Browsing by Author "Franz, Katherine J"

Copper was first released into the environment as the result of the mass generation of oxygen from photosynthetic bacteria roughly 2.7 billion years ago. While it proved to be poisonous to early life on Earth, those that met the evolutionary challenge utilized the metal as a cofactor in enzymes to perform biochemically significant functions, while controlling intracellular levels of copper with a sophisticated network of trafficking proteins. Proteins and enzymes that utilize copper as a cofactor have evolved significantly different coordination environments than copper trafficking proteins, as a result of the different functions they perform. Of particular interest was characterizing the unique Cu(I) binding events observed in some of these proteins, the extracellular N-terminal regions of eukaryotic high affinity copper transport proteins (Ctr), and the bacterial periplamsic CusF protein of the CusFBCA Cu(I)/Ag(I) efflux pathway.

Model peptides corresponding to the methionine rich binding motifs (Mets motifs) were characterized in terms of Cu(I) binding affinity, stoichiometry, and metal specificity, via an ascorbic acid oxidation assay and electrospray ionization mass spectrometry. Metal induced structural features and coordination environments were elucidated with NMR, CD and X-ray spectroscopy. A series of peptides was also examined to infer the relative Cu(I) binding affinities, and susceptibility to oxidation, of methionine, histidine, cysteine residues found in copper binding motifs. The resistance of Cu+ specific peptides to metal catalyzed oxidation is also described. Attempts were also made to model the Cu(I)/Ag(I) tryptophan cation-π interaction observed in CusF.

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.

Malonyl-CoA (malonyl-CoA) is a platform chemical that serves as a precursor for a wide range of commercial products and pharmaceutical intermediates. In E. coli, malonyl-CoA levels are tightly regulated to remain at low levels. Two Stage Dynamic Metabolic Control (DMC) is a tool previously demonstrated to improve desired metabolite flux for several products. This work leverages DMC to improve malonyl-CoA fluxes. Specifically, we demonstrate coordinated dynamic reductions in the activities of fabI (enoyl-ACP reductase), gltA (citrate synthase), zwf (glucose-6-phosphate dehydrogenase) and glnB (nitrogen regulatory protein PII-1), during stationary phase lead to synergistic improvements in malonyl-CoA flux and the production of malonyl-CoA dependent products, 1,3,6,8-tetrahydroxynaphthalene (THN), Triacetic Lactone (TAL), and Phloroglucinol (PG). We also discuss the unique set of limitations that were observed for both TAL and PG biosynthesis as well as the strategies that were tested to overcome them. Additionally, we provide a historical review of the challenges associated with the production of Phloroglucinol. Finally, we end with a critical review focused on the bioproduction of an Acetyl-CoA and Succinyl-CoA derived product, Adipic Acid, to give perspective to common challenges associated with biobased product development.

The emergence of resistance to existing antibiotic drugs necessitates the development of new strategies to treat bacterial infections. Copper (Cu) has been used since ancient times to inhibit bacterial growth and has recently experienced a resurgence in its clinical utility as an antimicrobial coating for surfaces in hospitals. Small molecule chelators that bind Cu have also been shown to have antibacterial activity and are believed to disrupt metal homeostasis within the microbes they kill. Molecules called ionophores shuttle Cu into the cell to poison it. However, the antibacterial modes of action behind Cu and small molecule ionophores are not well understood. In this work, we employ a variety of biological, spectrometric, and proteomic techniques to study how Cu and a small molecule ionophore called pyrithione (PT) kill bacteria. First, we present antibacterial susceptibility assays that demonstrate PT and a β-lactamase-activated prodrug of PT called PcephPT kill bacteria in a Cu-dependent manner. Cu hyperaccumulated in cells that were cotreated with low-micromolar Cu and either PT or PcephPT, demonstrating their activity as metal-shuttling ionophores. Next, proteome-wide protein expression level and stability measurements were used to probe treatment-induced cellular changes after E. coli were exposed to Cu in the absence and presence of PT or PcephPT. The stability-based study identified key protein targets such as the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase and isocitrate dehydrogenase, whose activities were confirmed to be inhibited by PT-induced copper toxicity in enzymatic assays. Finally, the impact of Cu on the proteome was further investigated in a metal-induced protein precipitation experiment. Unlike other divalent first row transition metals, low millimolar Cu induced complete protein precipitation from E. coli lysate. Protein solubility was restored by addition of Cu chelators, showing that Cu-induced protein precipitation is reversible. We then obtained Cu precipitation curves for over 800 proteins and saw that some were more sensitive while others were more tolerant to precipitation by Cu. Finally, we analyzed the data set to better understand what biophysical characteristics of the proteins may contribute to making them sensitive or tolerant to precipitation by Cu.

Mechanisms of copper homeostasis are of great interest partly due to their connection to debilitating genetic and neurological disorders. The family of high-affinity copper transporters (Ctr) is responsible for extracellular copper acquisition and internalization in yeast, plants, and mammals, including human. The extracellular domain of the human high-affinity copper transporter (hCtr1) contains essential Cu-binding methionine-rich MXXM and MXM (Mets) motifs that are important for copper acquisition and transport. The hCtr1 extracellular domain also contains potential copper binding histidine (His) clusters, including a high-affinity Cu(II) ATCUN site. As of yet, extracellular His clusters have no established significance for hCtr1 function. We have made model peptides based on the extracellular copper acquisition domain of hCtr1 that is rich in His residues and Mets motifs. The peptides' Cu(I) and Cu(II) binding properties have been characterized by UV-Vis and mass spectrometry. Our findings have been extended to a mouse cell model and we show that His residues are important for hCtr1 function likely because of their contribution to strong copper-binding sites in the hCtr1 extracellular domain responsible for copper acquisition.

Copper's pro-oxidant property is also medicinally promising if it can be harnessed to induce oxidative stress as a cancer chemotherapy strategy. Our lab has designed a photocleavable caged copper complex that can selectively release redox-active copper in response to light. The thermodynamic copper binding properties of these potential chemotherapeutics have been characterized

Transition metals such as iron and copper are valued in biology for their redox activities because they are able to access various oxidation states. However, these transition metals are also implicated in a number of human disease states and play a role in bacterial infections. The ability to manipulate and monitor metal ions has vast implications on the fields of biology and human health. As such, the research described here covers two related goals: to manipulate metals in specific biological circumstances and to visualize this disturbance in cellular metal homeostasis.

Antibiotic resistance necessitates the development of drugs that exploit new mechanisms of action such as the disruption of metal homeostasis. In order to manipulate metals at the site of bacterial infection, two prochelators were developed around a β-lactam core such that the active chelator is released in the presence of bacteria that produce the resistance-causing β-lactamase enzyme. Both prochelators display enhanced activity toward resistant bacteria compared to clinical antibiotics.

Fluorescent sensors are a powerful tool for detecting small concentrations of biological analytes. Two analogs of a ratiometric fluorescent sensor were designed and synthesized to monitor cellular concentrations of copper and iron. These sensors were found to operate as designed in vitro; however the fluorescence intensity necessary for quantification of cellular metal pools has not yet been achieved.

The concept of light-activated "caged" metal ions was first introduced for Ca2+. These high affinity coordination complexes are activated by UV light to release calcium ions intracellularly and have found widespread use in understanding the many roles of calcium in biological processes. There is an unmet need for photocaging ligands for biologically relevant transition metal ions. Described here are the first examples of uncaging biologically important d-block metal ions using photoactive ligands.

New nitrogen-donor ligands that contain a photoactive nitrophenyl group within the backbone have been prepared and evaluated for their metal binding affinity. Exposure of buffered aqueous solutions of apo-cage or metal-bound cage to UV light induces cleavage of the ligand backbone reducing the denticity of the ligands. Characterization of several caging compounds reveals that quantum efficiency and metal binding affinity can be tuned by modifications to the parent structure. The change in reactivity of caged vs. uncaged metal for promoting hydroxyl radical formation was demonstrated using the in vitro deoxyribose assay. The function of several of these compounds in vivo pre- and post-photolysis has been validated using MCF-7 cells. This strategy of caging transition metals ions is promising for applications where light can trigger the release of metal ions intracellularly to study metal trafficking and distribution, as well as, selectively impose oxidative stress and/or metal toxicity on malignant cells causing their demise.

Chelating agents that can passivate the redox reactivity of transgressing iron have shown promising potential to minimize damage associated with oxidative stress in diseases. Systemic administration of these chelating agents, however, raises safety concerns due to the potential risks of indiscriminate extraction of metals from critical metalloproteins and inhibition of metalloenzymes. To overcome this challenge of unselective metal chelation, we have pursued a prodrug strategy for two extensively studied iron chelators, salicylaldehyde isonicotinic hydrazone (SIH) and deferasirox, where a boronate masking group blocks metal binding in the resulting “prochelator” until peroxide activation release the parent chelator.

Based on the aroylhydrazone chelator SIH, our first prochelator BSIH was previously shown to have favorable therapeutic ratio of low inherent toxicity and profound cytoprotection against oxidative damage in cultured cells. However, it failed to yield a full complement of SIH upon reaction with hydrogen peroxide in aqueous solutions and cell contexts. In the current study, we report that BSIH undergoes rapid hydrolysis equilibrium with its two degradation components isoniazid and Bsal, which ultimately lower the content of intact SIH formation upon peroxide activation. In our search for boronate-masked prochelators with improved hydrolytic stability, a series of BSIH derivatives have been developed by modifying the parent aroylhydrazone framework with various functionalities. Among others, the para-methoxy derivative (p-OMe)BSIH and the meta-, para- double substituted MD-BSIH have shown improved hydrolytic stability, which ultimately maximizes the release of corresponding active chelators upon peroxide activation. Moreover, both prochelators provide profound cytoprotection to ARPE-19 retinal pigment epithelial cells stressed either exogenously with H2O2 addition, or endogenously with paraquat insult.

Deferasirox is a triazole-based chelator used clinically for iron overload, but also is cytotoxic to cells in culture. In order to test whether a prodrug version of deferasirox could minimize its cytotoxicity but retain its protective properties against iron-induced oxidative damage, we present here a prochelator version TIP that contains a self-immolative boronic ester masking group that is removed upon exposure to hydrogen peroxide to release the bis-hydroxyphenyltriazole ligand deferasirox. TIP does not coordinate to Fe3+ or Zn2+ and shows only weak affinity for Cu, in stark contrast to deferasirox, which avidly binds all three metal ions. TIP converts efficiently in vitro upon reaction with hydrogen peroxide to deferasirox. In cell culture, TIP protects retinal pigment epithelial cells from death induced by hydrogen peroxide; however, TIP itself is more cytotoxic than deferasirox in unstressed cells. These results imply that the cytotoxicity of deferasirox may not derive exclusively from its iron withholding properties, which encourages further investigation on identifying potential target proteins responsible for the toxicity of deferasirox.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects over 5 million people in the United States alone. This number is predicted to triple to by the year 2050 due to both increasing life expectancies and the absence of disease-attenuating drugs. The etiology of AD remains unclear, and although there are multiple theories implicating everything from oxidative stress to protein misfolding, misregulated metal ions appear as a common thread in disease pathology.

Chelation therapy has shown some effectiveness in clinical trials, but to date, there are no FDA-approved metal chelators for the treatment of AD. One of the biggest problems with general chelators is their inability to differentiate between the metal ions involved in disease progression verses those involved in normal metabolic function. To address this problem, we have developed a prochelator approach whereby the prochelator (SWH) does not bind metals with significant biological affinity. However, once activated to the chelator (CP) via enzymatic hydrolysis, the molecule is able to bind copper and reduce its toxicity both in vitro and in a cellular model of Alzheimer's Disease.

Central to this strategy is the site-specificity provided by enzymatic activation of the prochelator. In our system, SWH to CP conversion is mediated by beta-secretase, an enzyme involved in A-beta generation. However, in order to render SWH capable of hydrolysis in cells, we modified the prochelator to contain a dihydrocholesterol membrane anchor attached via a polyethylene glycol linker. From this construct, we created beta-MAP, which is an SWH-based FRET probe to demonstrate beta-secretase-mediated conversion of SWH to CP. beta-MAP was also used to confirm the efficacy of a known beta-secretase inhibitor without the need to for mutated cells lines or expensive antibodies. beta;-MAP and the associated microscopy method represent a significant advancement to the currently available ELISA assays for beta-secretase activity.

While activation of the prochelator by an enzyme in cells is encouraging, non-specific hydrolysis of the peptide prevents significant accumulation of the chelator on the cell membrane. Furthermore, attachment of the polyethylene glycol and sterol units induce cell toxicity not seen with the native CP peptide. These drawbacks prevent the current prochelator from effectively protecting cells from AD conditions. Structural modifications to overcome these problems, including implementation of a new peptide sequence are planned for future experiments.

Fluorescent sensors provide a powerful analytical tool for the intracellular detection of metal cations. In some cases, these fluorescent metal-chelating sensors have helped elucidate the function of metal cations within complicated cellular systems. However, most measure or sense changes in the bulk concentration of a metal species and do not respond to those involved in a specific cellular event. For instance, misregulated copper and iron are implicated in neurodegenerative disease and cancer because of their ability to catalytically propagate the formation of the hydroxyl radical through reaction with hydrogen peroxide. A fluorescent sensor that is unresponsive to metal binding until activation by intracellular hydrogen peroxide could potentially pinpoint the location of this oxidative reaction and provide an understanding of the relationship between copper/iron and hydrogen peroxide.

Described here is the development of two fluorescent prochelators that show a selective fluorescence response to iron or copper only in the presence of hydrogen peroxide. A boronic ester masked spirolactam-based prochelator displays a copper-selective turn-on response after oxidation with hydrogen peroxide in organic solvents as determined by absorbance and fluorescence spectroscopy. However, a competing mechanism occurs in aqueous solution due to hydrolytic instability of the masked prochelator and results in a separate copper-dependent turn-on response as verified by liquid chromatography-mass spectroscopy. A second fluorescent prochelator design relies on metal-dependent fluorescence quenching after oxidation of a self-immolative boronic ester in both organic and aqueous solvents. Cellular microscopy studies show that the sensor's fluorescence intensity is unchanged until incubation with exogenous hydrogen peroxide, which resulted in a decreased fluorescent signal that is restored upon competitive chelation. Both of these prochelators provide a template for future applications and designs with improved properties.

Two additional chapters describe the development of a UV-activated iron prochelator and a new fluorescently tagged metal chelator. The UV-activated prochelator is protected with two nitrophenyl groups that are photolyzed with 350 nm light within 10 minutes to reveal a high affinity iron triazole-base chelator. A chelator of this nature may provide protection from UV-induced iron liberation and oxidative stress. A second triazole-based chelator with an embedded coumarin fluorophore was prepared as a potential metal sensor. However, this design showed off-target fluorescence responses, thus it cannot be utilized in its current form for metal detection.

The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.

Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N′-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.

Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.

Histatins are a family of histidine-rich peptides that are found in saliva. This family of peptides has antifungal properties. In this family, histatin 5 is the most efficient antifungal peptide against Candida albicans. The mechanism for the antifungal activity of histatin 5 is still unknown. Previous studies suggest that the metal-binding properties of histatin 5 may have some connection to its antifungal activity. Although there is some evidence that histatin 5 can bind Cu(II), currently there is no conclusive data on the Cu(I) binding properties of histatin 5. This work focuses on investigating the copper-binding properties of histatin 5 and analyzing the role these copper-binding properties may play in the antifungal activity of histatin 5. Model peptides of histatin 5 have been synthesized and copper binding studies were performed by UV-Vis spectroscopy and mass spectrometry. Reactive oxygen species formation process was studied by fluorescence assay.

Transition metal ions are required nutrients for many organisms but also potent toxins if misappropriated. Iron (Fe) plays a central role in the transport of oxygen, and other transition metals, such as copper (Cu) or zinc (Zn), are found in enzymes. It is critical for the human body to maintain careful control on both the levels of metals and their distribution to maintain healthy function. An imbalance in these metals such as an overload often leads to organ failure, while deficiencies result in other medical conditions like anemia, neutropenia, leukopenia. Metal imbalances have been implicated in neurodegenerative diseases, cancer and infections.

This dissertation explores several strategies envisioned to alter the bioavailability of metal ions by using synthetic metal-binding agents targeted specifically for diseases where misappropriated metal ions are suspected of exacerbating cellular damage. In Chapter 1, we discuss chemical properties that influence the pharmacological outcome of a subset of metal-binding agents known as ionophores, and review several examples that have shown multiple pharmacological activities in metal-related diseases, with a particular focus on Cu. Chapter 2 describes results of a growth assay in which we screened small molecule Fe and Cu chelators to determine if altering the bioavailability of these essential metal ions inhibits growth of the fungal pathogen, Cryptococcus neoformans. Results show that select chelating agents that facilitate the increase of intracellular Cu levels inhibited growth of C. neoformans, while traditional metal sequestering agents had no effect on growth at the concentrations tested. In Chapter 3, various chemical properties of the select ligands that demonstrated Cu-dependent antifungal activity (8-hydroxyquinoline (8HQ), thiomaltol and pyrithione) were analyzed and compared to those of counterpart ligands that did not inhibit C. neoformans growth, namely clioquinol, maltol, pyridinol-n-oxide, deferiprone, and thiodeferiprone. The UV-vis spectroscopy of the Cu(II) and Cu(I) complexes of each ligand, along with calculation of their apparent binding affinities by competitive ligand titrations, are described. In addition, we determined octanol-water partition coefficients for the Cu(II) complexes and compare them with reported partition coefficients for the free ligands. An initial assessment of the reduction potential of Cu complexes of the select agents is also investigated in this chapter, along with an analysis of structural details from available crystallographic data.

After identifying chelating agents that inhibit C. neoformans in a Cu-dependent manner, we take a closer look in Chapter 4 at 8HQ as a model Cu ionophore. In spite of its promising biological activity of, the metal-dependent toxicity of 8HQ extends to mammalian cells as well. To overcome this challenge, Chapter 4 describes the application of a prochelator strategy to manipulate host Cu in innate immune cells to fight microbial infection. QBP is a nontoxic protected form of 8HQ in which a pinanediol boronic ester blocks metal ion coordination by 8HQ. The prochelator, QBP, is deprotected via reactive oxygen species produced by activated macrophages, creating 8HQ and eliciting Cu-dependent killing of C. neoformans in vitro. Finally, Chapter 5 outlines the synthesis of multifunctional metal chelators that contain a masking group on a metal binding moiety that has been incorporated onto the structural framework of Aβ aggregate-imaging agents. Masking the metal binding site should prevent non-specific metal binding of the prochelator. Once activated to its unmasked form under conditions that mimic early Alzheimer's disease, the released chelator should complex metal ions. The design and rates of oxidation in response to hydrogen peroxide exposure along with their ability to interact with Cu are described in Chapter 5.

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.

Maintenance of metal homeostasis is critical to cell survival due to the multitude of cellular processes that depend on one or more metal cofactors. We show that the opportunistic fungal pathogen Candida albicans becomes sensitized to both Cu limitation and Cu elevation during exposure in liquid culture to the antifungal drug fluconazole, a widely prescribed antifungal agent. Cu supplementation reduces tolerance of C. albicans to fluconazole in a way that does not require formation of a Cu–fluconazole complex. Rather, our data point to a less obvious relationship between drug stress and Cu availability that gives rise to metal-mediated outcomes of drug treatment. qRT-PCR, EPR, fluorescence, and ICP-MS studies demonstrate that C. albicans extensively remodels its metal homeostasis networks to respond to treatment with fluconazole. These adaptation strategies include increased Cu import and storage, increased retention of Fe, Mn, and Zn, altered utilization of Cu- and Mn-dependent enzymes, mobilization of intracellular Fe stores, and increased production of the heme prosthetic group utilized by the enzyme target of fluconazole. Furthermore, RNA-seq analysis reveals that co-treatment with fluconazole and Cu gives rise to unique patterns of gene expression that illustrate the profound impacts of small fluctuations in Cu availability on the transcriptomic response to fluconazole stress. Finally, we show that fluconazole causes substantial changes to the metalloproteome of C. albicans, most notably to the levels of Cu proteins. The findings offer a new perspective for thinking about fungal response to drug stress that pushes cells out of their metal homeostatic zones, leading them to enact metal-associated adaptation mechanisms to restore homeostasis to survive.

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.

In recent years, the demand for new antifungal drugs has increased, partly due to the rise of antifungal drug resistance. Previous research has shown that Candida albicans is hypersensitive to copper (Cu) dyshomeostasis in the presence of antifungal drug treatment by the popular triazole fluconazole. This work aims to investigate the ability of metal trafficking inhibitors to alter metal homeostasis in C. albicans. Toward this goal, we identified five compounds with demonstrated metal-trafficking protein inhibition from a literature review. One of the metal trafficking inhibitors tested, the Cu chelator ammonium tetrathiomolybdate (TTM), was found by C. albicans growth assays to have antifungal properties, an activity that has not been documented previously . Data from ion-coupled plasma mass spectrometry revealed significantly increased intracellular Mo levels, confirming that TTM internalizes into the cell. Utilizing bulk proteomic methods to measure protein stability changes, we found that TTM treatment results in altered stability of metalated proteins such as cytochrome c, Cu/Zn superoxide dismutase 1 (Sod1), the flavo-hemoglobin nitric oxide dioxygenase (Yhb1), and the multicopper oxidase Fet31. TTM treatment destabilizes Sod1 and increases the reactive oxygen species (ROS) burden while stabilizing and upregulating the compensatory Yhb1. Upon comparison of TTM’s antifungal activity to that of the extracellular Cu chelator bathocuproinedisulfonic acid (BCS), we determined that TTM’s activity is not due to Cu chelation and subsequent deprivation, as BCS does not inhibit C. albicans growth or impact the metalloproteome stability in the same way. Additionally, inhibiting the upregulated and stabilized Yhb1 during TTM treatment with azole antifungals with known Yhb1 inhibiting capabilities resulted in synergistic antifungal activity as determined by checkerboard growth assays. The combined inhibition of Yhb1 with TTM treatment is a promising antifungal development avenue.