Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action

BACKGROUND Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

Stereoselective Metabolism of the Sterol Biosynthesis Inhibitor Fungicides Fenpropidin, Fenpropimorph, and Spiroxamine in Grapes, Sugar Beets, and Wheat.

Metabolism of chiral pesticides in crops is typically studied using achiral analytical methods and, consequently, the stereoisomer composition of residues is unknown. In this study, we developed an enantioselective GC-MS/MS method to quantify residues of the fungicides fenpropidin, fenpropimorph, and spiroxamine in plant matrices. In field trials, the fungicides were applied to grapevines, sugar beets, or wheat. Fenpropidin was metabolized with no or only weak enantioselectivity. For fenpropimorph, slightly enantioselective metabolism was observed in wheat but more pronounced in sugar beets. This enantioselectivity was due to different rates of metabolism and not due to interconversion of enantiomers. The four stereoisomers of spiroxamine were also metabolized at different rates, but selectivity was only found between diastereomers and not between enantiomers. trans-Spiroxamine was preferentially degraded in grapes and cis-spiroxamine in wheat. These findings may affect the consumer dietary risk assessment because toxicological end points were determined using racemic test substances.

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.

Sensitivity of California Isolates of Uncinula necator to Trifloxystrobin and Spiroxamine, and Update on Triadimefon Sensitivity.

Sensitivities of Uncinula necator to spiroxamine and trifloxystrobin were established by assay of 36 and 35 isolates, respectively, recovered from California grape vineyards in 2002 and increased as single-spore lines for laboratory testing. Twenty-nine single-spore isolates also were evaluated for levels of sensitivity to the fungicide triadimefon to determine if there had been a reversion to sensitivity following the development of resistance in 1986. Although triadimefon use was limited after 1992, other demethylation inhibitor (DMI) fungicides (fenarimol and myclobutanil) were used extensively in California vineyards. For spiroxamine, the sample mean value of the median effective concentration (EC50 value) was 365 µg/liter (95% confidence interval [CI] from 251 to 531 µg/liter) and values were distributed log-normally. The corresponding mean for trifloxystrobin was 12.8 µg/liter bounded by 8.9 to 18.5 µg/liter for the 95% CI. State-wide, the triadimefon mean EC50 was 8.8 mg/liter, bounded by a 5.3 to 14.5 mg/liter 95% CI, and those values were significantly higher than those obtained in the last assay 12 years earlier. Significant differences in sensitivity of U. necator to triadimefon were detected at a regional scale by comparison of mean EC50 values of frequency distributions representative of regions within California, although the relations between those regions were different from the prior survey.