Ergosterol Biosynthesis Inhibitors Research Articles

Small molecules inhibit growth, viability and ergosterol biosynthesis in Candida albicans

The aim of this work was to evaluate the anti-Candida efficacy of twenty five molecules of plant origin. Based on their MICs, effective molecules were categorized into four categories. Susceptibility testing of test compounds was carried out by standard methodology (M27-A2) as per CLSI guidelines. Minimum Fungicidal Concentration (MFC) was determined as the lowest concentration of drug killing 99.9% cells. Effect on sterol profile was evaluated by sterol quantitation method. Among the screened molecules, cinnamaldehyde, piperidine, citral, furfuraldehyde and indole were potent inhibitors of growth and viability. Exposure of Candida cells to cinnamaldehyde, piperidine, citral, furfuraldehyde, indole, α- and β- pinene at MIC’s, altered ergosterol profile. Our results indicate that the molecules altering sterol profile may exert their antifungal effect through inhibition of ergosterol biosynthesis and could be good candidates for fungal specific drug development.

Clinical Efficacy of Topical Terbinafine Versus Topical Luliconazole in Treatment of Tinea Corporis/Tinea Cruris Patients

Aims: Tinea corporis & cruris of skin respond well to topical antifungal therapy, but there is a need to apply cream 2- 3 times daily for up to four weeks will impair compliance & lead to treatment failure. Luliconazole is one of those drugs offering good efficacy & tolerability with a short duration of treatment. Terbinafine, an allylamine antifungal agent, acts by selective inhibition of fungal squalene epoxidase. Luliconazole, an imidazole antifungal agent is considered to be more effective in inhibition of ergosterol biosynthesis and its reservoir property in stratum corneum is greater than that of terbinafine. As there are lack of studies between terbinafine & luliconazole, the present study was undertaken to compare the clinical efficacy in tinea corporis/tinea cruris patients. Study Design: Prospective parallel study. Place and Duration of Study: Study was conducted on 60 patients presenting to the Dermatology out-patient department of RL Jalapa Hospital, Kolar, from 1st December 30th April 2012. Methodology: Patients alternatively assigned to either terbinafine or luliconazole & advised to apply test drugs topically for 14 days. Clinical symptoms & signs were assessed using 4-point (pruritus, erythema, scaling) scale & 10% KOH mount at base line, end of treatment visit (15th day) & later 30th day. The data was analysed based on age, gender distribution, duration of lesion, clinical score & KOH mount. Results: Of the 60 patients recruited, all came for 1st follow up (14th day) & 51 patients for 2nd follow-up (30th day). Mean age of the patients was 33.80± 9.58 years in terbinafine & 33.90 ± 9.58 years luliconazole group. Majority of patients were in 12- 40 years aged in both group. Sixty patients and 51 patients were negative for KOH mount preparation on 15th & 30th day respectively. At the end of first follow-up, the clinical score was reduced from 3 to zero (P=0.0001) in both the treatment groups. Mycological cure was 100% in both the drug groups. There was no relapse in 51 patients who came for 2nd follow-up. Four in terbinafine and 5 in luliconazole group were lost to follow up. Conclusion: Only mild forms of tinea infections were included as compared to other studies where moderate to severe (pustules, incrustations, vesiculation). Hence the onset of illness, treatment duration and severity of illness were favorable in this study for two weeks. In both the treatment arms, clinical & mycological cure was comparable, hence once a day application for two weeks of terbinafine & luliconazole were equally effective for treatment of tinea corporis/cruris infection.

Sterol C-22 Desaturase ERG5 Mediates the Sensitivity to Antifungal Azoles in Neurospora crassa and Fusarium verticillioides

Antifungal azoles inhibit ergosterol biosynthesis by interfering with lanosterol 14α-demethylase. In this study, seven upregulated and four downregulated ergosterol biosynthesis genes in response to ketoconazole treatment were identified in Neurospora crassa. Azole sensitivity test of knockout mutants for six ketoconazole-upregulated genes in ergosterol biosynthesis revealed that deletion of only sterol C-22 desaturase ERG5 altered sensitivity to azoles: the erg5 mutant was hypersensitive to azoles but had no obvious defects in growth and development. The erg5 mutant accumulated higher levels of ergosta 5,7-dienol relative to the wild type but its levels of 14α-methylated sterols were similar to the wild type. ERG5 homologs are highly conserved in fungal kingdom. Deletion of Fusarium verticillioides erg5 also increased ketoconazole sensitivity, suggesting that the roles of ERG5 homologs in azole resistance are highly conserved among different fungal species, and inhibition of ERG5 could reduce the usage of azoles and thus provide a new target for drug design.

Phenyl aldehyde and propanoids exert multiple sites of action towards cell membrane and cell wall targeting ergosterol in Candida albicans

In the present study, two phyto-compounds phenyl aldehyde (cinnamaldehyde) and propanoid (eugenol) were selected to explore their modes of action against Candida albicans. Electron microscopy, flow cytometry and spectroscopic assays were employed to determine the targets of these compounds. Treatment of C. albicans (CA04) with sub-MICs of cinnamaldehyde (50 μg/mL) and eugenol (200 μg/mL) indicated multiple sites of action including damages to cell walls, cell membranes, cytoplasmic contents and other membranous structures as observed under electron microscopy. Concentration and time dependent increase in the release of cytoplasmic contents accompanied with change in extracellular K+ concentration was recorded. Exposure of Candida cells at 4 × MIC of cinnaamldehyde and eugenol resulted in 40.21% and 50.90% dead cells, respectively as revealed by flow cytometry analysis. Treatment of Candida cells by cinnamaldehyde and eugenol at 0.5 × MIC showed 67.41% and 76.23% reduction in ergosterol biosynthesis, respectively. The binding assays reflected the ability of compounds to bind with the ergosterol. Our findings have suggested that the membrane damaging effects of phenyl aldehyde and propanoids class of compounds is attributed to their ability to inhibit ergosterol biosynthesis and simultaneously binding with ergosterol. Indirect or secondary action of these compounds on cell wall is also expected as revealed by electron microscopic studies.

Steroid toxicity and detoxification in ascomycetous fungi

In the last couple of decades fungal infections have become a significant clinical problem. A major interest into fungal steroid action has been provoked since research has proven that steroid hormones are toxic to fungi and affect the host/fungus relationship. Steroid hormones were found to differ in their antifungal activity in ascomycetous fungi Hortaea werneckii, Saccharomyces cerevisiae and Aspergillus oryzae. Dehydroepiandrosterone was shown to be the strongest inhibitor of growth in all three varieties of fungi followed by androstenedione and testosterone. For their protection, fungi use several mechanisms to lower the toxic effects of steroids. The efficiency of biotransformation in detoxification depended on the microorganism and steroid substrate used. Biotransformation was a relatively slow process as it also depended on the growth phase of the fungus. In addition to biotransformation, steroid extrusion out of the cells contributed to the lowering of the active intracellular steroid concentration. Plasma membrane Pdr5 transporter was found to be the most effective, followed by Snq2 transporter and vacuolar transporters Ybt1 and Ycf1. Proteins Aus1 and Dan1 were not found to be involved in steroid import. The research of possible targets of steroid hormone action in fungi suggests that steroid hormones inhibit ergosterol biosynthesis in S. cerevisiae and H. werneckii. Results of this inhibition caused changes in the sterol content of the cellular membrane. The presence of steroid hormones most probably causes the degradation of the Tat2 permease and impairment of tryptophan import.

Investigation on mechanism of antifungal activity of eugenol againstTrichophyton rubrum

Trichophyton rubrum is a worldwide agent responsible for chronic cases of dermatophytosis which have high rates of resistance to antifungal drugs. Attention has been drawn to the antimicrobial activity of aromatic compounds because of their promising biological properties. Therefore, we investigated the antifungal activity of eugenol against 14 strains of T. rubrum which involved determining its minimum inhibitory concentration (MIC) and effects on mycelial growth (dry weight), conidial germination and morphogenesis. The effects of eugenol on the cell wall (sorbitol protect effect) and the cell membrane (release of intracellular material, complex with ergosterol, ergosterol synthesis) were investigated. Eugenol inhibited the growth of 50% of T. rubrum strains employed in this study at an MIC = 256 μg/ml, as well as mycelial growth and conidia germination. It also caused abnormalities in the morphology of the dermatophyte in that we found wide, short, twisted hyphae and decreased conidiogenesis. The results of these studies on the mechanisms of action suggested that eugenol exerts antifungal effects on the cell wall and cell membrane of T. rubrum. Eugenol act on cell membrane by a mechanism that seems to involve the inhibition of ergosterol biosynthesis. The lower ergosterol content interferes with the integrity and functionality of the cell membrane. Finally, our studies support the potential use of the eugenol as an antifungal agent against T. rubrum.

β-Asarone, an active principle of Acorus calamus rhizome, inhibits morphogenesis, biofilm formation and ergosterol biosynthesis in Candida albicans

Anti-Candida potential of Acorus calamus rhizome and its active principle, β-asarone, was evaluated against the human fungal pathogen, Candida albicans. β-Asarone exhibited promising growth inhibitory activity at 0.5mg/ml and it was fungicidal at 8mg/ml. Time dependant kill curve assay showed that MFC of β-asarone was highly toxic to C. albicans, killing 99.9% inoculum within 120min of exposure. β-Asarone caused significant inhibition of C. albicans morphogenesis and biofilm development at sub-inhibitory concentrations. Our data indicate that the growth inhibitory activity of β-asarone might be through inhibition of ergosterol biosynthesis. Hemolytic assay showed that β-asarone is non-toxic, even at concentrations approaching MIC value. Our results suggest that β-asarone may be safe as a topical antifungal agent.

The stepwise acquisition of fluconazole resistance mutations causes a gradual loss of fitness in Candida albicans

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1, Tac1 and Upc2, which result in constitutive overexpression of multidrug efflux pumps and ergosterol biosynthesis genes respectively. It is not known how the permanently changed gene expression program in resistant strains affects their fitness in the absence of drug selection pressure. We have systematically investigated the effects of activating mutations in Mrr1, Tac1 and Upc2, individually and in all possible combinations, on the degree of fluconazole resistance and on the fitness of C. albicans in an isogenic strain background. All combinations of different resistance mechanisms resulted in a stepwise increase in drug resistance, culminating in 500-fold increased fluconazole resistance in strains possessing mutations in the three transcription factors and an additional resistance mutation in the drug target enzyme Erg11. The acquisition of resistance mutations was associated with reduced fitness under non-selective conditions in vitro as well as in vivo during colonization of a mammalian host. Therefore, without compensatory mutations, the inability to appropriately regulate gene expression results in a loss of competitive fitness of drug-resistant C. albicans strains.

Synthesis and biological evaluation of some novel 1-indanone thiazolylhydrazone derivatives as anti-Trypanosoma cruzi agents

A series of novel 4-arylthiazolylhydrazones (TZHs) derived from 1-indanones were synthesized in good yields (66–92%) in a simple procedure using microwave irradiation and then characterized by spectroscopy studies. The compounds were evaluated for their in vitro anti-Trypanosoma cruzi activity against the epimastigote, trypomastigote and amastigote forms of the parasite. Most TZHs displayed excellent activity, and were more potent and selective than the reference drug Benznidazole, used in the current chemotherapy. Analysis of the free sterols from parasite incubated with the compounds showed that inhibition of ergosterol biosynthesis is a possible target for the action of these new TZHs. In particular, TZH 9 emerged as a promising antichagasic compound to be evaluated in animal models.

Calcofluor white combination antifungal treatments for Trichophyton rubrum and Candida albicans.

Superficial mycoses caused by dermatophyte fungi are among the most common infections worldwide, yet treatment is restricted by limited effective drugs available, drug toxicity, and emergence of drug resistance. The stilbene fluorescent brightener calcofluor white (CFW) inhibits fungi by binding chitin in the cell wall, disrupting cell wall integrity, and thus entails a different mechanism of inhibition than currently available antifungal drugs. To identify novel therapeutic options for the treatment of skin infections, we compared the sensitivity of representative strains of the dermatophyte Trichophyton rubrum and Candida albicans to CFW and a panel of fluorescent brighteners and phytoalexin compounds. We identified the structurally related stilbene fluorescent brighteners 71, 85, 113 and 134 as fungicidal to both T. rubrum and C. albicans to a similar degree as CFW, and the stilbene phytoalexins pinosylvan monomethyl ether and pterostilbene inhibited to a lesser degree, allowing us to develop a structure-activity relationship for fungal inhibition. Given the abilities of CFW to absorb UV365 nm and bind specifically to fungal cell walls, we tested whether CFW combined with UV365 nm irradiation would be synergistic to fungi and provide a novel photodynamic treatment option. However, while both treatments individually were cytocidal, UV365 nm irradiation reduced sensitivity to CFW, which we attribute to CFW photoinactivation. We also tested combination treatments of CFW with other fungal inhibitors and identified synergistic interactions between CFW and some ergosterol biosynthesis inhibitors in C. albicans. Therefore, our studies identify novel fungal inhibitors and drug interactions, offering promise for combination topical treatment regimes for superficial mycoses.

Sphingolipids Regulate the Yeast High-Osmolarity Glycerol Response Pathway

The yeast high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase pathway is activated in response to hyperosmotic stress via two independent osmosensing branches, the Sln1 branch and the Sho1 branch. While the mechanism by which the osmosensing machinery activates the downstream MAP kinase cascade has been well studied, the mechanism by which the machinery senses and responds to hyperosmotic stress remains to be clarified. Here we report that inhibition of the de novo sphingolipid synthesis pathway results in activation of the HOG pathway via both branches. Inhibition of ergosterol biosynthesis also induces activation of the HOG pathway. Sphingolipids and sterols are known to be tightly packed together in cell membranes to form partitioned domains called rafts. Raft-enriched detergent-resistant membranes (DRMs) contain both Sln1 and Sho1, and sphingolipid depletion and hyperosmotic stress have similar effects on the osmosensing machinery of the HOG pathway: dissociation of an Sln1-containing protein complex and elevated association of Sho1 with DRMs. These observations reveal the sphingolipid-mediated regulation of the osmosensing machinery of the HOG pathway.

The possible mechanism of rhapontigenin influencing antifungal activity onCandida albicans

Rhapontigenin, an aglycone of rhapontin, was produced by biotransformation and we investigated its antifungal activity against Candida albicans, one of the most important opportunistic fungal pathogens. Rhapontigenin is found to have, in vitro, inhibitory activity with a minimal inhibitory concentration (MIC) value against all test isolates of 128-256 μg/ml. We detected increased reactive oxygen species (ROS) levels in yeast cultures treated with rhapontigenin at the MIC. Rhapontigenin inhibited DNA, RNA, and protein synthesis, especially RNA synthesis, and induced morphological changes and apoptosis of C. albicans. The apoptotic effect of rhapontigenin on C. albicans at subinhibitory concentrations was higher in the stationary growth phase than in the exponential phase, while the opposite results were noted with amphotericin B. The mechanism of antifungal activity of rhapontigenin may be associated with the generation of ROS that might induce apoptosis and it may also involve the inhibition of ergosterol biosynthesis.

High potential bioactive oil based bimetallic polymer: anticandidal effect is via inhibition of ergosterol biosynthesis

Copper and zinc containing soybean oil based polymer was prepared by condensation polymerisation of oil based fatty amide polyole and sebacic acid in a process initiated by sodium ethoxide through microwave assisted synthesis. Fourier transform infrared and proton nuclear magnetic resonance have been used to study the structure of the polymers. Atomic absorption spectroscopy has been used for confirming the presence of metal in the bimetallic polymer. The thermal behaviour of the polymer was analysed by a thermogravimetry/differential thermal analyser. Inherent viscosity, iodine value, saponification values, specific gravity and refractive index of polymers were determined through standard laboratory methods. To explore the antifungal activity in vitro, a series of synthesised polymers were studied for their antifungal activity against different Candida isolates. The growth and sensitivity of the organisms were affected by the synthesised polymers at significantly reduced concentrations. Insight studies to the mechanism suggested that the synthesised polymers and fluconazole exert antifungal activity by targeting ergosterol biosynthesis. Furthermore, it was observed that metal incorporation increased the anticandidal activity of the synthesised polymers. The observed selective fungicidal characteristics of these synthesised polymers indicate that these compounds might be promising antifungal agents, defining a new class of antimycotics. The bimetallic polymer was found to be thermally stable and biologically active. It can be used as a biomedical material.

A Genomewide Screen in Schizosaccharomyces pombe for Genes Affecting the Sensitivity of Antifungal Drugs That Target Ergosterol Biosynthesis

We performed a genomewide screen for altered sensitivity to antifungal drugs, including clotrimazole and terbinafine, that target ergosterol biosynthesis using a Schizosaccharomyces pombe gene deletion library consisting of 3,004 nonessential haploid deletion mutants. We identified 109 mutants that were hypersensitive and 11 mutants that were resistant to these antifungals. Proteins whose absence rendered cells sensitive to these antifungals were classified into various functional categories, including ergosterol biosynthesis, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction, ribosome biosynthesis and assembly, regulation of transcription and translation, cell wall organization and biogenesis, mitochondrion function, amino acid metabolism, nucleic acid metabolism, lipid metabolism, meiosis, and other functions. Also, proteins whose absence rendered cells resistant to these antifungals were classified into functional categories including mitochondrion function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and some unknown functions. Furthermore, the 109 sensitive mutants were tested for sensitivity to micafungin, another antifungal drug that inhibits (1,3)-β-D-glucan synthase, and 57 hypersensitive mutants were identified, suggesting that these mutants were defective in cell wall integrity. Altogether, our findings in fission yeast have shed light on molecular pathways associated with the cellular response to ergosterol biosynthesis inhibitors and may provide useful information for developing strategies aimed at sensitizing cells to these drugs.

Enhancement of β-Carotene Production by Over-Expression of HMG-CoA Reductase Coupled with Addition of Ergosterol Biosynthesis Inhibitors in Recombinant Saccharomyces cerevisiae

In this study, the synergistic effect of overexpressing the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene and adding ergosterol synthesis inhibitor, ketoconazole, on β-carotene production in the recombinant Saccharomyces cerevisiae was investigated. The results showed that the over-expression of HMG-CoA reductase gene and adding 100 mg/l ketoconazole alone can result in 135.1 and 15.6% increment of β-carotene concentration compared with that of the control (2.05 mg/g dry weight of cells), respectively. However, the combination of overexpressing HMG-CoA reductase gene and adding ketoconazole can achieve a 206.8% increment of pigment content (6.29 mg/g dry weight of cells) compared with that of the control. Due to the fact that over-expression of the HMG-CoA reductase gene can simultaneously improve the flux of the sterol and carotenoid biosynthetic pathway, it can be concluded that under the circumstances of blocking sterol biosynthesis, increasing the activity of HMG-CoA reductase can result in more precursors FPP fluxing into carotenoid branch and obtain a high increment of β-carotene production. The results of this study collectively suggest that the combination of overexpressing HMG-CoA reductase gene and supplying ergosterol synthesis inhibitor is an effective strategy to improve the production of desirable isoprenoid compounds such as carotenoids.

The Transcription Factor Ndt80 Does Not Contribute to Mrr1-, Tac1-, and Upc2-Mediated Fluconazole Resistance in Candida albicans

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80Δ mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80Δ mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.

Antifungal activity of Coriaria nepalensis essential oil by disrupting ergosterol biosynthesis and membrane integrity against Candida

Fungal diseases in humans have increased significantly with the advent of an expanding population of immunosuppressed patients and with the introduction of sophisticated life-saving medical procedures. Plant extracts and products have been used in traditional medicine for centuries. Coriaria nepalensis essential oil (CNEO) is known to possess antimicrobial activity. This study was an attempt to examine CNEO against various fluconazole-sensitive and -resistant Candida isolates. Insight into the mechanism of action was elucidated by flow cytometric analysis and ergosterol biosynthesis studies. The susceptibility tests for CNEO were carried out in terms of MIC and by disc diffusion assays against all Candida isolates, employing standard protocols. Insight into the mechanism of action was elucidated by propidium iodide cell sorting (FACS) and by assessing ergosterol content in treated and untreated isolates with the test entity. CNEO was found effective against all Candida isolates, including the resistant strains. While CNEO inflicts fungal cell death by disrupting membrane integrity, significant impairment of ergosterol biosynthesis was induced by the test entity. CNEO showed a strong antifungal effect against all the Candida isolates. Mechanisms of action appear to originate from the inhibition of ergosterol biosynthesis and the disruption of membrane integrity. It can be concluded that the observed antimicrobial characteristics of C. nepalensis indicate that it might be a promising antimicrobial agent.

The pharmacological inhibition of sterol biosynthesis in Leishmania is counteracted by enhancement of LDL endocytosis

Leishmania parasites, despite being able to synthesize their own sterols, acquire and accumulate significant amounts of cholesterol through low density lipoprotein (LDL) particle endocytosis. The role of this system in Leishmania amazonensis promastigotes under pharmacological pressure by sterol biosynthesis inhibitors (SBIs) was investigated. First, thin layer chromatography demonstrated that L. amazonensis promastigotes, in response to ergosterol biosynthesis inhibition by treatment with 4.0 and 6.0μM ketoconazole or miconazole, accumulate up to two times more cholesterol than controls. The treatment of promastigotes with ketoconazole and simvastatin, two SBIs with non-related mechanisms of action, showed that both drugs induce increases in 125I-LDL endocytosis in a dose-dependent manner, indicating that the accumulation of exogenous cholesterol is due to the enhancement of LDL uptake. Finally, it was demonstrated that L. amazonensis promastigotes were rendered more susceptible to treatment with SBIs (ketoconazole, miconazole, simvastatin and terbinafine) in the absence of exogenous cholesterol sources, with a reduction of the IC50s of about 50% in three of the four tested drugs. These results show that the exogenous cholesterol uptake system in L. amazonensis plays a role as a compensatory mechanism in response to the presence of SBIs, suggesting that it may be a potential pharmacological target.

Scorpion Toxins Modify Phytopathogenic Fungus Physiology. A Possible Source of New Fungicides

Seven toxins (F1-F7) were purified from Tityus discrepans scorpion venom on a C18 HPLC column. The compounds were fungitoxic on Macrophomina phaseolina. The molecular masses of F1-F7 were (Da) 1061.1, 7328.8, 7288.3, 7268.5, 7104.6, 6924.6, and 6823.3, respectively. It is not known if F1 is a small peptide or some other kind of organic molecule. Compounds F2-F7 were peptides. The most potent was F7, with a minimal inhibition concentration of 0.4 μg/μL and a concentration for 50% inhibition of 0.13 μg/μL. Fungal esterase activity was abolished by F2, F3, and F5 and inhibited by 89, 60, 58, and 54% by F4, F6, F7, and F1, respectively. F1, F2, F5, and F7 induced an increase on hyphae chitin wall and septum thickness. Peptides F3-F6 induced efflux of the fluorescent dye Na-CoroNa Red complex from hyphae. Only F5 and F6 were inhibited by the prokaryote sodium channel blockers amiloride and mibefradil. Gas chromatography-mass spectrometry analysis suggested that F1, F5, F6, and F7 altered sterol biosynthesis either by inhibiting ergosterol biosynthesis or by producing ergosterol analogues. The peptides affect M. phaseolina viability by three mechanisms: decreasing esterase activity, altering Na(+) membrane permeability, and altering wall sterol biosynthesis. It seems that interfering with sterol synthesis is an important mechanism behind the effect of the fungicideal toxins. However, the antifungal effects at short times are indicative of a direct esterase inhibition, which, with the increased membrane leakiness to Na(+), makes the fungus inviable.

Antifungal activity of α-methyl trans cinnamaldehyde, its ligand and metal complexes: promising growth and ergosterol inhibitors

Antifungal effectivity and utility of cinnamaldehyde is limited because of its high MIC and skin sensitivity. In this study, α-methyl trans cinnamaldehyde, a less irritating derivative, have been self coupled and complexed with Co(II) and Ni(II) to generate N, N'-Bis (α-methyl trans cinnamadehyde) ethylenediimine [C(22)H(24)N(2)], [Co(C(44)H(48)N(4))Cl(2)] and [Ni(C(44)H(48)N(4))Cl(2)]. Ligand and complexes were characterized on the basis of FTIR, ESI-MS, IR and (1)HNMR techniques. Synthesized ligand [L] and complexes were investigated for their MICs, inhibition of ergosterol biosynthesis and H(+) extrusion against three strains of Candida: C. albicans 44829, C. tropicalis 750 and C. krusei 6258. Average of three species MIC of methyl cinnamaldehyde is 317 μg/ml (2168 μM). Compared to methyl cinnamaldehyde ligand [L], Co(II) and Ni(II) complex are found to be 4.48, 17.78 and 21.46 times more effective in liquid medium and 2.73, 8.93 and 10.38 times more effective in solid medium. At their respective MIC(90) average inhibition of ergosterol biosynthesis caused by methyl cinnamaldehyde, ligand [L], Co(II) and Ni(II) complex, respectively was 80, 78, 90 and 93%. H(+) extrusion was also significantly inhibited but did not co-relate well with MIC(90). Results indicate ergosterol biosynthesis as site of action of α-methyl cinnamaldehyde, synthesized ligand and complexes. α-methyl cinnamaldehyde and ligand did not show any toxicity against H9c2 rat cardiac myoblast cell, whereas Co(II) and Ni(II) complexes on an average produced 19% cellular toxicity.