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Metal cations have been exploited for their precipitation properties in a wide variety of studies, ranging from differentiating proteins from serum and blood to identifying the protein targets of drugs. Despite widespread recognition of this phenomenon, the mechanisms of metal-induced protein aggregation have not been fully elucidated. Recent studies have suggested that copper's (Cu) ability to induce protein aggregation may be a main contributor to Cu-induced cell death. Here, we provide the first proteome-wide analysis of the relative sensitivities of proteins across the Escherichia coli proteome to Cu-induced aggregation. We utilize a metal-induced protein precipitation (MiPP) methodology that relies on quantitative bottom–up proteomics to define the metal concentration–dependent precipitation properties of proteins on a proteomic scale. Our results establish that Cu far surpasses other metals in promoting protein aggregation and that the protein aggregation is reversible upon metal chelation. The bulk of the Cu bound in the protein aggregates is Cu1+, regardless of the Cu2+ source. Analysis of our MiPP data allows us to investigate underlying biophysical characteristics that determine a protein's sensitivity to Cu-induced aggregation, which is independent of the relative concentration of protein in the lysate. Overall, this analysis provides new insights into the mechanism behind Cu cytotoxicity, as well as metal cation–induced protein aggregation.

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. Previous reports on the effect of Zn(2+) on Hist-5 activity have been varied and seemingly contradictory. Here we present data elucidating the dynamic role Zn(2+) plays as an inhibitory switch to regulate Hist-5 fungicidal activity. A novel fluorescently labeled Hist-5 peptide (Hist-5*) was developed to visualize changes in internalization and localization of the peptide as a function of metal availability in the growth medium. Hist-5* was verified for use as a model peptide and retained antifungal activity and mode of action similar to native Hist-5. Cellular growth assays showed that Zn(2+) had a concentration-dependent inhibitory effect on Hist-5 antifungal activity. Imaging by confocal microscopy revealed that equimolar concentrations of Zn(2+) kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. However, the Zn-induced decrease in Hist-5 activity and uptake was rescued by decreasing Zn(2+) availability upon addition of a metal chelator EDTA or S100A12, a Zn-binding protein involved in the innate immune response. These results lead us to suggest a model wherein commensal C. albicans may exist in harmony with Hist-5 at concentrations of Zn(2+) that inhibit peptide internalization and antifungal activity. Activation of host immune processes that initiate Zn-sequestering mechanisms of nutritional immunity could trigger Hist-5 internalization and cell killing.

Histatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 has the ability to bind metals in vitro and metals have been shown to alter the fungicidal activity of the peptide. Previous reports on the effect of Zn2+ on Hist-5 activity have been varied and seemingly contradictory. Here we present data elucidating the dynamic role Zn2+ plays as an inhibitory switch to regulate Hist-5 fungicidal activity. A novel fluorescently labeled Hist-5 peptide (Hist-5*) was developed to visualize changes in internalization and localization of the peptide as a function of metal availability in the growth medium. Hist-5* was verified for use as a model peptide and retained antifungal activity and mode of action similar to native Hist-5. Cellular growth assays showed that Zn2+ had a concentration-dependent inhibitory effect on Hist-5 antifungal activity. Imaging by confocal microscopy revealed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. However, the Zn-induced decrease in Hist-5 activity and uptake was rescued by decreasing Zn2+ availability upon addition of a metal chelator EDTA or S100A12, a Zn-binding protein involved in the innate immune response. These results lead us to suggest a model wherein commensal C. albicans may exist in harmony with Hist-5 at concentrations of Zn2+ that inhibit peptide internalization and antifungal activity. Activation of host immune processes that initiate Zn-sequestering mechanisms of nutritional immunity could trigger Hist-5 internalization and cell killing.

Bacterial expression of β-lactamase enzymes that hydrolyze β-lactam antibiotics contributes to the growing threat of antibacterial drug resistance. Metallo-β-lactamases, such as NDM-1, use catalytic zinc ions in their active sites and hydrolyze nearly all clinically available β-lactam antibiotics. Inhibitors of metallo-β-lactamases are urgently needed to overcome this resistance mechanism. Zinc-binding compounds are promising leads for inhibitor development, as many NDM-1 inhibitors contain zinc-binding pharmacophores. Here, we evaluated thirteen chelating agents containing benzimidazole and benzoxazole scaffolds as NDM-1 inhibitors. Six of the compounds showed potent inhibitory activity with IC(50) values as low as 0.38 μM, and several compounds restored meropenem susceptibility of NDM-1-expressing E. coli. Spectroscopic and docking studies suggest ternary complex formation as the mechanism of inhibition, making these compounds promising for development as NDM-1 inhibitors.

Azole antifungals are an important class of antifungal drugs due to their low cost, ability to be administered orally, and broad-spectrum activity. However, their widespread and long-term use have given rise to adaptation mechanisms that render these compounds less effective against common fungal pathogens, including Candida albicans. New antifungals are desperately needed as drug resistant strains become more prevalent. We recently showed that copper supplementation potentiates the activity of the azole antifungal fluconazole against the opportunistic fungal pathogen C. albicans. Here, we report eight new azole analogues derived from fluconazole in which one triazole group has been replaced with a metal-binding group, a strategy designed to enhance potentiation of azole antifungal activity by copper. The bioactivity of all eight compounds was tested and compared to that of fluconazole. Three of the analogues showed activity against C. albicans and two had lower levels of trailing growth. One compound, Flu-TSCZ, was found to impact the levels, speciation, and bioavailability of cellular metals.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis. In the fungal pathogen Cryptococcus neoformans, Bim1 is a copper-binding lytic polysaccharide monooxygenase-like protein that participates in copper uptake in concert with the Ctr1 importer to drive virulence mechanisms during fungal meningitis.

Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu

Copper is an essential nutrient whose redox properties make it both beneficial and toxic to the cell. Recent progress in studying transition metal signalling has forged new links between researchers of different disciplines that can help translate basic research in the chemistry and biology of copper into clinical therapies and diagnostics to exploit copper-dependent disease vulnerabilities. This concept is particularly relevant in cancer, as tumour growth and metastasis have a heightened requirement for this metal nutrient. Indeed, the traditional view of copper as solely an active site metabolic cofactor has been challenged by emerging evidence that copper is also a dynamic signalling metal and metalloallosteric regulator, such as for copper-dependent phosphodiesterase 3B (PDE3B) in lipolysis, mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 in cell growth and proliferation and the kinases ULK1 and ULK2 in autophagy. In this Perspective, we summarize our current understanding of the connection between copper and cancer and explore how challenges in the field could be addressed by using the framework of cuproplasia, which is defined as regulated copper-dependent cell proliferation and is a representative example of a broad range of metalloplasias. Cuproplasia is linked to a diverse array of cellular processes, including mitochondrial respiration, antioxidant defence, redox signalling, kinase signalling, autophagy and protein quality control. Identifying and characterizing new modes of copper-dependent signalling offers translational opportunities that leverage disease vulnerabilities to this metal nutrient.

Maintenance of metal homeostasis is critical to cell survival due to the multitude of cellular processes that depend on one or more metal cofactors. Here, we show that the opportunistic fungal pathogen Candida albicans extensively remodels its metal homeostasis networks to respond to treatment with the antifungal drug fluconazole. Disruption of the ergosterol biosynthetic pathway by fluconazole requires C. albicans adaptation, including increased Cu import and storage, increased retention of Fe, Mn, and Zn, altered utilization of Cu- and Mn-dependent enzymes, mobilization of Fe stores, and increased production of the heme prosthetic group utilized by the enzyme target of fluconazole. 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.

To survive, fungal pathogens must acquire nutrient metals that are restricted by the host while also tolerating mechanisms of metal toxicity that are induced by the host. Given this dual vulnerability, we hypothesized that a pathogen’s access to and control of essential yet potentially dangerous metal ions would affect fungal tolerance to antifungal drug stress. Here, we show that 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. Spectroscopic data confirm that while fluconazole forms a complex with Cu(II) in water, interactions of fluconazole with neither Cu(II) nor Cu(I) are observed in the cell culture media used for the cellular assays. This result is further supported by growth assays in deletion strains that lack Cu import machinery. Overall, we establish that increases in Cu levels by as little as 40 nM over basal levels in the growth medium reduce 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 more complex relationship between drug stress and Cu availability that gives rise to metal-mediated outcomes of drug treatment.

The ability of pathogens to maintain homeostatic levels of essential biometals is known to be important for survival and virulence in a host, which itself regulates metal availability as part of its response to infection. Given this importance of metal homeostasis, we sought to address how the availability of copper in particular impacts the response of the opportunistic fungal pathogen Candida albicans to treatment with the antifungal drug fluconazole. The present study reports whole transcriptome analysis via time-course RNA-seq of C. albicans cells exposed to fluconazole with and without 10 µM supplemental CuSO4 added to the growth medium. The results show widespread impacts of small changes in Cu availability on the transcriptional response of C. albicans to fluconazole. Of the 2359 genes that were differentially expressed under conditions of cotreatment, 50% were found to be driven uniquely by exposure to both Cu and fluconazole. The breadth of metabolic processes that were affected by cotreatment illuminates a fundamental intersectionality between Cu metabolism and fungal response to drug stress. More generally, these results show that seemingly minor fluctuations in Cu availability are sufficient to shift cells’ transcriptional response to drug stress. Ultimately, the findings may inform the development of new strategies that capitalize on drug-induced vulnerabilities in metal homeostasis pathways.

Metal-complexed N-heterocyclic carbene (NHC) mechanophores are latent reactants and catalysts for a range of mechanically driven chemical responses, but mechanochemical scission of the metal-NHC bond has not been experimentally characterized. Here we report the single-molecule force spectroscopy of ligand dissociation from a pincer NHC-pyridine-NHC Pd(II) complex. The force-coupled rate constant for ligand dissociation reaches 50 s-1 at forces of approximately 930 pN. Experimental and computational observations support a dissociative, rather than associative, mechanism of ligand displacement, with rate-limiting scission of the Pd-NHC bond followed by rapid dissociation of the pyridine moiety from Pd.

The ability of metal ionophores to induce cellular metal hyperaccumulation endows them with potent antimicrobial activity; however, the targets and mechanisms behind these outcomes are not well understood. This work describes the first utilization of proteome-wide measurements of protein folding stability in combination with protein expression level analysis to identify protein targets of copper, thereby providing new insight into ionophore-induced copper toxicity in E. coli. The protein folding stability analysis employed a one-pot protocol for p ulse p roteolysis (PP) in combination with a s emi- t ryptic peptide e nrichment strategy for p roteolysis p rocedures (STEPP) to generate stability profiles for proteins in cell lysates derived from E. coli exposed to copper with and without two ionophores, the antimicrobial agent pyrithione and its β-lactamase-activated prodrug, PcephPT. As part of this work, the above cell lysates were also subject to protein expression level analysis using conventional quantitative bottom-up proteomic methods. The protein folding stability and expression level profiles generated here enabled the effects of ionophore vs copper to be distinguished and revealed copper-driven stability changes in proteins involved in processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (∼3×) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies.

Antibacterial drug resistance is a rapidly growing clinical threat, partially due to expression of β-lactamase enzymes, which confer resistance to bacteria by hydrolyzing and inactivating β-lactam antibiotics. The increasing prevalence of metallo-β-lactamases poses a unique challenge, as currently available β-lactamase inhibitors target the active site of serine β-lactamases but are ineffective against the zinc-containing active sites of metallo-β-lactamases. There is an urgent need for metallo-β-lactamase inhibitors and antibiotics that circumvent resistance mediated by metallo-β-lactamases in order to extend the utility of existing β-lactam antibiotics for treating infection. Here we investigated the antibacterial chelator-releasing prodrug PcephPT (2-((((6R,7R)-2-carboxy-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)thio)pyridine 1-oxide) as an inhibitor of New Delhi metallo-β-lactamase 1 (NDM-1). PcephPT is an experimental compound that we have previously shown inhibits growth of β-lactamase-expressing E. coli using a mechanism that is dependent on both copper availability and β-lactamase expression. Here, we found that PcephPT, in addition to being a copper-dependent antibacterial compound, inhibits hydrolysis activity of purified NDM-1with an IC(50) of 7.6 μM without removing zinc from the active site and restores activity of the carbapenem antibiotic meropenem against NDM-1-producing E. coli. This work demonstrates that targeting a metal binding pharmacophore to β-lactamase-producing bacteria is a promising strategy for inhibition of both bacterial growth and metallo-β-lactamases.

The unabated rise in bacterial resistance to conventional antibiotics, coupled with collateral damage to normal flora incurred by overuse of broad-spectrum antibiotics, necessitates the development of new antimicrobials targeted against pathogenic organisms. Here, we explore the antibacterial outcomes and mode of action of a prochelator that exploits the production of β-lactamase enzymes by drug-resistant bacteria to convert a non-toxic compound into a metal-binding antimicrobial agent directly within the microenvironment of pathogenic organisms. Compound PcephPT contains a cephalosporin core linked to 2-mercaptopyridine N-oxide (pyrithione) via one of its metal-chelating atoms, which minimizes its pre-activation interaction with metal ions and its cytotoxicity. Spectroscopic and chromatographic assays indicate that PcephPT releases pyrithione in the presence of β-lactamase-producing bacteria. The prochelator shows enhanced antibacterial activity against strains expressing β-lactamases, with bactericidal efficacy improved by the presence of lowmicromolar copper in the growth medium. Metal analysis shows that cell-associated copper accumulation by the prochelator is significantly lower than that induced by pyrithione itself, suggesting that the location of pyrithione release influences biological outcomes. Lowmicromolar (4–8 μg/mL) MIC values of PcephPT in ceftriaxone-resistant bacteria compared with LD(50) values greater than 250 μM in mammalian cells suggests favorable selectivity. Further investigation into the mechanisms of prochelators will provide insight for the design of new antibacterial agents that manipulate cellular metallobiology as a strategy against infection.

Pyrithione (2-mercaptopyridine-N-oxide) is a metal binding modified pyridine, the antibacterial activity of which was described over 60 years ago. The formulation of zinc-pyrithione is commonly used in the topical treatment of certain dermatological conditions. However, the characterisation of the cellular uptake of pyrithione has not been elucidated, although an unsubstantiated assumption has persisted that pyrithione and/or its metal complexes undergo a passive diffusion through cell membranes. Here, we have profiled specific membrane transporters from an unbiased interrogation of 532 E. coli strains of knockouts of genes encoding membrane proteins from the Keio collection. Two membrane transporters, FepC and MetQ, seemed involved in the uptake of pyrithione and its cognate metal complexes with copper, iron, and zinc. Additionally, the phenotypes displayed by CopA and ZntA knockouts suggested that these two metal effluxers drive the extrusion from the bacterial cell of potentially toxic levels of copper, and perhaps zinc, which hyperaccumulate as a function of pyrithione. The involvement of these distinct membrane transporters contributes to the understanding of the mechanisms of action of pyrithione specifically and highlights, more generally, the important role that membrane transporters play in facilitating the uptake of drugs, including metal–drug compounds.

The range of applications for photoswitching moieties is diverse, and the ability to design switches with variable photochemical and physical properties is consequently important for realizing their potential. Previously we reported on the photochromism of (E)-N'-(1-(2-hydroxyphenyl)ethylidene)isonicotinohydrazide (HAPI), an aroylhydrazone compound first developed as a transition metal chelator. Herein we report the synthesis of structurally related aroylhydrazone chelators and explore the effect of these modifications on their UVA, UVC and blue light photoreactivity, photostationary state composition, photoisomer thermal stability, and relative iron(iii) binding affinity. These findings will inform the next generation of aroylhydrazone photoswitches for metal-gated photoswitching applications.

The indispensable requirement for metals in life processes has led to the evolution of sophisticated mechanisms that allow organisms to maintain dynamic equilibria of these ions. This dynamic control of the level, speciation, and availability of a variety of metal ions allows organisms to sustain biological processes while avoiding toxicity. When functioning properly, these mechanisms allow cells to return to their metal homeostatic set points following shifts in the metal availability or other stressors. These periods of transition, when cells are in a state of flux in which they work to regain homeostasis, present windows of opportunity to pharmacologically manipulate targets associated with metal-trafficking pathways in ways that could either facilitate a return to homeostasis and the recovery of cellular function or further push cells outside of homeostasis and into cellular distress. The purpose of this Viewpoint is to highlight emerging opportunities for chemists and chemical biologists to develop compounds to manipulate metal-trafficking processes for therapeutic benefit.

Welcome to this Metallomics themed issue highlighting the work of emerging investigators in metallomics, Guest Edited by Metallomics Editorial Board member Katherine Franz (Duke University, USA).

Catecholamines may undergo iron-promoted oxidation resulting in formation of reactive intermediates (aminochromes) capable of redox cycling and reactive oxygen species (ROS) formation. Both of them induce oxidative stress resulting in cellular damage and death. Iron chelation has been recently shown as a suitable tool of cardioprotection with considerable potential to protect cardiac cells against catecholamine-induced cardiotoxicity. However, prolonged exposure of cells to classical chelators may interfere with physiological iron homeostasis. Prochelators represent a more advanced approach to decrease oxidative injury by forming a chelating agent only under the disease-specific conditions associated with oxidative stress. Novel prochelator (lacking any iron chelating properties) BHAPI [(E)-N′-(1-(2-((4-(4,4,5,5-tetramethyl-1,2,3-dioxoborolan-2-yl)benzyl)oxy)phenyl)ethylidene) isonicotinohydrazide] is converted by ROS to active chelator HAPI with strong iron binding capacity that efficiently inhibits iron-catalyzed hydroxyl radical generation. Our results confirmed redox activity of oxidation products of catecholamines isoprenaline and epinephrine, that were able to activate BHAPI to HAPI that chelates iron ions inside H9c2 cardiomyoblasts. Both HAPI and BHAPI were able to efficiently protect the cells against intracellular ROS formation, depletion of reduced glutathione and toxicity induced by catecholamines and their oxidation products. Hence, both HAPI and BHAPI have shown considerable potential to protect cardiac cells by both inhibition of deleterious catecholamine oxidation to reactive intermediates and prevention of ROS-mediated cardiotoxicity.

Herein we report the synthesis and investigation of the properties of two tris-cyclometalated luminescent iridium complexes. These complexes are the simple derivatives of fac-[Ir(ppy)3] bearing amino alkyl groups on one of the phenylpyridine rings. The complexes are highly emissive and exhibit structured emission peaks in aqueous solution while having only broad unstructured emission in organic solvents. The complexes have been shown to be taken up by NIH-3T3 and PC3 cells, where they localize in the lysosomes and remain emissive with lifetimes in the microsecond domain.

Disulfiram in conjunction with copper has been shown to be a potent anticancer agent. However, disulfiram’s therapeutic potential in prostate cancer is hindered by off-target effects due to its reactive and nucleophilic thiol-containing component, diethyldithiocarbamate (DTC). To minimize undesirable reactivity, we have strategically blocked the thiol moiety in DTC with a cleavable p-aminobenzyl (pAB) group linked to peptide substrates recognized by prostate specific antigen (PSA). Here we report the synthesis and evaluation in cancer cell models of two PSA-activatable prodrugs: HPD (Ac-HSSKLQL-pAB-DTC and RPD (RSSYYSL-pAB-DTC). In vitro exposure to PSA was found to trigger activation of HPD and RPD to release diethyldithiocarbamate, and both prodrugs were found to induce toxicity in prostate cancer cells, with HPD showing the most promising selectivity. With copper supplementation, the IC50 of HPD was 1.4 µM in PSA-expressing LNCaP cells, and 11 µM in PC3 cells that do not express PSA. These studies demonstrate the utility of using peptide recognition handles to direct the activity of dithiocarbamate prodrugs for selective cytotoxicity of cancer cells.

The avidity of prostate cancer cells for copper sensitizes them to cytotoxicity by disulfiram, a dithiocarbamate-containing drug approved for alcohol aversion therapy. While disulfiram has received attention as a possible therapy for various cancers, its lack of cancer specificity is a liability that limits this promise. Here we present a prodrug approach to direct copper-dependent cytotoxicity of dithiocarbamate pharmacophores to prostate cancer cells. The prochelator GGTDTC requires activation by gamma-glutamyl transferase (GGT) to release the metal chelator diethyldithiocarbamate from a linker that masks the thiol reactivity and metal binding properties of the pharmacophore prior to activation. In vitro studies demonstrated successful masking of its copper binding properties as well as clean liberation of the chelator by GGT. GGTDTC was found to be stable to non-specific degradation when incubated with a series of prostate cancer and normal cell lines, with selective release of diethyldithiocarbamate only occurring in cells with measurable GGT activity. The antiproliferative efficacy of the prochelator correlated with cellular GGT activity, with 24 h half maximal inhibitory concentrations ranging from 800 nM in prostate cancer lines 22Rv1 and LNCaP to over 15 μM in normal prostate PWR-1E cells. These findings underscore a new strategy to leverage the amplified copper metabolism of prostate cancer by conditional activation of a metal-binding pharmacophore.

Copper is a vital metal cofactor in enzymes that are essential to myriad biological processes. Cellular acquisition of copper is primarily accomplished through the Ctr family of plasma membrane copper transport proteins. Model peptide studies indicate that the human Ctr1 N-terminus binds to Cu(II) with high affinity through an amino terminal Cu(II), Ni(II) (ATCUN) binding site. Unlike typical ATCUN-type peptides, the Ctr1 peptide facilitates the ascorbate-dependent reduction of Cu(II) bound in its ATCUN site by virtue of an adjacent HH (bis-His) sequence in the peptide. It is likely that the Cu(I) coordination environment influences the redox behavior of Cu bound to this peptide; however, the identity and coordination geometry of the Cu(I) site has not been elucidated from previous work. Here, we show data from NMR, XAS, and structural modeling that sheds light on the identity of the Cu(I) binding site of a Ctr1 model peptide. The Cu(I) site includes the same bis-His site identified in previous work to facilitate ascorbate-dependent Cu(II) reduction. The data presented here are consistent with a rational mechanism by which Ctr1 provides coordination environments that facilitate Cu(II) reduction prior to Cu(I) transport.

A peptide has been designed so that its chelating affinity for one type of metal ion regulates its affinity for a second, different type of metal ion., A peptide has been designed so that its chelating affinity for one type of metal ion regulates its affinity for a second, different type of metal ion. The prochelator peptide (PCP), which is a fusion of motifs evocative of calcium loops and zinc fingers, forms a 1 : 2 Zn : peptide complex at pH 7.4 that increases its affinity for Zn2+ ∼3-fold in the presence of Tb3+ (log β2 from 13.8 to 14.3), while the 1 : 1 luminescent complex with Tb3+ is brighter, longer lived, and 20-fold tighter in the presence of Zn2+ (log K from 6.2 to 7.5). This unique example of cooperative, heterometallic allostery in a biologically compatible construct suggests the possibility of designing conditionally active metal-binding agents that could respond to dynamic changes in cellular metal status.

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 show that availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that co-treatment of Hist-5 with Cu improved the EC50 from ~5 µM to ~1 µM, whereas co-treatment with a high-affinity Cu-specific chelator abrogated antifungal activity. Spectrophotometric titrations revealed two previously unrecognized Cu(I) binding sites with apparent Kd values at pH 7.4 ~ 20 nM, and confirmed a high-affinity Cu(II) binding site at the Hist-5 N-terminus with apparent Kd ~ 8 pM. Evaluation of a series of His-to-Ala peptides containing the first 12 residues of Hist-5 identified adjacent His residues (bis-His) as critical anchors for Cu(I) binding, and the presence of a third ligand was revealed by X-ray absorption spectroscopy (XAS). On their own, the truncated peptides were ineffective at inhibiting growth of C. albicans, but treatment with supplemental Cu resulted in EC50 values down to ~ 5 µM, approaching that of full-length Hist-5. The efficacy of the peptides depended on an intact bis-His site and correlated with Cu(I) affinity. Together, these results establish new structure-function relationships linking specific histidine residues with Cu-binding affinity and antifungal activity, and provide further evidence for the involvement of metals in modulating the biological activity of these antifungal peptides.

The prochelator BSIH ((E)-N'-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) contains a boronate group that prevents metal coordination until reaction with peroxide releases the iron chelator SIH ((E)-N'-(2-hydroxybenzylidene)isonicotinohydrazide). BSIH exists in aqueous buffer and cell culture media in equilibrium with its hydrolysis products isoniazid and (2-formylphenyl)boronic acid (FBA). The relative concentrations of these species limit the yield of intact SIH available for targeted iron chelation. While the hydrolysis fragments are nontoxic to retinal pigment epithelial cells, these results suggest that modifications to BSIH that improve its hydrolytic stability yet maintain its low inherent cytotoxicity are desirable for creating more efficient prochelators for protection against cellular oxidative damage.

A panel of iron (Fe) and copper (Cu) chelators was screened for growth inhibitory activity against the fungal pathogen Cryptococcus neoformans. Select bidentate metal-binding ligands containing mixed O,S or O,N donor atoms were identified as agents that induce cell killing in a Cu-dependent manner. Conversely, structurally similar ligands with O,O donor atoms did not inhibit C. neoformans growth regardless of Cu status. Studies of Cu(II) and Cu(I) binding affinity, lipophilicity, and growth recovery assays of Cu-import deficient cells identified lipophilicity of thermodynamically stable CuIIL2 complexes as the best predictor of antifungal activity. These same complexes induce cellular hyperaccumulation of Zn and Fe in addition to Cu. The results described here present the utility of appropriate metal-binding ligands as potential antifungal agents that manipulate cellular metal balance as an antimicrobial strategy.

Metal ions are essential for a wide range of physiological processes, but they can also be toxic if not appropriately regulated by a complex network of metal trafficking proteins. Intervention in cellular metal distribution with small molecule or peptide chelating agents has promising therapeutic potential to harness metals to fight disease. Molecular outcomes associated with forming metal-chelate interactions in situ include altering concentration and subcellular metal distribution, inhibiting metalloenzymes, enhancing the reactivity of a metal species to elicit a favorable biological response, or passivating the reactivity of a metal species to prevent deleterious reactivity. The systemic administration of metal chelating agents, however, raises safety concerns due to the potential risks of indiscriminate extraction of metals from critical metalloproteins and inhibition of metalloenzymes. One can estimate that chelators capable of complexing metal ions with dissociation constants in the sub-micromolar range are thermodynamically capable of extracting metal ions from some metalloproteins and disrupting regular function. Such dissociation constants are easily attainable for multidentate chelators interacting with first-row d-block metal cations in relevant +1, +2, and +3 oxidation states. To overcome this challenge of indiscriminate metal chelation, we have pursued a prodrug strategy for chelating agents in which the resulting “prochelator” has negligible metal binding affinity until a specific stimulus generates a favorable metal binding site. The prochelator strategy enables conditional metal chelation to occur preferentially in spatial or temporal location initiated by disease- or therapy-associated stimuli, thereby minimizing off-target metal chelation. Our design of responsive prochelators encompasses three general approaches of activation: the “removal” approach operates by eliminating a masking group that blocks a potential metal chelation site to reveal the complete binding site under the desired conditions; the molecular “switch” approach involves a reversible conformational change between an inactive and active form of a chelator with differential metal binding affinity under specific conditions; while the “addition” approach adds a new ligand donor arm to the prochelator to constitute a complete metal chelation site. Adopting these approaches, we have created four categories of triggerable prochelators that respond to 1) reactive oxygen species, 2) light, 3) specific enzymes, and 4) biological regulatory events. This account highlights progress from our group on building prochelators that showcase these four categories of responsive metal chelating agents for manipulating cellular metals. The creation and chemical understanding of such stimulus-responsive prochelators enables exciting applications for understanding the cell biology of metals and for developing therapies based on metal-dependent processes in a variety of diseases.

Free cellular iron catalyzes the formation of toxic hydroxyl radicals and therefore chelation of iron could be a promising therapeutic approach in pathological states associated with oxidative stress. Salicylaldehyde isonicotinoyl hydrazone (SIH) is a strong intracellular iron chelator with well documented potential to protect against oxidative damage both in vitro and in vivo. Due to the short biological half-life of SIH and risk of toxicity due to iron depletion, boronate prochelator BSIH has been designed. BSIH cannot bind iron until it is activated by certain reactive oxygen species to active chelator SIH. The aim of this study was to examine the toxicity and cytoprotective potential of BSIH, SIH, and their decomposition products against hydrogen peroxide-induced injury of H9c2 cardiomyoblast cells. Using HPLC, we observed that salicylaldehyde was the main decomposition products of SIH and BSIH, although a small amount of salicylic acid was also detected. In the case of BSIH, the concentration of formed salicylaldehyde consistently exceeded that of SIH. Isoniazid and salicylic acid were not toxic nor did they provide any antioxidant protective effect in H9c2 cells. In contrast, salicylaldehyde was able to chelate intracellular iron and significantly preserve cellular viability and mitochondrial inner membrane potential induced by hydrogen peroxide. However it was consistently less effective than SIH. The inherent toxicities of salicylaldehyde and SIH were similar. Hence, although SIH - the active chelating agent formed following the BSIH activation - undergoes rapid hydrolysis, its principal decomposition product salicylaldehyde accounts markedly for both cytoprotective and toxic properties.

UVA radiation can damage cells and tissues by direct photodamage of biomolecules as well as by initiating metal-catalyzed oxidative stress. In order to alleviate both concerns simultaneously, we synthesized a multifunctional prochelator PC-HAPI (2-(( E )-1-(2-isonicotinoylhydrazono)ethyl)phenyl ( trans )-3-(2,4-dihydroxyphenyl)acrylate) that contains a trans -( o -hydroxy)cinnamate ester photocleavable protecting group that is cleaved upon UVA exposure to release a coumarin, umbelliferone, and an aroylhydrazone metal chelator, HAPI ( N ′-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide). While the prochelator PC-HAPI exhibits negligible affinity for iron, it responds rapidly to UVA irradiation and converts to an iron-binding chelator that inhibits iron-catalyzed formation of reactive oxygen species and protects cells from UVA damage.

Salicylaldehyde isonicotinoyl hydrazone (SIH) is an intracellular iron chelator with well documented potential to protect against oxidative injury both in vitro and in vivo. However, it suffers from short biological half-life caused by fast hydrolysis of the hydrazone bond. Recently, a concept of boronate prochelators has been introduced as a strategy that might overcome these limitations. This study presents two complementary analytical methods for detecting the prochelator BSIH (boronyl salicylaldehyde isonicotinoyl hydrazone) along with its active metal-binding chelator SIH in different solution matrices and concentration ranges. An LC-UV method for determination of BSIH and SIH in buffer and cell culture medium was validated over concentrations of 7 – 115 and 4 – 115 μM, respectively, and applied to BSIH activation experiments in vitro. An LC-MS assay was validated for quantification of BSIH and SIH in plasma over the concentration range of 0.06 – 23 and 0.24 – 23 μM, respectively, and applied to stability studies in plasma in vitro as well as analysis of plasma taken after i.v. administration of BSIH to rats. A Zorbax-RP bonus column and mobile phases containing either phosphate buffer with EDTA or ammonium formate and methanol/acetonitrile mixture provided suitable conditions for the LC-UV and LC-MS analysis, respectively. Samples were diluted or precipitated with methanol prior to analysis. These separative analytical techniques establish the first validated protocols to investigate BSIH activation by hydrogen peroxide in multiple matrices, directly compare the stabilities of the prochelator and chelator in plasma, and provide the first basic pharmacokinetic data of this prochelator. Experiments reveal that BSIH is stable in all media tested and is partially converted to SIH by H2O2. The observed integrity of BSIH in plasma samples from the in vivo study suggest that the concept of prochelation might be a promising strategy for further development of aroylhydrazone cytoprotective agents.