differential cell wall remodeling of two chitin synthase deletants Δchs3A and Δchs3B in the pathogenic yeast Candida glabrata.

It is known that cell wall remodeling and the salvaging pathway act to compensate for an impaired or a damaged cell wall. Lately, it has been indicated that this mechanism is partly required for resistance to the glucan synthesis inhibitor echinocandin. While cell wall remodeling has been described in mutants of glucan or mannan synthesis, it has not yet been reported in a chitin synthesis mutant. Here, we describe a novel cell wall remodeling and salvaging pathway in chitin synthesis mutants, Δchs3A and Δchs3B, of the pathogenic yeast Candida glabrata. Electron microscopic analysis revealed a thickened mannoprotein layer in Δchs3A cells and a thickened chitin-glucan layer of Δchs3B cells, and it indicated the hypothesis that mannan synthase and chitin-glucan synthase indemnify Δchs3A and Δchs3B cells, respectively. The double-mutant CHS3A and MNN10, encoding α-1,6-mannosyltransferase, showed synergistic stress sensitization, and the Δchs3B strain showed supersensitivity to echinocandins. Hence, these findings support the above hypothesis of remodeling. Furthermore, unlike Δchs3A cells, Δchs3B cells showed supersensitivity to calcineurin inhibitor FK506 and Tor1p kinase inhibitor rapamycin, indicating that the Δchs3B strain uses the calcineurin pathway and a Tor1p kinase for cell wall remodeling.

Effect of antiplaque compounds and mouthrinses on the activity of glucosyltransferases from Streptococcus sobrinus and insoluble glucan production.

INTRODUCTION: The development of therapeutic agents inhibiting the activity of glucosyltransferases (GTF) and their production of glucans is a potential strategy to reduce dental decay. The aim of this study was first to characterize a GTF preparation from Streptococcus sobrinus ATCC 33478 and then to evaluate the effects of select compounds and mouthrinses on insoluble glucan (ISG) formation by combined GTFs. METHODS: The purity of the crude GTF mixture was assessed by electrophoresis. The effects of pH, temperature, sucrose, and dextran T10 concentrations on GTF activity were analyzed and the chemical structure of the products was investigated. Finally, the inhibition of GTF by commercial mouthrinses used in oral hygiene and their active components (chlorhexidine, polyphenolic compounds, fluoride derivatives, polyols, cetylpyridinium chloride, and povidone iodine) was analyzed through the reductions in the overall reaction rate and the quantity of ISG synthesized. RESULTS: The S. sobrinus ATCC 33478 crude GTF preparation obtained contains a mixture of four different GTFs known for this species. For optimal adherent ISG formation, the reaction parameters were 37 degrees C, pH 6.5, sucrose 50 g/l, and dextran T10 2 g/l. Under these conditions, the most effective agents were chlorhexidine, cetylpyridinium chloride, and tannic acid. Eludril, Elmex, and Betadine were the most effective inhibitors of all the mouthrinses tested. CONCLUSION: As the formulation of commercial products considerably influences the efficiency of active components, the fast representative ISG inhibition test developed in this study should be of great interest.

In vitro anti-bacterial and anti-adherence effects of natural polyphenolic compounds on oral bacteria.

AIMS: To investigate the action of different polyphenolic compounds, extracted from red wine, grape marc and pine bark, on oral bacteria. METHODS AND RESULTS: The anti-microbial activity of extracts was examined by determining the Minimal Inhibitory Concentration and Minimal Bactericidal Concentration using the macro dilution broth technique. Their effect on the adherence was tested on growing cells of Streptococcus mutans on a glass surface and on a multi-species biofilm grown on saliva-coated hydroxyapatite discs. The effect on glucosyltransferase activity was analysed through the reductions in the overall reaction rate and the quantity of insoluble glucan (ISG) synthesized. Pine bark and grape marc extracts were the most effective inhibitors of the multi-species biofilm formation and of the ISG synthesis. CONCLUSION: The tested components showed an interesting anti-plaque activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is, to our knowledge, the first and the most complete report on the properties of wine and pine bark extracts that could be used for oral disease prevention purpose.

In vitro effect of Chinese herb extracts on caries-related bacteria and glucan.

The purpose of this study was to evaluate the inhibitory effects of herb extracts on caries-related bacteria and glucan of dental plaque in vitro. Bacterial sensitivity tests were done using broth dilution, and the phenol sulphate method was used to assess glucan inhibition. The results showed that tannic acid could inhibit bacterial growth more effectively than other herb extracts. Eugenol showed a 46.87 +/- 12.74 and 36.67 +/- 6.30% inhibitory effect on insoluble and soluble glucan synthesis, respectively. Cnidium, barbaloin, caryophyllin, and piperine had > 40.0% inhibitory effect on soluble glucan synthesis. Both insoluble and soluble glucan synthesis of S. sobrinus were inhibited by eugenol and piperine. Eugenol and piperine were efficacious in inhibiting glucan synthesis making them desirable agents for oral care products.

Recent advances in antifungal agents.

New antifungals are needed in the medicine because of more aggressive and invasive diagnostic and therapeutic methods used, rapid emergence of resistant and new opportunistic fungi, increasing number of patients suffering from immunosuppressive situations e.g., AIDS, transplantation, cancer, etc. Several classes of new antifungal agents are discussed here including some new members of known families. Voriconazole, posaconazole and ravuconazole, are novel triazoles that inhibit the ergosterol synthesis. These drugs overcome problems associated with the ineffectivity of fluconazole against some Aspergillus spp. or the variable bioavailability of itraconazole. Echinocandins (caspofungin, anidulafungin and micafungin) represent a new family of antifungal agents that inhibit 1,3-beta-glucan synthase. Nikkomycins targeting the chitin synthase, show activity against Histoplasma capsulatum and Blastomyces dermatitidis. Sordarin derivatives that block the fungal protein synthesis can be considered as a promising new class of antifungal agents for the treatment of Candida and Pneumocystis infections.

A novel cyclic isomaltooligosaccharide (cycloisomaltodecaose, CI-10) produced by Bacillus circulans T-3040 displays remarkable inclusion ability compared with cyclodextrins.

Cyclodextrans (CIs) are cyclic isomaltooligosaccharides and only CI-7, CI-8, and CI-9 were known. CI-7, CI-8, and CI-9, consisting of seven, eight, and nine glucoses, respectively, bound by alpha-(1-->6) linkages, are known to be produced by T-3040 strain of Bacillus circulans. However, we have found, using 13C NMR and mass spectrometry, that this strain also produces CI-10, CI-11 and CI-12. These large CIs are very soluble in water and inhibit the glucan synthesis of glucansucrases to the same degree as do the smaller CIs. The CIs were thought to be poor at forming inclusion complexes with chemical compounds, due to their flexible alpha-(1-->6)-glucosidic structure. Among these six CIs, CI-10 was much better at forming an inclusion complex, and it ability to do so was as good as cyclodextrins, as determined by its ability to stabilize the color of Victoria blue B. Therefore, CI-10 may be the most commercially useful CI.

In vitro effects of crude khat extracts on the growth, colonization, and glucosyltransferases of Streptococcus mutans.

Millions of Yemenites, East Africans, and immigrants to Western countries chew khat daily for its amphetamine-like effects. There is little information in the literature concerning the possible effects of the habit on oral microbiota. Our objective was to study in vitro crude khat extract effects on Streptococcus mutans growth and sucrose-dependent colonization, and on its glucosyltransferase (GTF) activity and production. Three khat cultivars were used. Lyophilized crude aqueous khat extracts were applied to the different assays at concentrations of 0-1% (w/v). Sucrose-dependent colonization was assessed as the ability of Streptococcus mutans UA159 to form adherent biofilms in glass culture tubes. Colony forming units (CFUs) in the planktonic phase served as a measure of bacterial growth, while CFUs in the biofilm phase were used to quantify viability in the biofilms. GTFs activity was tested by incubating a crude GTFs preparation with sucrose and determining the amount of water-soluble and water-insoluble glucans formed. GTFs production was assayed by comparing intensities of GTF bands in Western blots of extracts from control and khat-containing cultures. The khat extracts effectively inhibited biofilm formation. The minimum biofilm inhibitory concentration (MBIC) varied among the cultivars (0.25-1%). The extracts also inhibited synthesis of both glucan types, particularly insoluble glucans (average 85% inhibition at 1%), with significant differences among the cultivars. However, khat increased bacterial growth and at sub-MBIC also viability within biofilms; there were no inter-cultivar differences. It is shown that khat leaves contain water-soluble constituents that inhibit some cariogenic properties of S. mutans in vitro.

Caffeine inhibits callose deposition in the cell plate and the depolymerization of microtubules in the central region of the phragmoplast.

Treatment of tobacco BY-2 cells with 10 mM caffeine that was started after the cells had entered the mitotic phase did not completely inhibit the deposition of callose in the cell plate and allowed the centrifugal redistribution of phragmoplast microtubules. On the other hand, when treatment with caffeine was started before the cells entered the mitotic phase, the deposition of callose was completely inhibited and the redistribution of phragmoplast microtubules was also inhibited. As the inhibition of redistribution of phragmoplast microtubules seems to be caused by the inhibition of depolymerization of microtubules at the central region of the phragmoplast, these results strongly suggest that the deposition of callose in the cell plate is tightly linked with the depolymerization of phragmoplast microtubules. Callose deposition was observed in phragmoplasts isolated from caffeine-treated cells as well as in those isolated from non-caffeine-treated cells, and caffeine did not inhibit callose synthesis in isolated phragmoplast, indicating that caffeine neither inhibits the accumulation of callose synthase at the equatorial regions of the phragmoplast nor arrests callose synthase itself.