Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack.

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

The combination effects of phenolic compounds and fluconazole on the formation of ergosterol in Candida albicans determined by high-performance liquid chromatography/tandem mass spectrometry.

A liquid chromatography/tandem mass spectrometry (LC/MS) with atmospheric pressure chemical ionization (APCI) for the quantification of ergosterol, lanosterol, and squalene was developed to evaluate the combination effects of phenolic compounds with fluconazole on ergosterol biosynthesis in Candida albicans. The three analytes were separated by a column of C18 and were quantified without interference with each other using positive mode tandem mass spectrometry (MS/MS). Molecular ions of ergosterol and lanosterol were detected as the [M+H-H2O]+ ion species at m/z 380 and 410, whereas squalene appeared as the [M+H]+ ion species at m/z 412. On fragmentation of ergosterol, lanosterol, and squalene, the product ions at m/z 69, 149, and 109, respectively, were present as major fragments. These product ions were used for the quantification of them in multiple reaction monitoring acquisition mode. The relationship between signal intensity and the analytes' concentration was linear over the concentration range of 0.05-10 microg/ml. Following the treatment of C. albicans with fluconazole in combination with albicanyl caffeate, resveratrol, and 3,4'-difluorostilbene, respectively, the content of ergosterol in both the sensitive and resistant C. albicans showed depletion, whereas the squalene showed accumulation especially in the sensitive isolates determined with the method developed.

Antifungals: mechanism of action and resistance, established and novel drugs.

Serious fungal infections, caused mostly by opportunistic species, are increasingly common in immunocompromised and other vulnerable patients. The use of antifungal drugs, primarily azoles and polyenes, has increased in parallel. Yet, established agents do not satisfy the medical need completely: azoles are fungistatic and vulnerable to resistance, whereas polyenes cause serious host toxicity. Drugs in clinical development include echinocandins, pneumocandins, and improved azoles. Promising novel agents in preclinical development include several inhibitors of fungal protein, lipid and cell wall syntheses. Recent advances in fungal genomics, combinatorial chemistry, and high-throughput screening may accelerate the antifungal discovery process.

Assessment of the essentiality of ERG genes late in ergosterol biosynthesis in Saccharomyces cerevisiae.

Isogenic strains of yeast were constructed, differing only in insertionally inactivated genes for ergosterol biosynthesis. These and their allelic wild-types were grown in competition to ascertain growth differences and any selective advantage for organisms producing sterols with or without specific features of ergosterol. In every instance tested, the wild-type allele afforded a competitive advantage over the isogenic pair producing modified sterol structures instead of ergosterol. A general trend was seen in which the earlier in the biosynthetic pathway that a mutation occurred, the less able the strain producing the defective sterols could compete with the ergosterol-producing strains.