By: Chloe McClanahan, BioFiles 2009, 4.3, 10.
Antifungal compounds have been overshadowed by antibacterials in research interest and application due to the greater impact bacterial infections have had on health. Resistance to antibacterial drugs and the resultant clinical impact is of widespread concern regarding public health. However, resistance by pathogenic fungal infections to drug treatment has become more common in the last 20 years as well. Some mechanisms of the development of fungal resistance have similarities to those of the development of drug resistance in bacteria, and knowledge of those bacterial mechanisms is being applied to understanding fungal drug resistance.
Several key antibiotic compounds function by targeting the integrity of the cell. Many compounds increase the porosity of the cell wall or membrane, or interfere with key steps in the synthesis of cell walls. While prokaryotic bacteria and eukaryotic fungi do not have identical cell wall and membrane components, there are corresponding lipids and key structural molecules. As a result, similar to antibacterials, most antifungal compounds work because they directly or indirectly damage the cell wall or cell membrane.
The fungal cell wall is composed of multiple layers, with mannoproteins being predominantly expressed at the external surface (see Figure 1). An underlayer of β-glucan creates a supporting matrix for the mannoproteins and provides structural rigidity to the cell wall. The glucan structure is strengthened by frequent β(1→3) and additional β(1→6) linkages and by chitin interspersed with the β-glucan. Mannoproteins and glucan make up more than 80% of the cell wall composition, while chitin represents less than 2%. The plasma membranes of fungi are primarily composed of ergosterol, analogous to cholesterol in animal cells. Since ergosterol and cholesterol have sufficient structural differences, the majority of chemicals found to act as fungicides target ergosterol biosynthesis or cell membrane porosity and do not cross react with host cells.
Figure 1. Structure of the yeast cell wall. The wall is primarily composed of mannoproteins and β-glucan that is linked (1→3) and (1→6). Ergosterol is the major lipid component of the underlying plasma membrane.
Several reviews of antifungal compounds group them into structural classes and have associated certain structures with particular modes of actions. Examples of some of the key structures of fungicides are shown in Figure 2. The binding and synthesis of ergosterol, the major cell membrane component, are the targets for several antifungal structures. The azoles and triazoles interfere with the ergosterol biosynthesis pathway by inhibiting cytochrome P450-dependent 14α-demethylase and blocking the oxidative removal of 14α-methyl from lanosterol. This incomplete processing of lanosterol results in an increase in ergosterol precursors and a decrease in ergosterol, leading to structural changes in the lipid membrane. Azoles have also been reported to inhibit membrane-surface enzymes and lipid biosynthesis.
Figure 2. Examples of antifungal structure classes. a. Fluconazole (triazole) b. Terbinafine (allylamine) c. Nystatin A1 (polyene) d. Aculeacin A (echinocandin).
Allylamines, of which terbinafine (Cat. No. T8826) is the most common example, also block ergosterol biosynthesis, but at an earlier step. Terbinatine inhibits the enzyme squalene epoxidase, which participates in the conversion of squalene to lanosterol. The resulting build-up of squalene is toxic to the fungal cell. A third structural class, polyenes, increases the permeability of the plasma membrane. Amphotericin B (Cat. No. A4888), a polyene with high affinity for sterol binding, is one of the most potent antifungal drugs; its mechanism produces pores in the membrane surface of the yeast, resulting in leakage of the cell contents. (Odds, et al., 2003).
In addition to the plasma membrane and its lipid surface, fungicidal compounds may damage the cell wall of yeast. The major cell wall component β(1→3) glucan is the target of echinocandins and aculeacins such as aculeacin A (Cat. No. A7603). Echinocandins are semisynthetic lipopeptides that competitively inhibit β-glucan synthetase; the mechanism of action is not well defined but does not involve cytochrome P450 inhibition or P-glycoprotein transport (Kauffman and Carver, 2008).
Chitin is a trace, critical component of the fungal cell wall, and some inhibitors of chitin synthesis demonstrate antifungal activity. Members of this family of antifungals (i.e., polyoxins and nikkomycins) have structures analogous to UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). This nucleoside phosphate is a glycosyl donor substrate for chitin synthesis, and the antifungals act as competitive substrates to inhibit chitin synthetase (Hector, 1993).
Not all antifungal compounds have known mechanisms of action, and some of them are relatively unique. While there are several antibacterials that function by preventing DNA or RNA replication, 5-fluorocytosine (Cat. No. F7129) is a novel antifungal in that its mechanism of action involves blocking DNA synthesis and inhibiting thymidylate synthetase. Sordarin (Cat. No. S1442) is one of the few compounds that selectively inhibit fungal protein synthesis. Other antifungal antibiotics target sphingolipid biosynthesis and electron transport (Gupte, et al., 2002). The mode of action of griseofulvin (Cat. No. G4753) is not completely clear, but it has been speculated that griseofulvin inhibits microtubule binding within the mitotic spindle, weakening the cell structure (Odds, et al., 2003).
Drug resistance in fungi, especially to azoles, is becoming more prevalent clinically, and the mechanisms of drug resistance are similar to those present in bacteria. Several factors contribute to multidrug resistance in yeasts, including the mutation of genes and overexpression of proteins that act as efflux pumps (Monk and Goffeau, 2008). Fungi contain both ATP-binding cassette (ABC) transporter and major facilitator superfamily (MFS) transporter gene families.
The multidrug resistance process for fungi has been most analyzed for Saccharomyces cerevisiae, where it is called pleiotropic drug resistance (PDR) (Rogers, et al., 2001). In S. cerevisiae, point mutations occur in the genes for the transcription regulatory factors Pdr1p and Pdr3p. These mutations (called “gain-offunction” mutations) activated downstream target genes, including the ABC transporter genes and MFS transporter genes. The products of these genes are efflux pumps that transport drug compounds out of the cell, reducing the intracellular concentration to a sublethal level.
Some research reports on antifungals have found greater efficacy with combinations of antifungal drugs that use different mechanisms of action. The same process has been applied to other compounds in looking for ways to overcome fungal resistance. A variety of immunosuppressive compounds, including cyclosporin and D-octapeptides (Monk, et al., 2005), have been tested and found to counteract antifungal resistance due to efflux pumps. Cernicka, et al. screened a synthetic compound library and identified a chemical that increased the sensitivity of a drug-resistant strain of S. cerevisiae to fluconazole (Cernicka, et al., 2007). The compound also increased sensitivity of the pathogenic yeasts Candida albicans and Candida glabrata that expressed efflux pumps.
As drug resistance continues to develop in pathogenic fungi, there will be research to find ways to circumvent resistance and identify next-generation drugs. Understanding the cellular processes and resistance pathways can be applied to finding alternative compounds to the well-established azoles that are the prime targets of fungal efflux.
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