An antifungal benzimidazol reveals novel sterols after inhibition of Erg11.
ABSTRACT: Fungal infections are a serious health problem in clinics especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes and echinocandines. Due to emerging resistance to standard therapy and significant side effects and high costs for several antifungals.,there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities we previously screened a compound library, using a new type of activity-selectivity (AS) assay analysing both the antifungal activity and the compatibility with human cells at the same time. One compound, ((S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole (EMC120B12)), showed high antifungal activity against several species of pathogenic yeasts including C. glabrata and C. krusei, species which are highly refractory to antifungals, especially to the commonly used azoles. Here we could show by transcriptional profiling and sterol analysis that the target of this new antifungal compound is the ergosterol pathway. The effects of EMC120B12 on sterol biosynthesis mimic those of fluconazole, strongly indicating that EMC120B12 also targets ERG11 like the azols. But not only the marker sterol 14 methylergosta 8,24(28) dien 3β,6α diol accumulated in C. krusei under EMC120B12 treatment, but also hitherto unknown related sterols. The novel sterols have a 3β,6α diol structure. Furthermore, this is the first time that a benzimidazole structure has been shown to result in a block of the sterol pathway by accumulating marker sterols connected to ERG11 inactivation. In total, three biological replicates were performed. All experiments were performed as dye swaps. Thus, in total 18 arrays have been hybridzed. Hybridization experiments included an untreated reference sample and a sample of cells treated with either ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12), Fluconazole or Nocodazole. The array included one technical replicate of each probe.
Project description:Fungal infections are a serious health problem in the clinic especially in the immunocompromised patient. Disease ranges from widespread superficial vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses only a limited arsenal of antimycotica are available, including azoles, polyenes, echinocandines and amphothericin B. Due to emerging resistance to standard therapy and significant side effects for some antimycotica there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities we screened compound libraries, including combinatorial libraries as well as more than 30 000 pure compounds derived from organic synthesis for antimycotic activity. In total more than 100 000 compounds were screened using an innovative AS (activity-selectivity) assay analyzing both the antifungal activity and the compatability with human cells at the same time. One promising hit, a Benzimidazol-2-yl-alkylamine derivative, was developed in a series of lead compounds showing potent antifungal activity. ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12) showed the highest antifungal activity and best compatability with human cells in several cell culture models and against a number of different yeasts and clinical isolates. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol-pathway. In total, three biological replicates were performed. All experiments were performed as dye swaps. Thus, in total six arrays have been hybridzed. Hybridization experiments included an untreated reference sample and a sample of cells treated with ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12). The array included one technical replicate of each probe.
Project description:Sterols are essential nutrients for insects because, in contrast to mammals, no insect (or arthropod for that matter) can synthesize sterols de novo. Cholesterol is the most common sterol in insects, but it is not found in plants in large quantities; plant-feeding insects typically generate their cholesterol by metabolizing phytosterols. However, different plants species can contain different types of phytosterols, and some phytosterols are not readily converted to cholesterol. In this study we examined, using artificial diets containing single sterols, how typical (cholesterol and stigmasterol) and atypical (cholestanol and cholestanone) sterols/steroids affect the performance of a generalist caterpillar (Helicoverpa zea), restricting this analysis to midgut tissue because this is where sterol/steroid absorption occurs, and the midgut is the putative site of dietary sterol/steroid metabolism. In general, H. zea performed best on the cholesterol and stigmasterol treatments; performance was reduced on cholestanol, and was very poor on cholestanone. We compared the transcript profiles of larval guts in response to differentially suitable sterols, using the optimal sterol, cholesterol, as a control, using a two-color reference design microarray experiment. Midgut gene expression patterns differed between the treatments; relative to cholesterol, differences were lowest on the stigmasterol treatment, intermediate on the cholestanol treatment, and greatest on the cholestanone treatment. Transcriptional profiling comparing Helicoverpa zea gut tissue from third instar larvae exposed to four different dietary sterols, namely Cholesterol (CON), Cholestanol (ChStanol), Cholestan-3-one (Ch3one) and Stigmasterol (Stigma). Two-color reference design. Biological replicates: 4 (5 individuals per replicate). 12 samples total.
Project description:The present study describes a novel mechanism of antifungal resistance affecting the susceptibility of both the azole and echinocandin antifungals in an azole-resistant isolate from a matched pair of C. parapsilosis isolates obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate including upregulation of ERG1, ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, ERG27, DAP1 and UPC2, of the ergosterol biosynthesis pathway. Whole genome sequencing revealed a mutation in the ERG3 gene leading to a G111R amino acid substitution in the resistant isolate. Subsequent introduction of this allele in the native ERG3 locus in the susceptible isolate resulted in a fluconazole MIC of >64 mg/ml and a caspofungin MIC of 8 mg/ml. Corresponding allelic replacement of the wildtype allele for the mutant allele in the resistant isolate resulted in a drop in MIC to 1 mg/ml for both fluconazole and caspofungin. Sterol profiles indicated a loss of sterol demethylase activity as a result of this mutation. This work demonstrate that this G111R mutation is wholly responsible for the resistant phenotype in the C. parapsilosis resistant isolate and is the first report of this multidrug resistance mechanism. Overall design: RNA-sequencing of an antifungal drug-susceptible isolate and a multi-drug resistant isolate of C. parapsilosis.
Project description:A systematic approach allowing the identification of the molecular way-of-action of novel potential drugs represents the golden-tool for drug-discovery. While high-throughput screening technologies of large libraries is now well established, the assessment of the drug targets and mechanism of action is still under development. Taking advantage of the yeast model Saccharomyces cerevisiae, we herein applied BarSeq, a Next Generation Sequencing-based method to the analysis of both haploinsufficiency and homozygous fitness effects of a novel antifungal drug ('089') compared to the well-known antifungal ketoconazole. '089' was a novel compound identified in during a screen for antifungal drugs, as it was showing fungicidal effects, and able to affect the yeast fitness at the mitochondrial level (Stefanini et al., 2010. (Dissection of the Effects of Small Bicyclic Peptidomimetics on a Panel of Saccharomyces cerevisiae Mutants;.J Biol Chem, 285: 23477-23485.) Integrative bioinformatic analysis of BarSeq, whole genome expression analysis and classical biological assays identified the target and cell pathways affected by the novel antifungal. Confirmation of the effects observed in the yeast model and in pathogenic fungi further demonstrated the reliability of the multi-sided approach and the novelty of the targets and way-of-action of the new class of molecules studied representing a valuable source of novel antifungals.
Project description:Analyzing culture supernatants of yeast and hyphal cells of Candida albicans by mass spectrometry, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome of this human pathogen. By sequence homology, we assigned three yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, in addition to Rbe1p and Rbt4p to a novel family of proteins. Correspondent with our secretome analysis RBE1 was expressed in blastospores and opaque cells, whereas transcription was down-regulated in hyphae. On the contrary, RBT4 was up-regulated in hyphae and down-regulated in opaque cells. Remarkably, transcription of RBT4 and RBE1 was each up-regulated in blastospores of ∆rbe1 or hyphae of ∆rbt4 deletion strains, respectively, indicating a compensatory function of both proteins. In a ∆rbe1/∆rbt4 double deletion strain, genome-wide transcriptional analysis showed differential transcription of a limited set of genes that are also implicated in virulence and oxidative stress response. In this context, deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in the ∆rbe1/∆rbt4 double deletion strain, where virulence was strongly affected. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leukocytes (neutrophils). Therfore, our data suggest that the crucial contribution of both C. albicans pathogenesis-related proteins for in vivo virulence results at least partially from reduced survival in phagocytes. Experiments were performed under blastospore (YPD) as well as hyphae (alpha-MEM)-inducing conditions. In total, three biological replicates were performed for each condition. All experiments were performed as dye swaps. Thus, in total six arrays have been hybridzed for each comparison (alpha-MEM and YPD, respectively). Hybridization experiments included a reference strain (C. albicans SC5314) and a double deletion strain (C. albicans MRC27). The array included one technical replicate of each probe.
Project description:Mycobacterium neoaurum (Mn), an efficient sterol consumer, has been modified to transform sterols to produce valuable steroid intermediates, which can be widely used as precursors in the synthesis of steroid hormones.To deepen our understand of the underlying mechanisms of Mn to the in vitro and in vivo environment during growth on a critical carbon source of sterol and accumulation of valuable steroid intermediates, transcriptome studies were performed to characterize the differences between wide-type Mn and various mutant Mn strains for steroid accumulation. Totally, five Mn strains were investigated, including wide-type Mn ATCC25795 cultured without (Mn-C) and with sterol addition (Mn-CC), mutant Mn ATCC25795 strains producing 9OHAD (Mn-9OHAD), ADD (Mn-ADD), and 1,4-HBC (Mn-HBC), respectively.
Project description:All but a few eukaryotes die without oxygen and respond dynamically to changes in the level of oxygen available to them. One ancient oxygen-requiring biochemical pathway in eukaryotes is the pathway for the biosynthesis of sterols, leading to cholesterol in animals and ergosterol in fungi. Mutations in this pathway are a frequent cause of azole drug resistance in pathogenic fungi. The regulatory mechanism for the sterol pathway is also widely conserved between animals and fungi and is centred on a transcription activator, SREBP, that forms part of a sterol-sensing complex. However, in one group of yeasts – the Saccharomycotina, which includes the major pathogen Candida albicans – control of the sterol pathway has been taken over by an unrelated regulatory protein, Upc2. We show here by analysis of the yeast Yarrowia lipolytica that the evolutionary switch from SREBP to Upc2 was a two-step process in which Upc2 appeared in an ancestor of Saccharomycotina, and SREBP subsequently degenerated and lost its sterol-regulatory function while retaining an ancient role in filamentation. RNA was isolated from Y. lipolytica wildtype (JMY2900) in normoxia (21% oxygen, 4 biological replicates), wildtype (JMY2900) in hypoxia (1% oxygen, 5 biological replicates), upc2 deletion (SMY2) in hypoxia (1% oxygen, 3 biological replicates), and from sre1 (SMY5, SMY8) deletion in hypoxia (1% oxygen, 2 biological replicates). Gene expression was determined using strand-specific RNA-seq.
Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately up-regulated with ERG11 in a fluconazole resistant clinical isolate as compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug susceptible strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling Clinical isolates S1 (susceptible) and S2 (resistant), genome strain SC5314, UPC2 disruption strains (UPC2M4A and UPC2M4B), UPC2 re-integrant strains of non-mutated UPC2 allele (UPC2M2K21A and UPC2M2K21B), and UPC2 re-integrant strains of mutated UPC2 allele (UPC2M2K31A and UPC2M2K31B) were grown for 3 hours until log phase. RNA was extracted for each isolate/strain. Experiments were performed in duplicate.
Project description:A systematic approach allowing the identification of the molecular way of action of novel potential drugs still represents the golden-tool for drug-discovery researchers. While high-throughput screening technologies of large libraries is now well established, the assessment of the drug targets and mechanism of action is still under development. Taking advantage of the yeast model Saccharomyces cerevisiae, we herein applied BarSeq, a Next Generation Sequencing based method to the analysis of both haploinsufficiency and homozygous fitness effects of a novel antifungal drug compared to the well-known antifungal, ketoconazole. Integrative bioinformatic analysis of BarSeq, whole genome expression analysis and classical biological assays identified the target and cell pathways affected by the novel antifungal. Confirmation of the effects observed in the yeast model as well as in pathogenic fungi further demonstrated the reliability of the multi-sided approach and the novelty of the targets and mode of action of the new class of molecules studied that thus represent a valuable source of novel antifungals. Overall design: Two-condition experiments, drug vs. DMSO-treated S. cerevisiae cells. Analyzed drugs: two concentrations of ketoconazole (2.5 uM and 50 uM) and one concentration of the novel proposed antifungal 089. Biological replicates: 3 control replicates for each experiment.