Biomimetic Analogues of the Desferrioxamine E Siderophore for PET Imaging of Invasive Aspergillosis: Targeting Properties and Species Specificity.

The pathogenic fungus Aspergillus fumigatus utilizes a cyclic ferrioxamine E (FOXE) siderophore to acquire iron from the host. Biomimetic FOXE analogues were labeled with gallium-68 for molecular imaging with PET. [68Ga]Ga(III)-FOXE analogues were internalized in A. fumigatus cells via Sit1. Uptake of [68Ga]Ga(III)-FOX 2-5, the most structurally alike analogue to FOXE, was high by both A. fumigatus and bacterial Staphylococcus aureus. However, altering the ring size provoked species-specific uptake between these two microbes: ring size shortening by one methylene unit (FOX 2-4) increased uptake by A. fumigatus compared to that by S. aureus, whereas lengthening the ring (FOX 2-6 and 3-5) had the opposite effect. These results were consistent both in vitro and in vivo, including PET imaging in infection models. Overall, this study provided valuable structural insights into the specificity of siderophore uptake and, for the first time, opened up ways for selective targeting and imaging of microbial pathogens by siderophore derivatization.

Aspergillus fumigatus ZrfC Zn(II) transporter scavengers zincophore-bound Zn(II).

Aspergillus fumigatus is an opportunistic pathogen that is able to invade and grow in the lungs of immunosuppressed patients and cause invasive pulmonary aspergillosis. The concentration of free Zn(II) in living tissues is much lower than that required for optimal fungal growth; thus, to obtain Zn(II) from the host, Aspergillus fumigatus uses highly specified Zn(II) transporters: ZrfA, ZrfB and ZrfC. The ZrfC transporter plays the main role in Zn(II) acquisition from the host in neutral and mildly alkaline environment via interacting with the secreted Aspf2 zincophore. Understanding the Aspf2-ZrfC interactions is therefore necessary for explaining the process of Zn(II) acquisition by Aspergillus fumigatus, and identifying Zn(II) binding sites in its transporter and describing the thermodynamics of such binding are the fundamental steps to achieve this goal. We focus on two probable ZrfC Zn(II) binding sites and show that the Ac-MNCHFHAGVEHCIGAGESESGSSQ-NH2 region binds Zn(II) with higher affinity than the Ac-TGCHSHGS-NH2 one and that this binding is much stronger than the binding of Zn(II) to the Aspf2 zincophore, allowing efficient Zn(II) transport from the Aspf2 zincophore to the ZrfC transporter. The same ZrfC fragments also able to bind Ni(II), another metal ion essential for fungi that could also compete with Zn(II) binding, with comparable affinity.

-HH and -HAAAH motifs act as fishing nets for biologically relevant metal ions in metallopeptides.

Histidine are one of the most common residues involved in transition metal ion binding in the active sites of metalloenzymes. In order to mimic enzymatic metal binding sites, it is crucial to understand the basic coordination modes of histidine residues, distributed at different positions in the peptide sequence. We show that: (i) the separation of two histidines has a large effect on complex stability - a sequence with adjusting histidine residues forms more stable complexes with Zn(II) than the one in which the residues are separated, while the contrary is observed for Cu(II) complexes, in which amide nitrogens participate in metal binding. No pronounced effect is observed for Ni(II) complexes, where the amides participate in binding at higher pH; (ii) non-coordinating amino acid residues (basic, acidic and aromatic ones) have a significant impact on complex stability; charged and aromatic residues may enhance Zn(II) binding, while the contrary is observed for the amide-binding Cu(II); (iii) cysteine containing sequences are much more effective Zn(II) and Ni(II) binding motifs at pH above 8, while histidine containing ligands are more suitable for effective Zn(II) and Ni(II) binding at lower pH.

Fever-Range Hyperthermia Promotes Macrophage Polarization towards Regulatory Phenotype M2b.

Fever-range hyperthermia (FRH) is utilized in chronic disease treatment and serves as a model for fever's thermal component investigation. Macrophages, highly susceptible to heat, play a pivotal role in various functions determined by their polarization state. However, it is not well recognized whether this process can be modulated by FRH. To address this, we used two different macrophage cell lines that were treated with FRH. Next, to define macrophage phenotype, we examined their functional surface markers CD80 and CD163, intracellular markers such as inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1), and the expression of interleukin-10 (IL-10) and tumor necrosis factor α (TNF-α). Additionally, in FRH-treated cells, we analyzed an expression of Toll-like receptor 4 (TLR-4) and its role in macrophage polarization. We also checked whether FRH can switch the polarization of macrophages in pro-inflammatory condition triggered by lipopolysaccharide (LPS). FRH induced M2-like polarization, evident in increased CD163, IL-10, and Arg-1 expression. Notably, elevated COX-2, TNF-α, and TLR-4 indicated potential pro-inflammatory properties, suggesting polarization towards the M2b phenotype. Additionally, FRH shifted lipopolysaccharide (LPS)-induced M1 polarization to an M2-like phenotype, reducing antimicrobial molecules (ROS and NO). In summary, FRH emerged as a modulator favoring M2-like macrophage polarization, even under pro-inflammatory conditions, showcasing its potential therapeutic relevance.

Multistep synthesis of a novel copper complex with potential for Alzheimer's disease diagnosis.

Positron emission tomography (PET) imaging of Aß plaques, is recognized as a tool for the diagnosis of Alzheimer's disease. As a contribution to the development of new strategies for early diagnosis of the disease, using PET medical imaging technique, a new copper complex, the [Cu(TE1PA-ONO)]+ was synthesized in ten steps. The key step of our strategy is the coupling of a monopicolinate-N-alkylated cyclam-based ligand with a moiety capable of recognizing Aß plaques via a successful and challenging Buchwald-Hartwig coupling reaction. To our knowledge, it is the first time that such a strategy is used to functionalize polyazamacrocyclic derivatives. The thermodynamic stability constants determined in MeOH/H2O solvent indicate that the attachment of this moiety does not weaken the chelating properties of TE1PA-ONO ligand in relation to parent HTE1PA. The novel complex described here is able to recognize amyloid plaques in brain sections from Alzheimer's disease patients and shows low toxicity to human neuronal cells.

Fe(II), Mn(II), and Zn(II) Binding to the C-Terminal Region of FeoB Protein: An Insight into the Coordination Chemistry and Specificity of the Escherichia coli Fe(II) Transporter.

The interactions between two peptide ligands [Ac763CCAASTTGDCH773 (P1) and Ac743RRARSRVDIELLATRKSVSSCCAASTTGDCH773 (P2)] derived from the cytoplasmic C-terminal region of Eschericha coli FeoB protein and Fe(II), Mn(II), and Zn(II) ions were investigated. The Feo system is regarded as the most important bacterial Fe(II) acquisition system, being one of the key virulence factors, especially in anaerobic conditions. Located in the inner membrane of Gram-negative bacteria, FeoB protein transports Fe(II) from the periplasm to the cytoplasm. Despite its crucial role in bacterial pathogenicity, the mechanism in which the metal ion is trafficked through the membrane is not yet elucidated. In the gammaproteobacteria class, the cytoplasmic C-terminal part of FeoB contains conserved cysteine, histidine, and glutamic and aspartic acid residues, which could play a vital role in Fe(II) binding in the cytoplasm, receiving the metal ion from the transmembrane helices. In this work, we characterized the complexes formed between the whole cytosolic C-terminal sequence of E. coli FeoB (P2) and its key polycysteine region (P1) with Fe(II), Mn(II), and Zn(II) ions, exploring the specificity of the C-terminal region of FeoB. With the help of a variety of potentiometric, spectroscopic (electron paramagnetic resonance and NMR), and spectrometric (electrospray ionization mass spectrometry) techniques and molecular dynamics, we propose the metal-binding modes of the ligands, compare their affinities toward the metal ions, and discuss the possible physiological role of the C-terminal region of E. coli FeoB.

Metal coordination to solute binding proteins - exciting chemistry with potential biological meaning.

Zn(II) is essential for bacterial survival and virulence. In host cells, its abundance is extremely limited, thus, bacteria have evolved transport mechanisms that enable them to take up this essential metal nutrient. Paracoccus denitrificans encodes two solute binding proteins (SBPs) - ZnuA and AztC, which are responsible for zinc acquisition from the host cells. We focus on understanding the interactions of Zn(II) and Ni(II) (zinc's potential competitor, which is a biologically relevant metal ion essential for various bacterial enzymes) with the extracellular ZnuA and AztC's loops from P. denitrificans that are expected to be possible Zn(II) binding sites. In the case of Zn(II) complexes with ZnuA outercellular loop regions, the numerous histidines act as anchoring donors, forming complexes with up to four coordinated His residues, while in the AztC region, three imidazole nitrogens and one water molecule are involved in Zn(II) binding. In Zn(II) complexes with ZnuA His-rich loop regions, so-called polymorphic binding sites are observed. The large number of available imidazoles and carboxylic side chains also strongly affects the structure of Ni(II) complexes; the more histidines in the studied peptide, the higher the affinity to bind Ni(II) and the higher the pH value at which amide nitrogens start to participate in Ni(II) binding. Additionally, for Ni(II)-ZnuA complexes, a more rare octahedral geometry is observed and such complexes are more stable than the corresponding Zn(II) ones, in contrast to what was observed in the AztC region, suggesting that the numerous histidyl and glutamic acid side chains are more tempting for Ni(II) than for Zn(II).The general strong affinity of Zn(II)-zincophore complexes is also discussed.

Endotoxin Tolerance Creates Favourable Conditions for Cancer Development.

Endotoxin tolerance (ET) is an adaptive phenomenon of the immune system that protects the host from clinical complications due to repeated exposure of the body to endotoxins such as lipopolysaccharide (LPS). Since ET is an immunosuppressive mechanism in which a significant reprogramming of macrophages is observed, we hypothesized that it could influence cancer development by modifying the tumour environment. This study aimed to explore whether ET influences cancer progression by altering the tumour microenvironment. Endotoxin-tolerant macrophages (MoET) were examined for their impact on breast and colon cancer cells via direct interaction and conditioned media exposure. We characterized cancer cell behaviour by viability, clonogenic potential, motility, scratch assays, and 3D spheroidal assays. MoET-derived factors increased cancer cell viability, motility, and clonogenicity, suggesting a conducive environment for cancer development. Remarkably, despite reduced TNFα and IL-6 levels, MoET exhibited M1 polarization. These findings uncover an ET-associated macrophage reprogramming that fosters a favourable context for cancer progression across diverse tumours. Targeting ET could emerge as a promising avenue for cancer therapy and prevention.

Fever-range whole body hyperthermia leads to changes in immune-related genes and miRNA machinery in Wistar rats.

OBJECTIVE: Fever is defined as a rise in body temperature upon disease. Fever-range hyperthermia (FRH) is a simplified model of fever and a well-established medical procedure. Despite its beneficial effects, the molecular changes induced by FRH remain poorly characterized. The aim of this study was to investigate the influence of FRH on regulatory molecules such as cytokines and miRNAs involved in inflammatory processes. METHODS: We developed a novel, fast rat model of infrared-induced FRH. The body temperature of animals was monitored using biotelemetry. FRH was induced by the infrared lamp and heating pad. White blood cell counts were monitored using Auto Hematology Analyzer. In peripheral blood mononuclear cells, spleen and liver expression of immune-related genes (IL-10, MIF and G-CSF, IFN-γ) and miRNA machinery (DICER1, TARBP2) was analyzed with RT-qPCR. Furthermore, RT-qPCR was used to explore miRNA-155 levels in the plasma of rats. RESULTS: We observed a decrease in the total number of leukocytes due to lower number of lymphocytes, and an increase in the number of granulocytes. Furthermore, we observed elevated expressions of DICER1, TARBP2 and granulocyte colony-stimulating factor (G-CSF) in the spleen, liver and PBMCs immediately following FRH. FRH treatment also had anti-inflammatory effects, evidenced by the downregulation of pro-inflammatory macrophage migration inhibitor factor (MIF) and miR-155, and the increased expression of anti-inflammatory IL-10. CONCLUSION: FRH affects the expression of molecules involved in inflammatory processes leading to alleviated inflammation. We suppose these effects may be miRNAs-dependent and FRH can be involved in therapies where anti-inflammatory action is needed.

CH vs. HC-Promiscuous Metal Sponges in Antimicrobial Peptides and Metallophores.

Histidine and cysteine residues, with their imidazole and thiol moieties that deprotonate at approximately physiological pH values, are primary binding sites for Zn(II), Ni(II) and Fe(II) ions and are thus ubiquitous both in peptidic metallophores and in antimicrobial peptides that may use nutritional immunity as a way to limit pathogenicity during infection. We focus on metal complex solution equilibria of model sequences encompassing Cys-His and His-Cys motifs, showing that the position of histidine and cysteine residues in the sequence has a crucial impact on its coordination properties. CH and HC motifs occur as many as 411 times in the antimicrobial peptide database, while similar CC and HH regions are found 348 and 94 times, respectively. Complex stabilities increase in the series Fe(II) < Ni(II) < Zn(II), with Zn(II) complexes dominating at physiological pH, and Ni(II) ones-above pH 9. The stabilities of Zn(II) complexes with Ac-ACHA-NH2 and Ac-AHCA-NH2 are comparable, and a similar tendency is observed for Fe(II), while in the case of Ni(II), the order of Cys and His does matter-complexes in which the metal is anchored on the third Cys (Ac-AHCA-NH2) are thermodynamically stronger than those where Cys is in position two (Ac-ACHA-NH2) at basic pH, at which point amides start to take part in the binding. Cysteine residues are much better Zn(II)-anchoring sites than histidines; Zn(II) clearly prefers the Cys-Cys type of ligands to Cys-His and His-Cys ones. In the case of His- and Cys-containing peptides, non-binding residues may have an impact on the stability of Ni(II) complexes, most likely protecting the central Ni(II) atom from interacting with solvent molecules.

The diversity and utility of arylthiazoline and aryloxazoline siderophores: challenges of total synthesis.

Siderophores are unique ferric ion chelators produced and secreted by some organisms like bacteria, fungi and plants under iron deficiency conditions. These molecules possess immense affinity and specificity for Fe3+ and other metal ions, which attracts great interest due to the numerous possibilities of application, including antibiotics delivery to resistant bacteria strains. Total synthesis of siderophores is a must since the compounds are present in natural sources at extremely small concentrations. These molecules are extremely diverse in terms of molecular structure and physical and chemical properties. This review is focused on achievements and developments in the total synthesis strategies of naturally occurring siderophores bearing arylthiazoline and aryloxazoline units.

Poly-Gly Region Regulates the Accessibility of Metal Binding Sites in Snake Venom Peptides.

It is supposed that the presence of poly-His regions in close proximity to poly-Gly domains in snake venoms is related to their biological activity; poly-His/poly-Gly (pHpG) peptides inhibit the activity of metalloproteinases during venom storage via the chelation metal ions, necessary for their proper functioning. This work shows that only the histidyl residues from the N-terminal VDHDHDH motif (but not from the poly-His tag) were the primary Zn(II) binding sites and that the poly-Gly domain situated in the proximity of a central proline residue may play a regulatory role in venom gland protection. The proline induces a kink of the peptide, resulting in steric hindrance, which may modulate the accessibility of potential metal binding sites in the poly-His domain and may, in turn, be one of the regulators of Zn(II) accessibility in the venom gland and therefore a modulator of metalloproteinase activity during venom storage.

Specific Zn(II)-binding site in the C-terminus of Aspf2, a zincophore from Aspergillus fumigatus.

Aspergillus fumigatus, one of the most widespread opportunistic human fungal pathogens, adapts to zinc limitation by secreting a 310 amino acid Aspf2 zincophore, able to specifically bind Zn(II) and deliver it to a transmembrane zinc transporter, ZrfC. In this work, we focus on the thermodynamics of Zn(II) complexes with unstructured regions of Aspf2; basing on a variety of spectrometric and potentiometric data, we show that the C-terminal part has the highest Zn(II)-binding affinity among the potential binding sites, and Ni(II) does not compete with Zn(II) binding to this region. The 14 amino acid Aspf2 C-terminus coordinates Zn(II) via two Cys thiolates and two His imidazoles and it could be considered as a promising A. fumigatus targeting molecule.

Cyclic Analogs of Desferrioxamine E Siderophore for 68Ga Nuclear Imaging: Coordination Chemistry and Biological Activity in Staphylococcus aureus.

As multidrug-resistant bacteria are an emerging problem and threat to humanity, novel strategies for treatment and diagnostics are actively sought. We aim to utilize siderophores, iron-specific strong chelating agents produced by microbes, as gallium ion carriers for diagnosis, applying that Fe(III) can be successfully replaced by Ga(III) without losing biological properties of the investigated complex, which allows molecular imaging by positron emission tomography (PET). Here, we report synthesis, full solution chemistry, thermodynamic characterization, and the preliminary biological evaluation of biomimetic derivatives (FOX) of desferrioxamine E (FOXE) siderophore, radiolabeled with 68Ga for possible applications in PET imaging of S. aureus. From a series of six biomimetic analogs, which differ from FOXE with cycle length and position of hydroxamic and amide groups, the highest Fe(III) and Ga(III) stability was determined for the most FOXE alike compounds-FOX 2-4 and FOX 2-5; we have also established the stability constant of the Ga-FOXE complex. For this purpose, spectroscopic and potentiometric titrations, together with the Fe(III)-Ga(III) competition method, were used. [68Ga]Ga-FOXE derivatives uptake and microbial growth promotion studies conducted on S. aureus were efficient for compounds with a larger cavity, i.e., FOX 2-5, 2-6, and 3-5. Even though showing low uptake values, Fe-FOX 2-4 seems to be also a good Fe-source to support the growth of S. aureus. Overall, proposed derivatives may hold potential as inert and stable carrier agents for radioactive Ga(III) ions for diagnostic medical applications or interesting starting compounds for further modifications.

Metal specificity of the Ni(II) and Zn(II) binding sites of the N-terminal and G-domain of E. coli HypB.

HypB is one of the chaperones required for proper nickel insertion into [NiFe]-hydrogenase. Escherichia coli HypB has two potential Ni(II) and Zn(II) binding sites-the N-terminal one and the so-called GTPase one. The metal-loaded HypB-SlyD metallochaperone complex activates nickel release from the N-terminal HypB site. In this work, we focus on the metal selectivity of the two HypB metal binding sites and show that (i) the N-terminal region binds Zn(II) and Ni(II) ions with higher affinity than the G-domain and (ii) the lower affinity G domain binds Zn(II) more effectively than Ni(II). In addition, the high affinity N-terminal domain, both in water and membrane mimicking SDS solution, has a larger affinity towards Zn(II) than Ni(II), while an opposite situation is observed at basic pH; at pH 7.4, the affinity of this region towards both metals is almost the same. The N-terminal HypB region is also more effective in Ni(II) binding than the previously studied SlyD metal binding regions. Considering that the nickel chaperone SlyD activates the release of nickel and blocks the release of zinc from the N-terminal high-affinity metal site of HypB, we may speculate that such pH-dependent metal affinity might modulate HypB interactions with SlyD, being dependent on both pH and the protein's metal status.

Sometimes less is more-the impact of the number of His residues on the stability of Zn(II)-SmtB and BigR4 α-5 domain complexes.

The increasing number of antibiotic-resistant pathogens has become one of the major health problems of modern times, including infections caused by Mycobacterium tuberculosis. One of the possible mammalian immune system responses to mycobacterial infection is the increase of the zinc(II) concentration in phagosomes to a toxic level. The mycobacterial SmtB protein belongs to the family of ArsR/SmtB transcription regulators. In the presence of high concentrations of metals, SmtB dissociates from DNA and activates the expression of metal efflux proteins. In this work, we focus on the α5 zinc(II) binding domains of SmtB/BigR4 proteins (the latter being the SmtB homolog from non-pathogenic M. smegmatis) and two mutants of BigR4. We will be taking a closer look at the coordination modes and thermodynamic properties of their zinc(II) complexes. The study points out the specificity of metal-ligand interactions and describes the effect of mutations on the coordination properties of the studied systems. The stabilities of the zinc(II) complexes were determined by potentiometry. The coordination sites were determined by NMR experiments and DFT calculations. The comparison of complex stabilities reveals that the Zn(II)-BigR4 species are more stable than the Zn(II)-SmtB complexes. His mutations strongly affect the stability of the complexes and the coordination modes of the metal ion. Exchanging one of the histidines for alanine causes, surprisingly, an increase in the stability of zinc(II) complexes with the studied domain. This was confirmed by potentiometric and DFT methods. This work can be considered as a bioinorganic introduction to the discovery of new strategies in M. tuberculosis infection treatment based on zinc(II)-sensitive mechanisms.

Distinct Modulatory Effects of Fever-Range Hyperthermia on the Response of Breast Cancer Cells and Macrophages to Mistletoe (Viscum album L.) Extract.

Heat utility as a critical component of fever is often ignored, although the symptom is observed in many medical conditions. Mistletoe extract (ME) is an adjunctive medication prescribed to cancer patients. The increase in body temperature is frequently observed in patients following ME administration. Nevertheless, the impact of this fever on the effectiveness of therapy is unknown. Therefore, we aimed to investigate the effect of fever-range temperatures on ME-treated breast cancer cells and macrophages. The cells were simultaneously stimulated with ME and subjected to fever-range hyperthermia (FRH; 39 °C or 41 °C). After co-treatment, the cell viability, generation of reactive oxygen species (ROS), cell cycle distribution, and production of pro-inflammatory factors (interleukin (IL)-1ß, IL-6, and cyclooxygenase (COX)-2) were evaluated. The results showed that the exposure of ME-treated breast cancer cells to FRH at 39 °C resulted in a slight decrease in their viability, whereas FRH of 41 °C enhanced this effect. Only FRH of 41 °C induced minor changes in ROS level in ME-treated breast cancer cell lines. In ME-treated macrophages, FRH stimulated cell proliferation. The cell cycle distribution analysis showed a difference between cells cultured at 39 °C and 41 °C in all examined cell lines. Moreover, hyperthermia at 41 °C completely inhibited the ME-induced increase in IL-1ß and IL-6 expression in MCF-7 breast cancer cells, whereas this effect was not observed in 4T1 breast cancer cells. In contrast, in ME-treated macrophages, FRH of 41 °C strongly up-regulated expression of the pro-inflammatory factors. We conclude that fever is an important component of ME therapy that differentially affects cancer and immune cells.

Peptidomimetics - An infinite reservoir of metal binding motifs in metabolically stable and biologically active molecules.

The involvement of metal ions in interactions with therapeutic peptides is inevitable. They are one of the factors able to fine-tune the biological properties of antimicrobial peptides, a promising group of drugs with one large drawback - a problematic metabolic stability. Appropriately chosen, proteolytically stable peptidomimetics seem to be a reasonable solution of the problem, and the use of D-, ß-, γ-amino acids, unnatural amino acids, azapeptides, peptoids, cyclopeptides and dehydropeptides is an infinite reservoir of metal binding motifs in metabolically stable, well-designed, biologically active molecules. Below, their specific structural features, metal-chelating abilities and antimicrobial potential are discussed.

General Aspects of Metal Ions as Signaling Agents in Health and Disease.

This review focuses on the current knowledge on the involvement of metal ions in signaling processes within the cell, in both physiological and pathological conditions. The first section is devoted to the recent discoveries on magnesium and calcium-dependent signal transduction-the most recognized signaling agents among metals. The following sections then describe signaling pathways where zinc, copper, and iron play a key role. There are many systems in which changes in intra- and extra-cellular zinc and copper concentrations have been linked to important downstream events, especially in nervous signal transduction. Iron signaling is mostly related with its homeostasis. However, it is also involved in a recently discovered type of programmed cell death, ferroptosis. The important differences in metal ion signaling, and its disease-leading alterations, are also discussed.

Zinc(II)-The Overlooked Éminence Grise of Chloroquine's Fight against COVID-19?

Zn(II) is an inhibitor of SARS-CoV-2's RNA-dependent RNA polymerase, and chloroquine and hydroxychloroquine are Zn(II) ionophores-this statement gives a curious mind a lot to think about. We show results of the first clinical trials on chloroquine (CQ) and hydroxychloroquine (HCQ) in the treatment of COVID-19, as well as earlier reports on the anticoronaviral properties of these two compounds and of Zn(II) itself. Other FDA-approved Zn(II) ionophores are given a decent amount of attention and are thought of as possible COVID-19 therapeutics.