Antifungal Research Strategies Aiming for New Targets

Yeasts and filamentous fungi can be true pathogens infecting healthy persons or they can be opportunistic pathogens that cause infections in humans with compromised immune systems due to cancer chemotherapy, organ transplantation treatment protocols, or HIV-1 infection, among other causes. Fungal infections can be characterized as systemic, indicating that the infection is deep, or localized cutaneous, meaning that the infection is superficial as it occurs on the skin, nails, hair or mucous membranes. Skin, nails, and hair infections by dermatophytes (Trichophyton sp., Microsporum sp., Epidermophyton sp.) and skin, nails, mucous membranes and systemic infections by Candida albicans and other Candida sp. may occur. Also, deep fungal infections caused by Histoplasma capsulatum (Histoplasmosis), Blastomyces dermatitidis (Blastomycosis), and Aspergillus fumigatus (Aspergillosis) can affect the lungs and other internal organ systems. Over the years, the number of opportunistic fungal infections has increased dramatically due to the increase in immune-compromised patients, the use of broad-spectrum antibiotics, and the availability of more invasive medical procedures resulting in an increase in prescriptions of antifungal drugs and the emergence of drug-resistant pathogenic strains. Fungal infections are, therefore, an important modern medical problem causing morbidity and mortality among hospitalized patients. The increasing frequency of infections caused by C. albicans, A. fumigatus and Cryptococcus neoformans has also led to an expansion in the number of studies on the use of antifungal drugs in clinical practice (1-2). In the quest for new antifungal drugs, the budding yeast Saccharomyces cerevisiae has served as a valuable model system because many aspects of its genetics, cell wall construction, and stress signaling are also seen among other pathogenic fungal species, in particular C. albicans, a major opportunistic pathogen (2). Despite remarkable similarities between many fungal and mammalian metabolic and signal transduction pathways, promising antifungal targets are currently under study focusing on cell wall biosynthesis, lipid composition of the plasma membrane, and synthesis of DNA and protein.

Because of the limited success of traditional drug screening strategies and the mainstreaming of high throughput molecular genetic technologies, the search for more potent, specific, and non-toxic antifungal therapies has moved towards a wider use of applications such as gene expression profiling, insertional mutagenesis and RNA mediated gene silencing for identification of novel target proteins (3). The availability of DNA-microarrays for genome wide analysis of gene expression has provided a very important tool for research on host-pathogen interactions and the analysis of expression profiles of pathogens exposed to different physiological conditions (4-5). Gene expression profiling studies are currently being applied to the major pathogens C. albicans, Cr. neoformans and A. fumigatus, which can cause severe infections among the immune-compromised populations. These studies have been conducted using four main experimental strategies: 1) treatment of cells with an antifungal drug before profiling to obtain a drug-specific gene expression signature, 2) expression profiling of fungal strains containing gene deletions in suspected target genes or transcription factors as a strategy to identify cellular targets playing a central role in the control of growth and metabolism; 3) comparison of global expression profiles of phenotypic variants of the same pathogenic strain as a strategy to identify expression patterns of genes associated with drug resistance and virulence, and 4) RNA isolation for direct profiling of the pathogen while infecting the host (3). These methods have been applied in studies of differential gene expression in oral Candidiasis (6), identification of differentially expressed genes of the ergosterol pathway after treatment with itraconazole, and the role of transcription factors associated with virulence in C. albicans (3, 7).

Insertion mutagenesis by genetic recombination has been frequently employed to generate important information through genetic analysis of null mutant strains. In C. albicans the essentiality of the RAM2 gene in the prenylation pathway was demonstrated using this technique (8) and transient loss of genes has enabled researchers to select essential gene targets required for cell viability (9). Using these many different variations of mutagenesis, more than 600 potentially essential genes have been identified for A. fumigatus. Though less widely used, gene silencing using antisense RNA or RNA interference followed by gene expression profiling, has also helped researchers identify critical genes required for growth in C. albicans (10) and the discovery of potential novel drug targets in Cr. neoformans and Aspergillus (11-12). Bioinformatic tools are of vital importance to these molecular genetic approaches for mining of fungal genome databases, analysis of gene expression, and sequence databases (3).

We will review areas of research that have shown promise for identification of new drug targets. For the purpose of this discussion we will organize these areas into four categories: i) Cell wall architecture, ii) Plasma membrane composition, iii) Cellular machinery, and iv) Signaling pathways. Where appropriate, we will describe our own recent efforts and contributions to this research being conducted in our laboratory.

i. Cell wall architecture

The cell wall is an essential structure for the fungal cell, which participates in multiple cellular processes such as nutrient transport, metabolism, and communication with the extracellular environment. It is the principal barrier to the surrounding environment because it controls cell turgor which maintains the normal osmolarity necessary for cellular biochemical reactions (2) and it sustains cell integrity that is essential for budding and cytokinesis (13-14). This structure is composed of glucans, chitin and mannoproteins. Since the cell wall is essential for fungal cell survival and because human cells do not have walls, this structure continues to be considered a principal target for the development of antifungal agents (2). Caspofungin, anidulafungin, and micafungin are echinocandins approved by the FDA in 2001 after several decades of intensive research (15-17). Echinocandins are natural cyclic lipopeptides that interfere with cell-wall synthesis by noncompetitive inhibition of β-1, 3-glucan synthase, an enzyme present in most pathogenic fungi (18). As a result, this inhibition leads to a depletion of cell wall glucan and its resultant osmotic instability. The echinocandin compounds LY 303366, MK 0991, and FK 463 are typically administered in combination with available standard antifungal agents because of their distinct mechanism of action (19). Another antifungal agent, nikkomycin Z (NZ), is a pyrimidine nucleoside linked to a peptide moiety that is a potent competitive inhibitor of chitin synthase III leading to inhibition of growth and osmotic fragility (19-20). Although the clinical importance of NZ is currently limited because high doses are required for effectiveness, we continue to explore its usefulness as a sensitizing agent for standard antifungal compounds. Our laboratory is currently analyzing the global expression profile of S. cerevisiae cells containing mutations that cause cell wall stress (21) in order to apply this knowledge in understanding the stress-response in clinically important fungi. Our approach involves genetic deletion of two components of the cytokinetic machinery, myosin II and chitin synthase II that cause cell wall stress and sensitize yeast cells to NZ (22). We have reported that sensitivity to NZ is also enhanced in a dose-dependent manner by co-treatment with the compound 2,3-Butanedione monoxime (BDM) (22), a disruptor of the cortical cytoskeleton in mammalian and fungal cells (23). As we will discuss later, the PKC1 signaling pathway is essential for cell survival under these conditions (21). Inhibitors of PKC1 or downstream effectors of this pathway may therefore have potential as sensitizing agents for standard antifungal agents.

ii. Plasma membrane composition

The plasma membrane is the permeability barrier of the cell composed mainly of sterols and phospholipids that determine its fluidity. Fungal plasma membranes are similar to mammalian plasma membranes in their structure except for having ergosterol rather than cholesterol as the principal sterol. Ergosterol is the site of interaction for polyene antibiotics while azoles interfere with ergosterol synthesis. The polyene antibiotics are produced by Streptomyces species and bind directly to ergosterol to form complexes that increase membrane permeability. As a result, there is a rapid leakage of cytoplasmic content including cellular potassium, other ions, and small molecules leading to the inhibition of glycolysis, respiration and cell death. Amphotericin B, nystatin, and pimaricin are polyene antibiotics widely used clinically because of their high affinity for ergosterol and relatively low affinity for its mammalian counterpart, thereby limiting their toxicity in mammalian cells (20). The azole antifungal agents are classified as imidazoles (ketoconazole and miconazole) or triazoles (itraconazole and fluconazole) as determined by the number of nitrogens (either two or three respectively) in the five-membered azole ring (19). Their mechanism of action consists in the inhibition of the ergosterol synthesis by interfering with the cytochrome P-450-dependent enzyme lanosterol demethylase (18-19). The resulting ergosterol depletion leads to plasma membrane permeability changes and inhibition of growth. A limitation of the azole antifungal drugs is their frequent interactions with co-administered drugs, which may result in adverse clinical consequences (24).

Besides targeting the ergosterol in the membrane, current efforts are also focusing on enzymes involved in the biosynthesis of phospholipids or sphingolipids that are exclusive of fungi (25). Fungal sphingolipids synthesized from phytoceramide are essential for growth and cell viability. Although the metabolism of sphingolipids is similar among fungi and animals, certain enzymes involved in sphingolipids synthesis are considered potential antifungal drug targets due to specific differences with their mammalian counterparts (1). Serine palmitoyltransferase is an enzyme that catalyzes the committed step in sphingolipid biosynthesis conserved from fungi to humans. Inhibition of this enzyme by the natural compounds myriosins, sphingofungins, lipoxamycins, viridiofungins, and the non-natural product cycloserine block the production of all sphingolipids resulting in fungal cell death (1). Inositol phosphorylceramide synthase (IPC-synthase) is the enzyme catalyzing the first fungus-specific step in sphingolipids synthesis and is therefore an ideal target of antifungal drugs (25). There are several natural compounds described that inhibit the activity of this enzyme, including aureobasidin A, khafrefungin, and rustmycin (1). Moreover, the AUR1 gene of S. cerevisiae encodes a protein that is necessary for IPC synthase activity and confers resistance to the antifungal drug aureobasidin-A when mutated (26). Aur1 protein may represent a new target for screening for IPC synthase inhibitor compounds.

a. Novel fatty acids

Though not yet proven to interact with the plasma membrane, novel fatty acids derived from marine sponges have been found to be fungitoxic, either by plasma membrane disruption that eventually disintegrates the cell (27) or by inhibiting fatty acids biosynthesis (28). Acetylenic fatty acids (8-16 carbons) are one of the most promising groups of fatty acids to exert antifungal effects (29), while α-methoxylated fatty acids are less toxic (30). These represent a group of compounds with antifungal drug-sensitizing properties that will be investigated in order to elucidate their mechanism of action.

iii. Cellular machinery

Components of the cellular machinery have been proposed as potential drug targets in fungi. Specific examples are topoisomerases that belong to the nuclear DNA replication and RNA transcription machinery, where they are key components in DNA-RNA synthesis. Topoisomerases are enzymes that act in several biochemical processes such as chromosome replication, transcription, recombination, and chromosome segregation by controlling the topological state of DNA (31). Camptothecin, a topoisomerase-specific inhibitor, stabilizes topoisomerase/DNA complexes, leading to DNA damage and cell death (31). Several studies have demonstrated that Topoisomerase 1 (TOP1) is essential for life and is a virulence factor for some fungi (32). Its deletion in C. albicans induces slow cellular growth and aberrant cell morphology (32). The fungal TOP1 gene has a considerable amount of coding sequence not present in human homologs (33) suggesting differences in their protein structure and presenting the possibility of exploring these topoisomerases as drug targets.

The elongation factors required for mRNA translation in eukaryotes are promising targets for antimycotic drugs. For example, the elongation factor III (EF3) is unique to fungi and is essential for protein synthesis (31). Sordarin and sordarin-like compounds act by blocking the protein elongation cycle at the initial steps of ribosomal translocation, prior to GTP hydrolysis. They have in vitro activity against a wide range of pathogenic fungi, including Aspergillus spp, Candida spp, Cr. neoformans, other filamentous fungi, (34-35) and Saccharomyces cerevisiae, making them promising antimycotic agents.

Pyrimidine analogs also have been used as antifungal agents. The fluoropyrimidine 5-fuorocytosine (5-FC) is an FDA-approved drug in this class although it has a limited spectrum of activity and significant potential for toxic effects (36-37). The mechanism of action of 5-FC involves intracellular deamination to 5-fuorouracil followed by its incorporation into RNA that causes miscoding and blocking of RNA and DNA biosynthesis (20).

The ubiquitin (Ub) system is involved in protein degradation in eukaryotic cells including fungi. It was reported that ubiquitins (Ubs) in the fungus Aspergillus nidulans were strongly expressed in the presence of antifungal drugs like amphotericin B and miconazole (38). Another study suggested that the expression of Ub genes in fungi might be enhanced by different kinds of antifungal agents, correlating with resistance to drugs (39). Investigators have suggested that Ub might be associated with different receptor-like components at the cell surface and these could be related to antifungal drug resistance (39-40). We have accumulated substantial experimental evidence to confirm that resistance to nikkomycin Z can be correlated to an elevated expression of the ubiquitin conjugating enzyme UBC4 and the ubiquitin gene UBI4 (41) while the actual mechanism for this resistance is not defined.

Sumoylation is a post-translational modification pathway composed of SUMO (Small Ubiquitin-like Modifiers) enzymes that regulate diverse cellular processes such as intracellular trafficking, cell cycle, DNA repair and replication, RNA metabolism, and cell signaling through its reversible and covalent attachment to target proteins (42). Among the SUMO substrates are a large number of regulators of gene expression, particularly transcription factors, co-activators or repressors (43). An analysis of SUMO-mediated stress response in S. cerevisiae revealed that ethanol stress increases sumoylation while affecting cell growth (44). Further studies on the regulation of this pathway in pathogenic conditions can offer insights into sumoylation as a potential drug target.

Lindquist and colleagues have established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance (45). For Candida albicans, Hsp90 mediates resistance to azoles while for Aspergillus terreus; Hsp90 is required for basal resistance to echinocandins. These investigators employed combination therapy with an Hsp90 inhibitor successfully as an experimental therapeutic strategy against these fungi in model systems. They have concluded from this work that the use of Hsp90 inhibitors “enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broad-spectrum activity against diverse fungal pathogens.”

iv. Signaling pathways

a. PKC1-dependent cell wall integrity pathway (CWIP)

Integrity of the cell wall during fungal growth is essential for survival and adaptation to environmental stresses. Although several signaling pathways may contribute to the maintenance of cellular integrity, the CWIP is considered essential for sensing cell wall perturbations under many cell stress conditions. The signaling activity of this pathway is initiated at the cell surface through sensor-transducer proteins and is transmitted to downstream effectors through Rom2p-Rho1p complexes culminating with the activation of the MAPK/Slt2p. A family of sensors constituted by members of the Wsc-family, Mid2p, and its homologue Mtl1p, are localized upstream of the MAP kinase cascade (46). This signaling cascade is a linear pathway comprised of calcium-dependent protein kinase (PKC1), a MEKK (BCK1), a pair of redundant MEKs (MKK1/2) and a MAP kinase (MPK1/SLT2) (Figure 1) (2). The targets of Mpk1p include two transcription factors, Rlm1p and Swi4/6p, involved in the regulation of multiple cell wall components and cell cycle genes respectively.

Figure 1.

The stress-response signaling pathways in Saccharomyces cerevisiae contain many targets for current and future antifungal agents. Inhibitors for specific components of these signal transduction cascades are shown in italics.

Antifungal drugs such as caspofungin, block cell wall synthesis by inhibiting β-1, 3-glucan synthases (47). Synergistic effects are observed in vivo and in vitro when this echinocandin is combined with other antifungal drugs such as fluconazole or amphotericin B (48). Research has shown that caspofungin activates the PKC1 pathway leading to Slt2p phosphorylation, and has identified Wsc1p cell surface protein as the sensor for the stress induced by caspofungin (46). Like the inactivation of β-1, 3-glucan synthases, a PKC1 deletion mutant causes loss of osmotic integrity (49). The antifungal natural product cercosporamide is a highly selective and potent PKC1 inhibitor (50) that in combination with caspofungin has a synergistic effect against the opportunistic pathogen C. albicans, as well as against budding yeast S. cerevisiae. In our own studies with S. cerevisiae, we have found that loss of myosin type ii function by genetic deletion (myo1Δ) activates the PKC1 pathway. Function of the PKC1 pathway was shown to be essential because a null mutation in SLT2, the final MAP kinase effector of this pathway, exhibited a synthetic lethality phenotype in a myo1Δ strain (21). In this light, one of our current working hypotheses is that inhibition of myosin II function can sensitize yeast cells to antifungal compounds that interfere with cell wall biogenesis like caspofungin and nikkomycin Z.

b. TOR signaling pathway

The targets of rapamycin (TOR) proteins are members of a ubiquitous family of signaling proteins. These proteins have essential conserved roles in transducing signals in response to exogenous or endogenous cellular stimuli (1). TOR proteins were discovered by mutations that control cell growth by conferring resistance to the growth inhibitory effect of the potent antifungal agent rapamycin. Since this drug interferes with TOR signaling in eukaryotic cells, it has been used as a chemical probe to delineate TOR-dependent responses in these cells (51). Rapamycin is toxic to some pathogenic yeast and fungi, including C. albicans, Cr. neoformans, and A. fumigatus (52). In S. cerevisiae, rapamycin inhibits the TOR pathway by disrupting the stable complex formed between the phosphatase regulator Tap42p and the protein phosphatase Sit4p which results in cell cycle arrest and antiproliferative effects (53), downregulation of translation initiation (54), and repression of ribosome biogenesis (Figure 1) (55). Our ongoing studies have shown that myo1Δ cells are hypersensitive to the inhibitory effects of rapamycin suggesting that combined defects in cell wall biogenesis and cytokinesis can sensitize cells to this drug. The amino acid analog cispentacin that inhibits homoserine dehydrogenase (56) has been considered for use as an inhibitor of amino acid biosynthesis in fungi. A novel use of this compound may provide a strategy to inactivate the TOR pathway in fungal cells by inducing nutrient starvation while avoiding the immunosuppressive action of rapamycin observed in mammalian model organisms (Figure 1).

c. PKA signaling pathway

The signaling cascade mediated by cyclic AMP (cAMP)-dependent protein kinase A (PKA) is involved in the regulation of virulence, morphogenesis, and development in various fungi (57). This pathway is composed of Ras proteins (encoded by RAS1 and RAS2 GTPases) that consecutively activate adenylate cyclase (encoded by CDC35 in C. albicans and CYR1 in S. cerevisiae), which generates cAMP resulting in PKA activation (58). The regulatory subunit of PKA is encoded by BCY1 while the PKA catalytic subunit is encoded by two functionally redundant genes TPK1 and TPK2 (58). The PKA pathway was also implicated in fungal resistance to certain antibiotics, including polymyxin B, dicarboximide, and azoles (59). CDC35 and CYR1 mutations demonstrated a hyper-susceptibility to azole and other sterol biosynthesis inhibitors (59). Down regulation of the adenylate cyclase regulator RAS1 was observed in myosin II-deficient (myo1Δ) cells (21). We are currently analyzing the correlation between hypersensitivity to nikkomycin Z and rapamycin, and the activation status of the RAS/PKA signaling pathway in this mutant.

Conclusions

Development of new antifungal strategies requires detailed knowledge of the fundamental controls governing fungal cell division, intracellular signaling, and growth. We have discussed diverse approaches used to develop new compounds into viable drugs yet the mechanisms of action of many compounds with development potential remain undetermined. The use of non-pathogenic model systems such as Saccharomyces cerevisiae for such studies can contribute a great deal to our knowledge of basic fungal biology which can then be extrapolated to clinically relevant fungi and accelerate the development of translational applications.