Pyridines and Pyrimidines Mediating Activity against an Efflux-Negative Strain of Candida albicans through Putative Inhibition of Lanosterol Demethylase

Abstract

The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased ERG10 transcription results in elevated specific ␤-galactosidase activity, was used to find novel inhibitors of ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major fungal pathogen Candida albicans (MICs > 64 g ⅐ ml ؊1 ). However, a strain of C. albicans lacking the Cdr1p and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 g ⅐ ml ؊1 ), suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol. Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively mediate their antifungal activity against C. albicans via lanosterol demethylase.The sterol biosynthesis pathway, which is taken here to include the mevalonate pathway, converts acetyl coenzyme A (acetyl-CoA) into farnesyl-diphosphate, which subsequently leads to the synthesis of ergosterol. This metabolic pathway has many putative targets that vary in their degrees of genetic conservation relative to fungal and human orthologs. Furthermore, the exploitation of many of these targets has led to therapeutics for the treatment of human disease, and these targets are therefore considered proper objects of drugs. The therapeutics include drugs used for the treatment of fungal infection (azoles, allylamines, thiocarbamates, and morpholines, which all act against fungal targets that have human homologs [21]) and also for the treatment of osteoporosis (2) and hypercholesterolemia (e.g., reference 19).Dimster-Denk and Rine (5) and Dixon et al. (7) developed virtually identical gene reporter assays for Saccharomyces cerevisiae for the identification of fungal sterol biosynthesis inhibitors that could serve as chemical starting points for new drug discovery programs. The attractiveness of this assay resides in the fact that it can in principle identify inhibitors of any of the essential steps in the pathway. Furthermore, since this is a cell-based assay, all of these inhibitors are expected to have at least some degree of antifungal activity. The use of this assay has led to the identification of a new class of antifungal pyri...

Production of plant sesquiterpenes in Saccharomyces cerevisiae: Effect of ERG9 repression on sesquiterpene biosynthesis

“…Nevertheless, this strategy did not improve appreciably the production of the target sesquiterpenes but resulted in further production of farnesol as an FPP-derived by-product. Consistent with this finding are accumulation of farnesol in Candida albicans (Buurman et al, 2004;Hornby et al, 2003) as a consequence of treatment with zaragozic acid, which is potent inhibitor of squalene synthase. Furthermore, deletion of ERG9 gene in S. cerevisiae was reported to lead to accumulation of farnesol in S. cerevisiae (Song, 2003).…”

Candida dubliniensis in a Brazilian family with an HIV 1- infected child: identification, antifungal susceptibility, drug accumulation and sterol composition

“…In this report C. dubliniensis isolates showed a reduced ergosterol level in comparison to C. albicans reference strains (ATCC 90028 and 28516). The ergosterol level has shown values up to 80% to both C. albicans reference strains (ATCC 90028 and 28516), these results are similar to that described previously (3,9). The sterol profile in this work suggests an interesting difference between C. dubliniensis and C. albicans, in regarding that both species are closely related phylogenetically further investigation about the sterol profiles in other C. dubliniensis isolates is required.…”

Utilization of Target-Specific, Hypersensitive Strains of Saccharomyces cerevisiae To Determine the Mode of Action of Antifungal Compounds

“…1C and D). The compound concentrations at which these changes occur, with ergosterol IC 50 s of 10 to 20 g ml Ϫ1 , and their MICs are in line with the previously established quantitative relationship between antifungal activity and inhibition of sterol synthesis in C. albicans DSY654 in this assay (1). This strongly suggests that compound A mediates its antifungal activity via inhibition of Erg7p whereas compound B seems to inhibit both Erg7p and Erg11p.…”

“…However, each compound showed an MIC of 16 g ml Ϫ1 against C. albicans DSY654, a strain from which genes encoding efflux pump CDR1 and CDR2 have been deleted (27). Therefore the mode of action of both compounds was assessed by analyzing the alterations that occurred in the sterol composition of C. albicans DSY654 upon treatment with these inhibitors (1,25). Compound A resulted in a decrease of ergosterol level and accumulation of 2,3-oxidosqualene, whereas compound B showed an accumulation of both lanosterol and 2,3-oxidosqualene ( Fig.…”