Browsing by Subject "Antifungal"

An increase in drug-resistant infections and the paucity of new antibiotics present a major world health problem. Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised individuals and occasionally in those with no known immune impairment. The Franz lab recently identified several copper (Cu) chelators containing O, S and O, N donor atoms that exhibit Cu-dependent antifungal activity against C. neoformans. Interestingly, the O, O analogs of these chelators do not exhibit anti-fungal activity. Here, using UV-visible spectroscopy the Cu(I) binding properties of these ligands are determined. The lipophilic properties of these ligands and their bis-Cu(II) complexes were also determined using the traditional shake flask method. Lipophilicity and binding studies indicate that ligands exhibiting Cu-dependent antifungal activity are able to bind Cu(II) with a binding affinity, Log K_(〖CuL〗_2)^', greater than 19 and they form hydrophobic bis-Cu(II) complexes. Further, inductively coupled plasma-mass spectrometry (ICP-MS) was used to analyze total metal content of C. neoformans fungal cells treated with these ligands and Cu(II). This analysis revealed that the ligands displaying antifungal activity increased Cu, zinc (Zn), and iron (Fe) levels in the fungal cells dramatically compared to the ligand or Cu only treatment. Lastly, a new group of linear and cyclic thiohydroxamic acids (O,S donor atoms) was screened for their effect on C. neoformans’ growth, in the presence and absence of Cu(II). These studies indicate that cyclic thiohydroxamic acids are able to elicit Cu-dependent antifungal activity opening the possibility of a new class of metallo-antifungals. Further initial attempts were made to understand the Cu(II) binding properties of these thiohydroxamic acids using calcein fluorescence competition assays. The results from this work suggest that small molecules, capable of binding Cu(II) to form hydrophobic complexes, can deliver Cu to fungal cells altering not only their Cu but also intracellular Zn and Fe levels. This hypothesis about Cu delivery agents sets the stage for future work in genome-wide approaches to probe how alteration in metal levels affects different biochemical pathways to induce Cu-dependent antifungal activity.

!Drug discovery typically involves structure-based drug design based on three-dimensional protein structures and hit/lead compound identification and optimization. Herein, this technique was used to overcome several obstacles associated with the developing of antibiotics, anticancer agents, and antifungals and reveal critical insights into the corresponding structure-activity relationships (SARs).Phospho-N-acetyl-muramyl-pentapeptide translocase (MraY) is an important membrane enzyme involved in the early-stage biosynthesis of bacterial peptidoglycans. As the inhibition of MraY leads to bacterial cell lysis, such MraY inhibitors (e.g., muraymycin) hold great promise for antibiotic development. However, the structural complexity of muraymycin makes its synthesis and practical applications challenging. Hence, we synthesized several muraymycin analogs with reduced structural complexity and better synthetic tractability and identified the moieties responsible for their biological activity to facilitate the development of muraymycin-derived antibiotics. Translesion synthesis (TLS) is a major mechanism that enables bypass replication over DNA lesions and promotes the formation of mutagenic DNA. Rev1/Pol ζ–mediated TLS plays an important role in cisplatin-induced mutations, and thus, the Rev1/Pol ζ interface is an attractive target for small-molecule TLS inhibitors. Herein, we aimed to develop TLS inhibitors as potential anticancer agents based on the recently reported inhibitor of the Rev1-Rev7 interaction, JH-RE-06. Despite its high potency, JH-RE-06 is poorly soluble in aqueous media and is therefore a limitation for further development. To overcome this limitation and identify novel anticancer agents, we prepared various JH-RE-06 analogs and studied the related SARs, to determine the critical functional groups for improving the biological activity improvement and aqueous solubility. Currently, fungal infections, which are particularly dangerous to immunocompromised patients, are a frequent cause of a death. However, the similarities between the eukaryotic physiologies of fungal pathogens and their hosts render targeting of the pathogen without causing side effects in the host challenging. Calcineurin (CN) plays a major role in invasive fungal diseases and is therefore a promising target for antifungal drug development. FK506, which is an approved CN inhibitor, exhibits promising activity but an insufficient selectivity because of its strong immunosuppressive effect. Therefore, in developing antifungal agents, we exploited the major structural differences between the CN-FK506-FKBP12 ternary complexes of humans and fungi and developed FK506/520 analogs targeting these complexes. The synthesized analogs retained the parent antifungal efficacy while exhibiting lower immunosuppressive activities and improved therapeutic efficacies both in vivo and in vitro.

The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro, but the details of these interactions are poorly understood and their implications on in vivo antifungal activity have not been established. Here, we explore how the coordination environment of Cu influences chemical and biological activity of the antifungal peptide Hist-5. Antifungal susceptibility assays and Cu-binding experiments reveal how the efficacy of Hist-5 against the commensal organism Candida albicans depends on the availability of Cu in the growth environment. Further, this biological activity correlates with the presence of adjacent histidine residues (bis-His) within the histatin peptide that support Cu(I) binding in the low nM range. Evaluation of oxygen reactivity of the Histatin Cu(I)-bis-His complexes indicates the PCu(I) complex is reactive towards H2O2. EPR, UV-Vis and HPLC studies demonstrate that exposure to H2O2 results in the formation of a metalloradical complex reminiscent of radical copper oxidases. Additional exploration of the coordination environment conducive to metalloradical formation exposes the importance of the third ligand (His3) of the Cu(I)-bis-His Complex for H2O2. His3 mutant peptides also disclose the tunability of the H2O2 reactivity. Furthermore, substrate evaluation assays offer evidence of the capability of the Cu-Hist-5 to specifically chemically modify a cell wall component. Together, these results provided compelling evidence supporting that Cu-coordination plays a critical role in the biological and chemical activity of Hist-5.

Histatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 can bind metals in vitro, and metals have been shown to alter the fungicidal activity of the peptide. The goal of this work is to gain insight into the role of metals in the biological activity Hist-5. Toward this goal, we developed and characterized a novel fluorescently labeled Hist-5 peptide (Hist-5*) to visualize changes in internalization and localization of the peptide in fungal cells as a function of metal availability in the growth medium. Here, we provide evidence for Zn-modulated antifungal activity of Hist-5 in which the availability of Zn2+ in the surrounding environment inhibits Hist-5 cellular uptake and cidality. Cellular growth assays revealed a concentration-dependent inhibitory effect of Zn2+ on Hist-5 antifungal activity. Imaging by confocal microscopy showed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. We found that modulation of extracellular Zn2+ concentration by metal chelating molecules or proteins reversed Zn-induced surface adhesion of Hist-5, leading us to propose a dynamic role for Zn2+ as an inhibitory switch to regulate Hist-5 fungicidal activity. We next present data to support the hypothesis that Hist-5 interacts with intracellular Cu to increase the fungicidal activity Hist-5. Combined fluorescence spectroscopy and microscopy experiments showed reversible Cu-dependent quenching of Hist-5* fluorescence, indicating a direct interaction between Hist-5 and intracellular Cu. X-ray fluorescence microscopy images revealed peptide-induced changes to cellular Cu distribution and cell-associated Cu content. Finally, we present progress towards expanding the scope in which we understand and assess Hist-5 biological activity by investigating the activity of the peptide under biologically relevant conditions and testing its fungicidal activity against other fungal species.

Although the majority of the population carries Candida spp as normal components of their microflora, these species are important human pathogens that have the ability to cause disease under conditions of immunosuppression or altered host defenses. The spectrum of disease caused by these species ranges from cutaneous infections of the skin, mouth, esophagus and vagina, to life-threatening systemic disease. Despite increases in drug resistance, the antifungal armamentarium has changed little over the past decade. Thus increasing our understanding of the life cycles of these organisms, not only how they propagate themselves, but also how genetic diversity is created within the population is of considerable import. Additionally expanding our knowledge of key signal transduction cascades that are important for cell survival and response to stress will add in developing new antifungal therapies and strategies.

This thesis addresses both of these key areas of fungal pathogenesis. In the first chapter, we use genome comparisons between parasexual, asexual, and sexual species of pathogenic Candida as a first approximation to answer the question of whether examining genome content alone can allow us to understand why species have a particular life cycle. We start by examining the structure of the mating type locus (MAT) of two sexual species C. lusitaniae and C. guilliermondii. Interestingly, both species are missing either one or two (respectively) canonical transcription factors suggesting that the control of sexual identity and meiosis in these organisms has been significantly rewired. Mutant analysis of the retained transcription factors is used to understand how sexual identity and sporulation are controlled in these strains. Secondly, based on the observation that these species are missing many key genes involved in mating and meiosis, we use meiotic mapping, SPO11 mutant analysis, and comparative genome hybridization to demonstrate that these species are indeed meiotic, but that the meiosis that occurs is occasional unfaithful generating aneuploid and diploid progeny.

In the second and third chapters we examine the calcineurin signaling pathway, which is crucial for mediated tolerance to cellular stresses including cations, azole antifungals, and passage through the host bloodstream. First, we show that clinical use of calcineurin inhibitors in combination with azole antifungals does not result in resistance to the combination, suggesting that if non-immunosuppressive analogs could be further developed this combinatorial strategy may have great clinical efficacy. Second, we use previous studies of the calcineurin signaling pathway in S. cerevisiae to direct a candidate gene approach for elucidating other components of this pathway in C. albicans. Specifically, we identify homologs of the RCN1, MID1, and CCH1 genes, and use a combination of phenotypic assays and heterologous expression studies to understand the roles of these proteins in C. albicans. Although the mutant strains share some phenotypic properties with calcineurin deletion strains, none completely recapitulate a calcineurin mutant.

In the last chapter, we examine the plausibility of targeting the homoserine dehyrogenase (Hom6) protein in C. albicans and C. glabrata as a novel antifungal strategy. Studies in S. cerevisiae had demonstrated a synthetic lethality between hom6 and fpr1, the gene encoding FKBP12 a prolyl-isomerase that is the binding target of the immunosuppressant FK506. Thiss synthetic lethality was due to the buildup of a toxic intermediate in the methionine and threonine biosynthetic pathway as a result of deletion of hom6 and inhibition of FKBP12. We deleted HOM6 from both C. albicans and the more highly drug-resistant species C. glabrata. Studies suggest that regulation of the threonine and methionine biosynthetic pathway in C. albicans has been rewired such that the synthetic lethality between hom6 and FKBP12 inhibition no longer exists. However, in C. glabrata preliminary analysis suggest that similarly to S. cerevisiae hom6 and inhibition of FKBP12 can result in cell death.

Fungal cells sense the amount and quality of external nutrients through multiple interconnected signaling networks, which allow them to adjust their metabolism, transcriptional profiles and developmental programs to adapt readily and appropriately to changing nutritional states. In organisms ranging from yeasts to humans, the Tor signaling pathway responds to nutrient-derived signals and orchestrates cell growth. While in the baker's yeast Saccharomyces cerevisiae Tor responds to nutrient-derived signals and orchestrates cell growth and proliferation, in Schizosaccharomyces pombe Tor signaling modulates sexual differentiation in response to nutritional cues. Thus, these differences provide a framework to consider the roles of Tor in other fungal organisms, in particular those that are pathogens of humans.

In this dissertation, I demonstrate that in the human fungal pathogen Candida albicans, Tor signaling also functions to promote growth. This study also uncovered a novel role for the Tor molecular pathway in promoting hyphal growth of C. albicans on semi-solid surfaces and in controlling cell-cell adherence. Gene expression analysis and genetic manipulations identified several transcriptional regulators (Bcr1, Efg1, Nrg1, and Tup1) that together with Tor compose a regulatory network governing adhesin gene expression and cellular adhesion. While the Tor kinases are broadly conserved, these studies further demonstrate the contrasting strategies employed by fungal organism in utilizing the Tor signaling cascade.

While extensive studies have focused on elucidating functions for the Tor signaling cascades among ascomycetes, little is known about the pathway in basal fungal lineages, in particular among zygomycetes and chytrids. Moreover, given that the Tor pathway is the target of several small molecule inhibitors including rapamycin, a versatile pharmacological drug used in medicine, there is considerable interest in Tor signaling pathways and their function. Capitalizing on emerging genome sequences now available for several basal fungal species, we show a remarkable pattern of conservation, duplication, and loss of the Tor signaling cascade among basal fungal lineages. Targeting the pathway with rapamycin results in growth arrest of several zygomycete species, indicating a conserved role for this pathway in regulating fungal growth. In addition, we show a potential therapeutic advantage of using rapamycin in a heterologous model of zygomycosis. Taken together, the Tor signaling cascade and its inhibitors provide robust platforms from which to develop novel antimicrobial therapies, which may include less immunosuppressive rapamycin analogs.