Loss-of-function ROX1 mutations suppress the fluconazole susceptibility of upc2AΔ mutation in Candida glabrata, implicating additional positive regulators of ergosterol biosynthesis

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress induced transcription factors such as Upc2 and Upc2A as well as homologs of Sterol Response Element Binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14α methyl-ergosta-8,24(28)-dien-3β, 6α-diol, relative to upc2AΔ. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata.

Glutamine metabolism modulates azole susceptibility in Trypanosoma cruzi amastigotes

The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the role of metabolic heterogeneity. We found that limiting exogenous glutamine protects actively dividing amastigotes from ergosterol biosynthesis inhibitors (azoles), independent of parasite growth rate. The antiparasitic properties of azoles are derived from inhibition of lanosterol 14α-demethylase (CYP51) in the endogenous sterol synthesis pathway. We find that carbons from 13C-glutamine feed into amastigote sterols and into metabolic intermediates that accumulate upon CYP51 inhibition. Incorporation of 13C-glutamine into endogenously synthesized sterols is increased with BPTES treatment, an inhibitor of host glutamine metabolism that sensitizes amastigotes to azoles. Similarly, amastigotes are re-sensitized to azoles following addition of metabolites upstream of CYP51, raising the possibility that flux through the sterol synthesis pathway is a determinant of sensitivity to azoles and highlighting the potential role for metabolic heterogeneity in recalcitrant T. cruzi infection.

Glutamine metabolism modulates azole susceptibility in Trypanosoma cruzi amastigotes

The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the potential role of metabolic heterogeneity in modulating susceptibility of intracellular amastigotes to trypanocidal compounds. We found that limiting exogenous glutamine protects actively dividing amastigotes from ergosterol biosynthesis inhibitors (azoles), independent of parasite growth rate. The antiparasitic properties of azoles are derived from inhibition of lanosterol 14α-demethylase (CYP51) in the endogenous sterol synthesis pathway. We find that carbons from 13C-glutamine feed into amastigote sterols and into metabolic intermediates that accumulate upon CYP51 inhibition. Consistent with a model that decreased flux through the sterol biosynthetic pathway is protective for intracellular amastigotes exposed to azoles, we find that amastigotes become re-sensitized to azoles following addition of metabolites upstream of CYP51. Our results highlight the potential role of metabolic heterogeneity in recalcitrant T. cruzi infection, an avenue that is currently underexplored.

Polypharmacology in the Treatment of Chagas Disease

The current treatment of Chagas disease is based on monopharmacology where the used drugs have limited efficacy and severe side effects. In order to overcome these limitations, some tools have been described including the development or isolation of new drugs, drug repositioning, and polypharmacology. Here, we review the polypharmacology strategy where compounds belonging to different structural chemotypes were combined in order to affect different biochemical pathways of T. cruzi parasite. Therefore ergosterol biosynthesis inhibitors, anti-inflammatory agents, cardiac dysfunction drugs, trypanothione reductase inhibitors, vitamins, between others, were combined looking for new anti-Chagas treatment. Natural products were also used in the application of this strategy.

Linalool modulates dermatophyte susceptibility to azole drugs

This study investigated the monoterpene linalool and its resistance modulating activity involving ergosterol biosynthesis inhibitors (ketoconazole, fluconazole, and itraconazole) in strains of Microsporum spp. and Trichophyton spp. The minimum inhibitory concentration (MIC) of test-drugs were determined by microdilution. The modulating effect of linalool was evaluated by determining the MIC of the antifungals in the presence of subinhibitory concentrations of linalool. We also investigated the association effect (checkerboard) of linalool together with ketoconazole and itraconazole. The fungi became more sensitive to ketoconazole and itraconazole in the presence of linalool. The linalool and azole drug associations presented synergism.

PotentIn VitroAntiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis

ABSTRACTLeishmaniases comprise a spectrum of diseases caused by protozoan parasites of theLeishmaniagenus. 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 thatLeishmania amazonensisis 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 againstL. 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.