Use of cilofungin as direct fluorescent probe for monitoring antifungal drug-membrane interaction

Abstract

Cilofungin is an antifungal cyclopeptide which inhibits cell wall (1,3)-p1-glucan biosynthesis in fungal organisms, and its action against Candida albicans (1,3)-Il-glucan synthase has been widely studied. Since glucan synthase inactivation is thought to partially result from perturbations of the membrane lipid environment, the interaction of cilofungin with fungal membranes and phosphatidylcholine membrane vesicles was studied. Cilofungin, which contains two independent aromatic groups, has an excitation maximum of 270 nm and an emission maximum of 317 nm in aqueous solution. Comparison of the fluorescence properties of cilofungin with those of the analogs pneumocandin Bo, N-acetyl-tyrosinamide, and 4-hydroxybenzamide indicated that the emission of cilofungin largely derived from the p-octyloxybenzamide side chain. Microsomal membranes from Saccharomyces cerevisiae, C. albicans, and phosphatidylcholine membrane vesicles induced a blue shift in the cilofungin emission spectrum and increased the cilofungin steady-state emission anisotropy, providing direct evidence for a cilofungin-membrane interaction. Cilofungin interacted more strongly with membranes of C. albicans than with those of S. cerevisiae, correlating with previous findings that C. albicans is far more susceptible than S. cerevisiae to the action of cilofungin. These findings support the hypothesis that drug-induced inhibition of the (1,3)-,B-glucan synthesis results from the perturbation of the membrane environment and the interaction with the glucan synthase complex combined. The study demonstrated ways in which the fluorescence properties of drugs can be used to directly evaluate drug-membrane interactions and structure-activity relationships.New antifungal drugs that are directed against specific fungal targets and that cause minimal toxicity to the host have attracted considerable interest in recent years. One such target is the plasma membrane-localized (1,3)-3-glucan synthase (GS; EC 2.4.1.34), which catalyzes the biosynthesis of (1,3)-1-glucan, an essential structural polymer of the cell wall (17,36). A number of antifungal drugs have been targeted against GSs from various fungi including the lipopeptide echinocandin-like class of inhibitors (12,30,31,35), aculeacin A (23, 24), cilofungin (8, 10, 11), pneumocandins (12, 31), and the papulacandins (2, 16, 26, 37), which are lipid-like saccharides. Each of these compounds contains fatty acid side chains, which have been shown to be essential for the actions of cilofungin (34,38) and papulacandin B (37). Although various lipophilic compounds are known to rapidly inactivate fungal glucan (18,25) and chitin synthases (25), the mechanism by which these compounds interfere with GS has only recently begun to attract considerable attention (17,18,36).Drug-membrane interactions can be probed directly by fluorescence spectroscopic techniques, provided that the drug contains a structural moiety exhibiting fluorescence properties (6, 7). In the present study, we demonstrated that cilofungin, which contains...

The Candida albicans Cdr2p ATP‐binding cassette (ABC) transporter confers resistance to caspofungin

“…The increase in cell wall chitin and mannan may therefore provide a salvage pathway to bypass inhibition of the glucan synthase (Popolo et al ., 2001). Likewise, echinocandins interfere with the plasma membrane properties (Ko et al ., 1994), and ABC transporters such as Cdr1p and Cdr2p have been implicated in changes in plasma membrane permeability (Kohli et al ., 2002; Smriti et al ., 2002). Thus, we propose that clinical caspofungin resistance in vivo may perhaps be the consequence of a combination of altered plasma membrane features, candin target gene mutations, as well as Cdr2p‐mediated active drug efflux.…”

Antifungal agents: chemotherapeutic targets and immunologic strategies

“…Echinocandins, on the other hand, have fungicidal activity both in vitro and in animal models (48,272,283). It has been suggested that the limited spectrum of echinocandins and other ␤-(1,3)-glucan synthase inhibitors may be due in part to their interaction with the fungal membranes (150,151). Echinocandins have been chemically modified to produce semisynthetic analogs with improved pharmacological properties.…”

Membrane-Anchored Cyclic Peptides as Effectors of Mitochondrial Oxidative Phosphorylation

“…CLPs with novel structures continue to be discovered, 1 and structural variants can be produced by genetic and chemical modifications. 2,3 The hydrophobic tails of CLPs insert into biological membranes, 4 where they have the ability to interact with the transmembrane surfaces of integral membrane proteins loosely, like annular lipid, or more tightly, by binding at specific lipid binding sites. 5−8 Once tethered to the membrane, CLP headgroups can interact with those regions of protein normally in contact with phospholipid headgroups and immediately above.…”

“…5−8 Once tethered to the membrane, CLP headgroups can interact with those regions of protein normally in contact with phospholipid headgroups and immediately above. 4 Major areas of research on CLPs focus on the surfactant properties of some CLPs 9 and on their development for use as antibiotics and antifungals. 1,2 However, CLPs also represent a largely untapped resource of compounds with the potential to modify the activity of integral membrane proteins, including enzymes, pumps, transporters, ion channels, and receptors.…”