RESUMEN
Geranylgeranyltransferase I (GGTase I) catalyzes the post-translational transfer of lyophilic diterpenoid geranylgeranyl to the cysteine residue of proteins terminating with a CaaX motif such as Rho1p and Cdc42p. It has been shown that GGTase I activity is essential for viability of Saccharomyces cerevisiae and hence its inhibition is a potential antifungal target. From natural product screening, a number of azaphilones including one novel analog were isolated as broad-spectrum inhibitors of GGTase I. Isolation, structure elucidation, GGTase I inhibitory activities and antifungal activities of these compounds are described.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Antifúngicos/farmacología , Benzopiranos/farmacología , Inhibidores Enzimáticos/farmacología , Pigmentos Biológicos/farmacología , Saccharomyces cerevisiae/enzimología , Transferasas Alquil y Aril/antagonistas & inhibidores , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidoresRESUMEN
[structure: see text] Screening of natural products extracts led to the discovery of citrafungins A and B, two new fungal metabolites of the alkylcitrate family that are inhibitors of GGTase I of various pathogenic fungal species with IC(50) values of 2.5-15 microM. These compounds exhibited antifungal activities with MIC values of 0.40-55 microM. The isolation, structure elucidation, relative and absolute stereochemistry, and biological activities of citrafungins are described.
Asunto(s)
Alquenos/química , Transferasas Alquil y Aril/antagonistas & inhibidores , Antifúngicos/farmacología , Inhibidores Enzimáticos/farmacología , Lactonas/química , Alquenos/farmacología , Transferasas Alquil y Aril/metabolismo , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Lactonas/farmacología , Estructura Molecular , EstereoisomerismoRESUMEN
All previously characterized protein geranylgeranyltransferases I (GGTase I) are heterodimeric zinc metalloenzymes which catalyse geranylgeranylation of a cysteine residue in proteins containing a C-terminal CaaL motif (C, Cys; a, aliphatic amino acid; L, Leu). The alpha and beta subunits of GGTase I of Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and are essential for yeast viability. The authors are therefore investigating the role of geranylgeranylation in the related pathogenic yeast, Candida albicans, which is the most prevalent human fungal pathogen. GGTase I was purified to near homogeneity and also found to be a heterodimeric magnesium-dependent, zinc metalloenzyme displaying selectivity for CaaL-containing protein substrates. GGTase I peptide sequences were obtained from the purified protein and used to clone the genes encoding both subunits. CaRAM2 and CaCDC43 encode proteins that are 42 and 34% identical to their corresponding S. cerevisiae homologues, respectively, and 30% identical to their human homologues. Despite the limited overall homology, key zinc- and substrate-binding residues of the beta subunit (Cdc43p) are conserved. A unique feature of CaCdc43p is a tract of polyasparagine whose length varies from 6 to 17 residues among C. albicans strains and between alleles. Coexpression of both CaCDC43 and CaRAM2 under their native promoters complemented the ts defect of a S. cerevisiae cdc43 mutant but expression of the beta-subunit alone did not correct the growth defect, suggesting that hybrid GGTase I heterodimers are nonfunctional.