RESUMO
Three quinuclidine-based squalene synthase (SQS) inhibitors (BPQ-OH, E5700, and ER-119884) were evaluated against five Candida tropicalis strains with different susceptibility profiles to fluconazole (FLC), itraconazole (ITC), terbinafine (TRB), and amphotericin B (AMB). Although the quinuclidine derivatives were inactive against most C. tropicalis strains tested at concentrations up to 16 µg/ml, E5700 and ER-119884 showed antifungal activity against C. tropicalis ATCC 28707, a strain resistant to FLC, ITC, and AMB, with IC(50) and IC(90) values (i.e., the minimum inhibitory concentrations of the drugs determined as the lowest drug concentrations leading to a 50 and 90% of reduction in turbidity at 492 nm, respectively, after 48 h of incubation) of 1 and 4 µg/ml, respectively. Analysis of free sterols showed that non-treated C. tropicalis ATCC 28707 cells contained only 14-methylated sterols and that treatment with E5700 or ER-119884 led to a marked reduction of squalene content and the complete disappearance of the endogenous sterols. The fatty acid and phospholipid profiles in C. tropicalis ATCC 28707 cells grown in the presence of E5700 and ER-119884 were also markedly altered, with a large increase in the content of linolenic acid (C18:3), associated with a reduction in the content of linoleic (C18:2) and oleic (C18:1) acids. Treatment of C. tropicalis ATCC 28707 with E5700 or ER-119884 IC(50) values induced several ultrastructural alterations, including a marked increase in the thickness of the cell wall and the appearance of a large number of electron-dense vacuoles. In conclusion, our results indicated that E5700 and ER-119884 inhibited the growth and altered the lipid prolife and the ultrastructure of a multiple drug-resistant C. tropicalis strain. Therefore, such compounds could act as leads for the development of new treatment options against multidrug resistant Candida species.
Assuntos
Candida tropicalis/efeitos dos fármacos , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Ácidos Graxos/metabolismo , Piridinas/farmacologia , Quinuclidinas/farmacologia , Anfotericina B/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Candida tropicalis/química , Candida tropicalis/citologia , Candida tropicalis/metabolismo , Proliferação de Células/efeitos dos fármacos , Farmacorresistência Fúngica Múltipla , Ácidos Graxos/química , Ácidos Graxos/classificação , Fluconazol/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Histocitoquímica , Concentração Inibidora 50 , Itraconazol/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Piridinas/química , Quinuclidinas/químicaRESUMO
BACKGROUND: Sterol biosynthesis is an essential pathway for fungal survival, and is the biochemical target of many antifungal agents. The antifungal drugs most widely used to treated fungal infections are compounds that inhibit cytochrome P450-dependent C14α-demethylase (CYP51), but other enzymes of this pathway, such as squalene synthase (SQS) which catalyses the first committed step in sterol biosynthesis, could be viable targets. The aim of this study was to evaluate the antifungal activity of SQS inhibitors on Candida albicans, Candida tropicalis and Candida parapsilopsis strains. METHODS: Ten arylquinuclidines that act as SQS inhibitors were tested as antiproliferative agents against three ATCC strains and 54 clinical isolates of Candida albicans, Candida tropicalis and Candida parapsilopsis. Also, the morphological alterations induced in the yeasts by the experimental compounds were evaluated by fluorescence and transmission electron microscopy. RESULTS: The most potent arylquinuclidine derivative (3-[1'-{4'-(benzyloxy)-phenyl}]-quinuclidine-2-ene) (WSP1267) had a MIC50 of 2 µg/ml for all species tested and MIC90 varying from 4 µg/ml to 8 µg/ml. Ultrathin sections of C. albicans treated with 1 µg/ml of WSP1267 showed several ultrastructural alterations, including (a) loss of cell wall integrity, (b) detachment of the plasma membrane from the fungal cell wall, (c) accumulation of small vesicles in the periplasmic region, (d) presence of large electron-dense vacuoles and (e) significantly increased cell size and cell wall thickness. In addition, fluorescence microscopy of cells labelled with Nile Red showed an accumulation of lipid droplets in the cytoplasm of treated yeasts. Nuclear staining with DAPI revealed the appearance of uncommon yeast buds without a nucleus or with two nuclei. CONCLUSION: Taken together, our data demonstrate that arylquinuclidine derivatives could be useful as lead compounds for the rational synthesis of new antifungal drugs.