Fluconazole-COX Inhibitor Hybrids: A Dual-Acting Class of Antifungal Azoles.

When used in combination with azole antifungal drugs, cyclooxygenase (COX) inhibitors such as ibuprofen improve antifungal efficacy. We report the conjugation of a chiral antifungal azole pharmacophore to COX inhibitors and the evaluation of activity of 24 hybrids. Hybrids derived from ibuprofen and flurbiprofen were considerably more potent than fluconazole and comparable to voriconazole against a panel of Candida species. The potencies of hybrids composed of an S-configured azole pharmacophore were higher than those with an R-configured pharmacophore. Tolerance, defined as the ability of a subpopulation of cells to grow in the presence of the drug, to the hybrids was lower than to fluconazole and voriconazole. The hybrids were active against a mutant lacking CYP51, the target of azole drugs, indicating that these agents act via a dual mode of action. This study established that azole-COX inhibitor hybrids are a novel class of potent antifungals with clinical potential.

Identification of Thiazoyl Guanidine Derivatives as Novel Antifungal Agents Inhibiting Ergosterol Biosynthesis for Treatment of Invasive Fungal Infections.

Invasive fungal infections (IFIs) are fatal infections, but treatment options are limited. The clinical efficacies of existing drugs are unsatisfactory because of side effects, drug-drug interaction, unfavorable pharmacokinetic profiles, and emerging drug-resistant fungi. Therefore, the development of antifungal drugs with a new mechanism is an urgent issue. Herein, we report novel aryl guanidine antifungal agents, which inhibit a novel target enzyme in the ergosterol biosynthesis pathway. Structure-activity relationship development and property optimization by reducing lipophilicity led to the discovery of 6h, which showed potent antifungal activity against Aspergillus fumigatus in the presence of serum, improved metabolic stability, and PK properties. In the murine systemic A. fumigatus infection model, 6h exhibited antifungal efficacy equivalent to voriconazole (1e). Furthermore, owing to the inhibition of a novel target in the ergosterol biosynthesis pathway, 6h showed antifungal activity against azole-resistant A. fumigatus.

Eugenol loaded chitosan nanoemulsion for food protection and inhibition of Aflatoxin B1 synthesizing genes based on molecular docking.

The present investigation entails the fabrication and characterization of nanometric emulsion of eugenol (Nm-eugenol) encompassed into chitosan for assessing bio-efficacy in terms of in vitro antifungal actions, antiaflatoxigenic potential, and in situ preservative efficacy against Aspergillus flavus infestation and aflatoxin B1 (AFB1) mediated loss of dietary minerals, lipid triglycerides and alterations in composition of important macronutrients in stored rice. Nm-eugenol characterized by SEM, XRD, and FTIR exhibited biphasic burst release of eugenol. Reduction in ergosterol and methylglyoxal (AFB1-inducer) content after Nm-eugenol fumigation depicted biochemical mechanism of antifungal and antiaflatoxigenic activities. In silico 3D homology docking of eugenol with Ver-1 gene validated molecular mechanism of AFB1 inhibition. Further, significant protection of rice seeds from fungi, AFB1 contamination and preservation against loss of rice minerals, macronutrients and lipids during storage suggested deployment of chitosan as a biocompatible wall material for eugenol encapsulation and application as novel green preservative for food protection.

Eficacia y seguridad de isavuconazol oral, como terapia de mantenimiento, en pacientes adultos con aspergilosis invasiva y con falla o intolerancia a voriconazol oral y posaconazol oral / Efficacy and safety of oral isavuconazole, as maintenance therapy, in adult patients with invasive aspergillosis and with failure or intolerance to oral voriconazole and oral posaconazole

INTRODUCCIÓN: El presente dictamen preliminar expone la evaluación de la eficacia y seguridad de isavuconazol oral, en comparación con itraconazol oral, en pacientes adultos con aspergilosis invasiva con falla o intolerancia a voriconazol oral y posaconazol oral, que requieren terapia de mantenimiento posterior a la terapia de inducción con anfotericina B deoxicolato. La aspergilosis invasiva (AI) es la infección por hongos más común en huéspedes inmunodeprimidos. A pesar de los avances en el diagnóstico y el tratamiento de la AI, las tasas de mortalidad siguen siendo altas. A nivel mundial, se estima una incidencia acumulada anual de 250,000 casos de AI, con una tasa de mortalidad de aproximadamente 30 a 80 %. En el Perú, en el 2014 se estimó una prevalencia de 1,621 casos de AI. El tratamiento antimicótico de la AI suele ser prolongado, con una duración de varios meses a más de un año. En EsSalud, los pacientes con AI son tratados inicialmente con anfotericina B deoxicolato (terapia de inducción intravenosa) y luego, una vez estables, con azoles orales (voriconazol posaconazol o itraconazol) (terapia de mantenimiento). Sin embargo, basado en la opinión de expertos, es común que los pacientes con tratamiento prolongando presenten falla o intolerancia a la terapia de mantenimiento. Así, en un contexto clínico, en el que un paciente presenta falla o intolerancia a la terapia de mantenimiento con voriconazol oral y posaconazol oral, la única opción disponible a nivel institucional es el itraconazol oral. Sin embargo, la Red Prestacional Sabogal ha propuesto el uso de isavuconazol oral como alternativa al tratamiento con itraconazol oral, basándose en una mayor biodisponibilidad1 y menor tasa de efectos adversos serios asociados a interacciones farmacológicas, lo que podría llevar a una mejor eficacia y seguridad comparativa. Cabe mencionar que para este grupo específico de pacientes con AI, no se disponen de datos epidemiológicos a nivel institucional. METODOLOGÍA: Se realizó una búsqueda sistemática de literatura con el objetivo de identificar evidencia sobre la eficacia y seguridad de isavuconazol oral, en comparación con itraconazol oral, en pacientes adultos con aspergilosis invasiva con falla o intolerancia a voriconazol oral y posaconazol oral, que requieren terapia de mantenimiento posterior a la terapia de inducción con anfotericina B deoxicolato. Se utilizaron las bases de datos PubMed, Cochrane Library y LILACS, priorizándose la evidencia proveniente de ensayos clínicos controlados aleatorizados. Asimismo, se realizó una búsqueda dentro de bases de datos pertenecientes a grupos que realizan evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC), incluyendo el Healthcare Improvement Scotland, el National Institute for Health and Care Excellence (NICE), la Canadian Agency for Drugs and Technologies in Health (CADTH), la Haute Autorité de Santé (HAS), el Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen (IQWiG), además de la Base Regional de Informes de Evaluación de Tecnologías en Salud de las Américas (BRISA) y páginas web de sociedades especializadas en aspergilosis como American Thoracic Society (ATS), Infectious Diseases Society of America (IDSA), European Society for Clinical Microbiology and Infectious Diseases (ESCMID) y European Conference on Infections in Leukaemia (ECIL). Se hizo una búsqueda adicional en la página web del registro administrado por la Biblioteca Nacional de Medicina de los Estados Unidos (https://clinicaltrials.gov/) e International Clinical Trial Registry Platform (ICTRP) (https://apps.who.int/trialsearch/), para poder identificar ensayos clínicos en curso o que no hayan sido publicados para, de este modo, disminuir el riesgo de sesgo de publicación. La búsqueda sistemática se basó en una metodología escalonada, la cual consistió en la búsqueda inicial de estudios secundarios (tipo revisiones sistemáticas de ensayos clínicos) que respondan a la pregunta PICO, seguido de la búsqueda de estudios primarios (tipo ensayos clínicos aleatorizados). RESULTADOS: Se realizó una búsqueda de la literatura con respecto a la eficacia y seguridad de isavuconazol oral, en comparación con itraconazol oral, en pacientes adultos con aspergilosis invasiva con falla o intolerancia a voriconazol oral y posaconazol oral, que requieren terapia de mantenimiento posterior a la terapia de inducción con anfotericina B deoxicolato. A continuación, se describe la evidencia disponible según el orden jerárquico del nivel de evidencia o pirámide de Haynes 6S9, siguiendo lo indicado en los criterios de elegibilidad. CONCLUSIONES: El presente dictamen preliminar tuvo como objetivo evaluar la mejor evidencia sobre la eficacia y seguridad de isavuconazol oral, en comparación con itraconazol oral, en pacientes adultos con aspergilosis invasiva con falla o intolerancia a voriconazol oral y posaconazol oral, que requieren terapia de mantenimiento posterior a la terapia de inducción con anfotericina B deoxicolato. En la presente evaluación no se encontró evidencia que sustentara un mayor beneficio con isavuconazol oral en lugar de itraconazol oral en pacientes adultos con aspergilosis invasiva con falla o intolerancia a voriconazol oral y posaconazol oral, que requieren terapia de mantenimiento posterior a la terapia de inducción con anfotericina B deoxicolato. Sin embargo, se identificaron recomendaciones vigentes basadas en la opinión de expertos y la experiencia clínica que abordaron el tema en cuestión. Así, tanto en la GPC de la ATS como en DynaMed se realizaron recomendaciones a favor del uso de itraconazol oral como terapia de mantenimiento luego de la inducción con anfotericina B intravenosa (deoxicolato o liposomal). Además, en el contexto específico de falla o intolerancia a azoles orales, se identificaron recomendaciones de GPC a favor del cambio de un azol a otro en pacientes con aspergilosis tratados a largo plazo con azoles orales. Por otro lado, no se encontraron recomendaciones sobre el uso de isavuconazol oral en el contexto clínico de interés. Así, considerando la ausencia de evidencia que apoye la superioridad de isavuconazol oral sobre itraconazol oral en la población de interés, las recomendaciones de GPC a favor del uso de itraconazol oral en el contexto clínico de interés y la disponibilidad de itraconazol oral en la institución, cuyo costo es 150 veces menor que el costo de isavuconazol oral, no es posible hacer una recomendación a favor del uso de isavuconazol oral. Por lo expuesto, el IETSI no aprueba el uso de isavuconazol oral en pacientes adultos con aspergilosis invasiva con falla o intolerancia a voriconazol oral y posaconazol oral, que requieren terapia de mantenimiento posterior a la terapia de inducción con anfotericina B deoxicolato.

Tolnaftate inhibits ergosterol production and impacts cell viability of Leishmania sp.

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. The treatment of all forms of leishmaniasis relies on first-line drug, pentavalent antimonial, and in cases of drug failure, the second-line drug amphotericin B has been used. Besides the high toxicity of drugs, parasites can be resistant to antimonial in some areas of the World, making it necessary to perform further studies for the characterization of new antileishmanial agents. Thus, the aim of the present work was to evaluate the leishmanicidal activity of tolnaftate, a selective reversible and non-competitive inhibitor of the fungal enzyme squalene epoxidase, which is involved in the biosynthesis of ergosterol, essential to maintain membrane physiology in fungi as well as trypanosomatids. Tolnaftate eliminated promastigote forms of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum (EC50 ~ 10 µg/mL and SI ~ 20 for all leishmanial species), and intracellular amastigote forms of all studied species (EC50 ~ 23 µg/mL in infections caused by dermatotropic species; and 11.7 µg/mL in infection caused by viscerotropic species) with high selectivity toward parasites [SI ~ 8 in infections caused by dermatotropic species and 17.4 for viscerotropic specie]. Promastigote forms of L. (L.) amazonensis treated with the EC50 of tolnaftate displayed morphological and physiological changes in the mitochondria and cell membrane. Additionally, promastigote forms treated with tolnaftate EC50 reduced the level of ergosterol by 5.6 times in comparison to the control parasites. Altogether, these results suggest that tolnaftate has leishmanicidal activity towards Leishmania sp., is selective, affects the cell membrane and mitochondria of parasites and, moreover, inhibits ergosterol production in L. (L.) amazonensis.

Biological exploration of a novel 1,2,4-triazole-indole hybrid molecule as antifungal agent.

(2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(1,2,4-1H-triazol-1-yl)propan-2-ol (8 g), a new 1,2,4-triazole-indole hybrid molecule, showed a broad-spectrum activity against Candida, particularly against low fluconazole-susceptible species. Its activity was higher than fluconazole and similar to voriconazole on C. glabrata (MIC90 = 0.25, 64 and 1 µg/mL, respectively), C. krusei (MIC90 = 0.125, 64 and 0.125 µg/mL, respectively) and C. albicans (MIC90 = 0.5, 8 and 0.25 µg/mL, respectively). The action mechanisms of 8 g were also identified as inhibition of ergosterol biosynthesis and phospholipase A2-like activity. At concentration as low as 4 ng/mL, 8g inhibited ergosterol production by 82% and induced production of 14a-methyl sterols, that is comparable to the results obtained with fluconazole at higher concentration. 8 g demonstrated moderate inhibitory effect on phospholipase A2-like activity being a putative virulence factor. Due to a low MRC5 cytotoxicity, this compound presents a high therapeutic index. These results pointed out that 8 g is a new lead antifungal candidate with potent ergosterol biosynthesis inhibition.

Inhibition of ergosterol synthesis in Candida albicans by novel eugenol tosylate congeners targeting sterol 14α-demethylase (CYP51) enzyme.

This study is a continuation and extension of our previous study in which we synthesized seven novel eugenol tosylate congeners (ETC-1 to ETC-7) from a natural compound eugenol and checked their antifungal activity against different isolates of Candida albicans. All these ETCs showed potent antifungal activity to varying degrees. In this study, the aim is to evaluate the effect of most active compounds (ETC-5, ETC-6 and ETC-7) on ergosterol biosynthesis pathway and cellular viability in C. albicans by applying combined approach of in silico and in vitro methodologies. In silico studies were done through all atom molecular mechanics approach and free binding energy estimations, and in vitro study was done by estimating total intracellular sterol content and effect on expression of ERG11 gene. Furthermore, effect on cell viability by these compounds was also tested. Our results demonstrated that these ETCs target ergosterol biosynthesis pathway in C. albicans by inhibiting the lanosterol 14-α demethylase enzyme and also downregulates expression of its related gene ERG11. Furthermore, these ETCs exhibit potent fungicidal effect in cell viability assay, thus overall results advocating the claim that these tosylates have potential to be taken to next level of antifungal drug development.

Dehydrozingerone Inspired Discovery of Potential Broad-Spectrum Fungicidal Agents as Ergosterol Biosynthesis Inhibitors.

A series of dehydrozingerone derivatives were synthesized, and their fungicidal activities and action mechanism against Colletotrichum musae were evaluated. The bioassay result showed that most compounds exhibited excellent fungicidal activity in vitro at 50 µg mL-1. Compounds 13, 16, 18, 19, and 27 exhibited broad-spectrum fungicidal activity; especially, compounds 19 and 27 were found to have more potent fungicidal activity than azoxystrobin. The EC50 values of compounds 19 and 27 against Rhizoctonia solani were 0.943 and 0.161 µg mL-1 respectively. Moreover, compound 27 exhibited significant in vitro bactericidal activity against Xanthomonas oryzae pv. oryzae, with an EC50 value of 11.386 µg mL-1, and its curative effect (49.64%) and protection effect (51.74%) on rice bacterial blight disease was equivalent to that of zhongshengmycin (42.90%, 40.80% respectively). Compound 27 could also effectively control gray mold (87.10%, 200 µg mL-1) and rice sheath blight (100%, 200 µg mL-1; 82.89%, 100 µg mL-1) in vivo. Preliminary action mechanism study showed that compound 27 mainly acted on the cell membrane and significantly inhibited ergosterol biosynthesis in Colletotrichum musae.

The yeast pantothenate kinase Cab1 is a master regulator of sterol metabolism and of susceptibility to ergosterol biosynthesis inhibitors.

In fungi, ergosterol is an essential component of the plasma membrane. Its biosynthesis from acetyl-CoA is the primary target of the most commonly used antifungal drugs. Here, we show that the pantothenate kinase Cab1p, which catalyzes the first step in the metabolism of pantothenic acid for CoA biosynthesis in budding yeast (Saccharomyces cerevisiae), significantly regulates the levels of sterol intermediates and the activities of ergosterol biosynthesis-targeting antifungals. Using genetic and pharmacological analyses, we show that altered pantothenate utilization dramatically alters the susceptibility of yeast cells to ergosterol biosynthesis inhibitors. Genome-wide transcription and MS-based analyses revealed that this regulation is mediated by changes both in the expression of ergosterol biosynthesis genes and in the levels of sterol intermediates. Consistent with these findings, drug interaction experiments indicated that inhibition of pantothenic acid utilization synergizes with the activity of the ergosterol molecule-targeting antifungal amphotericin B and antagonizes that of the ergosterol pathway-targeting antifungal drug terbinafine. Our finding that CoA metabolism controls ergosterol biosynthesis and susceptibility to antifungals could set the stage for the development of new strategies to manage fungal infections and to modulate the potency of current drugs against drug-sensitive and -resistant fungal pathogens.

Synthesis of new bis-pyrazole linked hydrazides and their in vitro evaluation as antimicrobial and anti-biofilm agents: A mechanistic role on ergosterol biosynthesis inhibition in Candida albicans.

Literature reports suggest that pyrazoles and hydrazides are potential antimicrobial pharmocophores. Considering this fact, a series of nineteen conjugates containing hybrids of bis-pyrazole scaffolds joined through a hydrazide linker were synthesized and further evaluated for their antimicrobial activity against a panel of Gram-positive and Gram-negative bacteria along with Candida albicansMTCC 3017 strain. Although the derivatives exhibited good antibacterial activity, some of the derivatives (13d, 13j, 13l, 13p, and 13r) showed excellent anti-Candida activity with MICs values of 3.9 µg/ml, which was equipotent to that of the standard Miconazole (3.9 µg/ml), which has inspired us to further explore their anti-Candida activity. The same compounds were also tested for anti-biofilm studies against various Candida strains and among them, compounds 13l and 13r efficiently inhibited the formation of fungal biofilms. Field emission scanning electron micrographs revealed that one of the promising compound 13r showed cell damage and in turn cell death of the Candida strain. These potential conjugates (13l and 13r) also demonstrated promising ergosterol biosynthesis inhibition against some of the strains C. albicans, which were further validated through molecular docking studies. In silico computational studies were carried out to predict the binding modes and pharmacokinetic parameters of these conjugates.

Effect of pseudolaric acid B on biochemical and physiologic characteristics in Colletotrichum gloeosporioides.

In our previous study on natural products with fungicidal activity, pseudolaric acid B (PAB) isolated from Pseudolarix amabilis was examined to inhibit significantly mango anthracnose (Colletotrichum gloeosporioides) in vivo and in vitro. In the current study, sensitivity of 17 plant pathogenic fungi to PAB was determined. Mycelial growth rate results showed that PAB possessed strong antifungal activities to eleven fungi with median effective concentration (EC50) values ranging from 0.087 to 1.927µg/mL. EC50 of PAB against spore germination was greater than that of mycelium growth inhibition, which suggest that PAB could execute antifungal activity through mycelial growth inhibition. Further action mechanism of PAB against C. gloeosporioides was investigated, in which PAB treatment inhibited mycelia dry weight, decreased the mycelia reducing sugar and soluble protein. Furthermore, PAB induced an increase in membrane permeability, inhibited the biosynthesis of ergosterol, caused the extreme alteration in ultrastructure as indicated by the thickened cell wall and increased vesicles. These results will increase our understanding of action mechanism of PAB against plant pathogenic fungi.

Traditionally practiced medicinal plant extracts inhibit the ergosterol biosynthesis of clinically isolated dermatophytic pathogens.

OBJECTIVE: Dermatophytes are resistant to some available antibiotics. Development of new plant drugs to control drug resistant microbes is urgently needed. This study evaluates the antidermatophytic potential of 18 selected medicinal plants used by traditional healers in Theni and Virudhunagar Districts of Tamil Nadu, India. MATERIALS AND METHODS: Selected plant parts were collected, shade dried and powdered. Plant powders were extracted with ethanol and their antifungal potency was investigated against and clinical dermatophytes. The antioxidant effect of the extracts was screened using DPPH assay. Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) were estimated for the extracts. Ten plant extracts showed maximum MFC and they were selected to study their efficacy in interfering with ergosterol biosynthesis. Fluconazole-35µg/mL known fungicide was used as control. The most active extracts were taken for biocompatibility studies using 3T3-L1 fibroblast cell lines. RESULTS: The ethanol extract of Phyllanthus reticulates leaves showed good antifungal activity compared to other plant extracts. The MIC and MFC for Phyllanthus reticulatus were 62.5 and 250µg/mL against M. pachydermatitis and T. rubrum respectively. The ethanol extracts of Phyllanthus reticulatus, Coldenia procumbens, Thespesia populnea and Senna alata significantly lowered the release of ergosterol by 16.37, 19.53, 24.79, and 21.44%, respectively. The ethanol extract of Phyllanthus reticulatus leaves was more biocompatible to host cells than other active extracts. CONCLUSION: Our study indicated that the ethanol extract of Phyllanthus reticulates leaves showed promising activity against dermatophytes. It could be a potential material for future development of antidermatophytic agents.

Low molecular weight chitosan is an effective antifungal agent against Botryosphaeria sp. and preservative agent for pear (Pyrus) fruits.

Antifungal activity and preservative effect of a low molecular weight chitosan (LMWC) sample, derived from chitosan by enzymatic hydrolysis, were investigated in vitro and in vivo. A pathogenic fungal strain was isolated from decayed pear (Pyrus bretschneideri cv. "Huangguan") fruit and identified as Botryosphaeria sp. W-01. LMWC was shown to strongly inhibit W-01 growth based on studies of minimum inhibitory concentration (MIC) and effects on mycelial biomass and radial growth of the fungus. LMWC treatment of W-01 cells reduced ergosterol synthesis and mitochondrial membrane potential (ΔY), early events of apoptosis. Transmission electron microscopy and confocal laser scanning microscopy studies revealed that LMWC penetrated inside W-01 hyphae, thereby inducing ultrastructural damage. LMWC coating had a significant preservative effect on wounded and nonwounded pear fruits, by inhibiting postharvest decay and browning processes. LMWC activated several defense-related enzymes (polyphenol oxidase, peroxidase, chitinase), maintained nutritional value, and slowed down weight loss. Our findings indicate the strong potential of LMWC as a natural preservative agent for fruits and vegetables.

Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral.

BACKGROUND: Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood. RESULTS: In this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral. CONCLUSION: Our present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.

Antifungals: Mechanism of Action and Drug Resistance.

There are currently few antifungals in use which show efficacy against fungal diseases. These antifungals mostly target specific components of fungal plasma membrane or its biosynthetic pathways. However, more recent class of antifungals in use is echinocandins which target the fungal cell wall components. The availability of mostly fungistatic antifungals in clinical use, often led to the development of tolerance to these very drugs by the pathogenic fungal species. Thus, the development of clinical multidrug resistance (MDR) leads to higher tolerance to drugs and its emergence is helped by multiple mechanisms. MDR is indeed a multifactorial phenomenon wherein a resistant organism possesses several mechanisms which contribute to display reduced susceptibility to not only single drug in use but also show collateral resistance to several drugs. Considering the limited availability of antifungals in use and the emergence of MDR in fungal infections, there is a continuous need for the development of novel broad spectrum antifungal drugs with better efficacy. Here, we briefly present an overview of the current understanding of the antifungal drugs in use, their mechanism of action and the emerging possible novel antifungal drugs with great promise.

Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei.

Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min(-1)/0.24 min(-1); partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ(24)-sterol is a promising strategy for developing a new treatment for trypanosomiasis.

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols.

Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3ß,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3ß,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.

Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis.

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.

Synthesis and antimicrobial potential of nitrofuran-triazole congeners.

A series of 5-nitrofuran-triazole congeners were designed and synthesized by carrying out suitable structural modifications of the previously reported counterparts and were evaluated for their antimicrobial potential against both Gram-positive and Gram-negative bacterial strains. The compounds exhibited promising inhibition towards different Gram-positive pathogenic strains, while mild inhibitory effects were observed towards Gram-negative bacterial strains. Some of the compounds 9f, 9g, 9l and 9m were most active among the series, exhibiting a MIC value of 1.9 µg mL(-1) against different bacterial strains. The bactericidal activity was found to be in coherence with the bacterial growth inhibition data. The compounds were tested against fourteen different fungal strains and were found to possess excellent antifungal activities. Interestingly, all the compounds were equipotent to miconazole against one or more of the tested fungal strains and showed good activity against the other counterparts. A similar trend was observed in the case of their minimum fungicidal concentration values. Moreover, compound 9f exhibited two fold superior antifungal activity (MIC = 3.9 µg mL(-1)) than the standard miconazole (MIC = 7.8 µg mL(-1)) against C. albicans and C. parapsilosis. These compounds also effectively inhibited biofilm formation and compound 9f exhibited excellent anti-biofilm activity demonstrating a biofilm inhibitory concentration (BIC) as low as 0.8 µg mL(-1). A brief mechanistic study carried out on the most effective conjugate 9f indicated that it inhibits the ergosterol biosynthesis, thereby exhibiting antifungal effects. Molecular modelling studies carried out to study the binding modes of 9f correlates well with the antifungal activity and supported by ergosterol biosynthesis inhibition assay data. Most of these compounds exhibited ten times lower cytotoxicity toward the normal cells compared to the antimicrobial activity.