Design of Tetrazolium Cations for the Release of Antiproliferative Formazan Chelators in Mammalian Cells.

Cancer cells generally present a higher demand for iron, which plays crucial roles in tumor progression and metastasis. This iron addiction provides opportunities to develop broad spectrum anticancer drugs that target iron metabolism. In this context, prochelation approaches are investigated to release metal-binding compounds under specific conditions, thereby limiting off-target toxicity. Here, we demonstrate a prochelation strategy inspired by the bioreduction of tetrazolium cations widely employed to assess the viability of mammalian cells. We designed a series of tetrazolium-based compounds for the intracellular release of metal-binding formazan ligands. The combination of reduction potentials appropriate for intracellular reduction and an N-pyridyl donor on the formazan scaffold led to two effective prochelators. The reduced formazans bind as tridentate ligands and stabilize low-spin Fe(II) centers in complexes of 2:1 ligand-to-metal stoichiometry. The tetrazolium salts are stable in blood serum for over 24 h, and antiproliferative activities at micromolar levels were recorded in a panel of cancer cell lines. Additional assays confirmed the intracellular activation of the prochelators and their ability to affect cell cycle progression, induce apoptotic death, and interfere with iron availability. Demonstrating the role of iron in their intracellular effects, the prochelators impacted the expression levels of key iron regulators (i.e., transferrin receptor 1 and ferritin), and iron supplementation mitigated their cytotoxicity. Overall, this work introduces the tetrazolium core as a platform to build prochelators that can be tuned for activation in the reducing environment of cancer cells and produce antiproliferative formazan chelators that interfere with cellular iron homeostasis.

Supramolecular Arrangement of Lignosulfonate-Based Iron Heteromolecular Complexes and Consequences of Their Interaction with Ca2+ at Alkaline pH and Fe Plant Root Uptake Mechanisms.

Previous studies have shown that natural heteromolecular complexes might be an alternative to synthetic chelates to correct iron (Fe) deficiency. To investigate the mechanism of action of these complexes, we have studied their interaction with Ca2+ at alkaline pH, Fe-binding stability, Fe-root uptake in cucumber, and chemical structure using molecular modeling. The results show that a heteromolecular Fe complex including citric acid and lignosulfonate as binding ligands (Ls-Cit) forms a supramolecular system in solution with iron citrate interacting with the hydrophobic inner core of the lignosulfonate system. These structural features are associated with high stability against Ca2+ at basic pH. Likewise, unlike Fe-EDDHA, root Fe uptake from Ls-Cit implies the activation of the main root responses under Fe deficiency at the transcriptional level but not at the post-transcriptional level. These results are consistent with the involvement of some plant responses to Fe deficiency in the plant assimilation of complexed Fe in Ls-Cit under field conditions.

Synthesis of temporin L hydroxamate-based peptides and evaluation of their coordination properties with iron(III ).

Ferric iron is an essential nutrient for bacterial growth. Pathogenic bacteria synthesize iron-chelating entities known as siderophores to sequestrate ferric iron from host organisms in order to colonize and replicate. The development of antimicrobial peptides (AMPs) conjugated to iron chelators represents a promising strategy for reducing the iron availability, inducing bacterial death, and enhancing simultaneously the efficacy of AMPs. Here we designed, synthesized, and characterized three hydroxamate-based peptides Pep-cyc1, Pep-cyc2, and Pep-cyc3, derived from a cyclic temporin L peptide (Pep-cyc) developed previously by some of us. The Fe3+ complex formation of each ligand was characterized by UV-visible spectroscopy, mass spectrometry, and IR and NMR spectroscopies. In addition, the effect of Fe3+ on the stabilization of the α-helix conformation of hydroxamate-based peptides and the cotton effect were examined by CD spectroscopy. Moreover, the antimicrobial results obtained in vitro on some Gram-negative strains (K. pneumoniae and E. coli) showed the ability of each peptide to chelate efficaciously Fe3+ obtaining a reduction of MIC values in comparison to their parent peptide Pep-cyc. Our results demonstrated that siderophore conjugation could increase the efficacy and selectivity of AMPs used for the treatment of infectious diseases caused by Gram-negative pathogens.

The synthesis and properties of mitochondrial targeted iron chelators.

Iron levels in mitochondria are critically important for the normal functioning of the organelle. Abnormal levels of iron and the associated formation of toxic oxygen radicals have been linked to a wide range of diseases and consequently it is important to be able to both monitor and control levels of the mitochondrial labile iron pool. To this end a series of iron chelators which are targeted to mitochondria have been designed. This overview describes the synthesis of some of these molecules and their application in monitoring mitochondrial labile iron pools and in selectively removing excess iron from mitochondria.

Identification of high iron-chelating peptides with unusual antioxidant effect from sea cucumbers and the possible binding mode.

Iron deficiency anemia (IDA) is a common nutritional disease affecting 2 billion people. To develop a new iron-fortified food, we designed a novel type of iron-chelating peptide [Sea cucumbers peptides (SCP)-Fe] from sea cucumbers. SCP can chelate ferrous ions. The neutral protease hydrolysate have the highest iron chelating activity (117.17 ± 2.62 mg/g). Single factors including pH, material ratio, and molecular weight, had a significant effect on the iron chelating activity. The characterization of the SCP-Fe chelate revealed a loose and blocky structure with increased particle size. The amino acid composition, peptide identification and molecular docking indicated that Asp, Glu, Gly and Pro played an important role in binding to ferrous ions. After chelation, SCP-Fe chelate had dual nutrition effects of stronger radical scavenging ability and potential high-efficiency iron supplementation ability. These results might provide insights into the methods for developing functional foods such as iron-fortified seafood.

Thiol-Reactive Arylsulfonate Masks for Phenolate Donors in Antiproliferative Iron Prochelators.

Tridentate thiosemicarbazones, among several families of iron chelators, have shown promising results in anticancer drug discovery because they target the increased need for iron that characterizes malignant cells. Prochelation strategies, in which the chelator is released under specific conditions, have the potential to avoid off-target metal binding (for instance, in the bloodstream) and minimize unwanted side effects. We report a prochelation approach that employs arylsulfonate esters to mask the phenolate donor of salicylaldehyde-based chelators. The new prochelators liberate a tridentate thiosemicarbazone intracellularly upon reaction with abundant nucleophile glutathione (GSH). A 5-bromo-substituted salicylaldehyde thiosemicarbazone (STC4) was selected for the chelator unit because of its antiproliferative activity at low micromolar levels in a panel of six cancer cell lines. The arylsulfonate prochelators were assessed in vitro with respect to their stability, ability to abolish metal binding, and reactivity in the presence of GSH. Cell-based assays indicated that the arylsulfonate-masked prochelators present higher antiproliferative activities relative to the parent compound after 24 h. The activation and release of the chelator intracellularly were corroborated by assays of cytosolic iron binding and iron supplementation effects as well as cell cycle analysis. This study introduces the 1,3,4-thiadiazole sulfonate moiety to mask the phenolate donor of an iron chelator and impart good solubility and stability to prochelator constructs. The reactivity of these systems can be tuned to release the chelator at high glutathione levels, as encountered in several cancer phenotypes.

Safety and efficacy of a novel iron chelator (HBED; (N,N'-Di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid)) in equine (Equus caballus) as a model for black rhinoceros (Diceros bicornis).

While iron overload disorder (IOD) and related disease states are not considered a common occurrence in domestic equids, these issues appear prevalent in black rhinoceroses under human care. In addressing IOD in black rhinos, altering dietary iron absorption and excretion may be the most globally practical approach. A main option for treatment used across other species such as humans, is chelation therapy using iron-specific synthetic compounds. As horses may serve as an appropriate digestive model for the endangered rhinoceros, we evaluated the potential use of the oral iron chelator N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) in horses for safety and efficacy prior to testing in black rhinoceros. Health and iron digestibility and dynamics were assessed in horses (n = 6) before, and after treatment with HBED (50 mg/kg body weight) for 8 days using a crossover design with serum, faecal and urine collection. A preliminary pharmacokinetic trial was also performed but no trace of HBED was found in serially sampled plasma through 8 h post-oral dosing. HBED increased urinary iron output in horses compared to control by 0.7% of total iron intake (p < 0.01), for an average of 27 mg urinary iron/day, similar to human chelation goals. Blood chemistry, blood cell counts and overall wellness were not affected by treatment. As healthy horses are able to regulate iron absorption, the lack of change in iron balance is unsurprising. Short-term HBED administration appeared to be safely tolerated by horses, therefore it was anticipated it would also be safe to administer to black rhinos for the management of iron overload.

Mechanistic Insights of Chelator Complexes with Essential Transition Metals: Antioxidant/Pro-Oxidant Activity and Applications in Medicine.

The antioxidant/pro-oxidant activity of drugs and dietary molecules and their role in the maintenance of redox homeostasis, as well as the implications in health and different diseases, have not yet been fully evaluated. In particular, the redox activity and other interactions of drugs with essential redox metal ions, such as iron and copper, need further investigation. These metal ions are ubiquitous in human nutrition but also widely found in dietary supplements and appear to exert major effects on redox homeostasis in health, but also on many diseases of free radical pathology. In this context, the redox mechanistic insights of mainly three prototype groups of drugs, namely alpha-ketohydroxypyridines (alpha-hydroxypyridones), e.g., deferiprone, anthraquinones, e.g., doxorubicin and thiosemicarbazones, e.g., triapine and their metal complexes were examined; details of the mechanisms of their redox activity were reviewed, with emphasis on the biological implications and potential clinical applications, including anticancer activity. Furthermore, the redox properties of these three classes of chelators were compared to those of the iron chelating drugs and also to vitamin C, with an emphasis on their potential clinical interactions and future clinical application prospects in cancer, neurodegenerative and other diseases.

Thymosin ß4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis.

Thymosin ß4 (Tß4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tß4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tß4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tß4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tß4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tß4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tß4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tß4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tß4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.

Fluorescent Egg White-Based Carbon Dots as a High-Sensitivity Iron Chelator for the Therapy of Nonalcoholic Fatty Liver Disease by Iron Overload in Zebrafish.

Iron overload is the direct cause of many ferroptosis diseases, and it is essential to maintain iron homeostasis. In this paper, we report the Fe3+ chelation and therapy of the iron overload nonalcoholic fatty liver disease (NAFLD) by the fluorescent egg white-based carbon dots (EWCDs) obtained through the microwave-assisted pyrolysis method. As a high-sensitivity sensor, EWCDs show a high correlation between fluorescence emission and the concentration of Fe3+ (R2 = 0.993) in low concentration ranges of 0-25 µM. In vivo and in vitro, the EWCDs show characteristics of high biocompatibility and specific binding of Fe3+. As a novel type of the nano-iron-chelator, EWCDs can successfully attenuate the production of lethal reactive oxygen species. EWCDs not only alleviate the endoplasmic reticulum stress response but also regulate the NF-κB signaling pathway downstream of the Nrf2 signaling pathway. EWCDs prevent hepatocyte apoptosis, regulate fatty acid metabolism, and alleviate inflammation. Ultimately, they alleviate NAFLD induced by iron overload in zebrafish. This work may provide a new idea and method for the application of carbon dots in the field of disease detection and treatment.

An Untargeted Metabolomics Approach for Correlating Pulse Crop Seed Coat Polyphenol Profiles with Antioxidant Capacity and Iron Chelation Ability.

Pulse crop seed coats are a sustainable source of antioxidant polyphenols, but are typically treated as low-value products, partly because some polyphenols reduce iron bioavailability in humans. This study correlates antioxidant/iron chelation capabilities of diverse seed coat types from five major pulse crops (common bean, lentil, pea, chickpea and faba bean) with polyphenol composition using mass spectrometry. Untargeted metabolomics was used to identify key differences and a hierarchical analysis revealed that common beans had the most diverse polyphenol profiles among these pulse crops. The highest antioxidant capacities were found in seed coats of black bean and all tannin lentils, followed by maple pea, however, tannin lentils showed much lower iron chelation among these seed coats. Thus, tannin lentils are more desirable sources as natural antioxidants in food applications, whereas black bean and maple pea are more suitable sources for industrial applications. Regardless of pulse crop, proanthocyanidins were primary contributors to antioxidant capacity, and to a lesser extent, anthocyanins and flavan-3-ols, whereas glycosylated flavonols contributed minimally. Higher iron chelation was primarily attributed to proanthocyanidin composition, and also myricetin 3-O-glucoside in black bean. Seed coats having proanthocyanidins that are primarily prodelphinidins show higher iron chelation compared with those containing procyanidins and/or propelargonidins.

Characterization of Protein Hydrolysates from Fish Discards and By-Products from the North-West Spain Fishing Fleet as Potential Sources of Bioactive Peptides.

Fish discards and by-products can be transformed into high value-added products such as fish protein hydrolysates (FPH) containing bioactive peptides. Protein hydrolysates were prepared from different parts (whole fish, skin and head) of several discarded species of the North-West Spain fishing fleet using Alcalase. All hydrolysates had moisture and ash contents lower than 10% and 15%, respectively. The fat content of FPH varied between 1.5% and 9.4% and had high protein content (69.8-76.6%). The amino acids profiles of FPH are quite similar and the most abundant amino acids were glutamic and aspartic acids. All FPH exhibited antioxidant activity and those obtained from Atlantic horse mackerel heads presented the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and Cu2+ chelating activity. On the other hand, hydrolysates from gurnard heads showed the highest ABTS radical scavenging activity and Fe2+ chelating activity. In what concerns the α-amylase inhibitory activity, the IC50 values recorded for FPH ranged between 5.70 and 84.37 mg/mL for blue whiting heads and whole Atlantic horse mackerel, respectively. α-Glucosidase inhibitory activity of FPH was relatively low but all FPH had high Angiotensin Converting Enzyme (ACE) inhibitory activity. Considering the biological activities, these FPH are potential natural additives for functional foods or nutraceuticals.

N-Propargylamine-hydroxypyridinone hybrids as multitarget agents for the treatment of Alzheimer's disease.

AD is a progressive brain disorder. Because of the lack of remarkable single-target drugs against neurodegenerative disorders, the multitarget-directed ligand strategy has received attention as a promising therapeutic approach. Herein, we rationally designed twenty-nine hybrids of N-propargylamine-hydroxypyridinone. The designed hybrids possessed excellent iron-chelating activity (pFe3+ = 17.09-22.02) and potent monoamine oxidase B inhibitory effects. Various biological evaluations of the optimal compound 6b were performed step by step, including inhibition screening of monoamine oxidase (hMAO-B IC50 = 0.083 ± 0.001 µM, hMAO-A IC50 = 6.11 ± 0.08 µM; SI = 73.5), prediction of blood-brain barrier permeability and mouse behavioral research. All of these favorable results proved that the N-propargylamine-hydroxypyridinone scaffold is a promising structure for the discovery of multitargeted ligands for AD therapy.

A Friendly Complexing Agent for Spectrophotometric Determination of Total Iron.

Iron, one of the most common metals in the environment, plays a fundamental role in many biological as well as biogeochemical processes, which determine its availability in different oxidation states. Its relevance in environmental and industrial chemistry, human physiology, and many other fields has made it necessary to develop and optimize analysis techniques for accurate determination. Spectrophotometric methods are the most frequently applied in the analytical determination of iron in real samples. Taking advantage of the fact that desferrioxamine B, a trihydroxamic acid used since the 1970s in chelation therapy for iron overload treatment, forms a single stable 1:1 complex with iron in whichever oxidation state it can be found, a smart spectrophotometric method for the analytical determination of iron concentration was developed. In particular, the full compliance with the Lambert-Beer law, the range of iron concentration, the influence of pH, and the interference of other metal ions have been taken into account. The proposed method was validated in terms of LoD, LoQ, linearity, precision, and trueness, and has been applied for total iron determination in natural water certified material and in biological reference materials such as control human urine and control serum.

New Era in the Treatment of Iron Deficiency Anaemia Using Trimaltol Iron and Other Lipophilic Iron Chelator Complexes: Historical Perspectives of Discovery and Future Applications.

The trimaltol iron complex (International Non-proprietary Name: ferric maltol) was originally designed, synthesised, and screened in vitro and in vivo in 1980-1981 by Kontoghiorghes G.J. following his discovery of the novel alpha-ketohydroxyheteroaromatic (KHP) class of iron chelators (1978-1981), which were intended for clinical use, including the treatment of iron deficiency anaemia (IDA). Iron deficiency anaemia is a global health problem affecting about one-third of the world's population. Many (and different) ferrous and ferric iron complex formulations are widely available and sold worldwide over the counter for the treatment of IDA. Almost all such complexes suffer from instability in the acidic environment of the stomach and competition from other dietary molecules or drugs. Natural and synthetic lipophilic KHP chelators, including maltol, have been shown in in vitro and in vivo studies to form stable iron complexes, to transfer iron across cell membranes, and to increase iron absorption in animals. Trimaltol iron, sold as Feraccru or Accrufer, was recently approved for clinical use in IDA patients in many countries, including the USA and in EU countries, and was shown to be effective and safe, with a better therapeutic index in comparison to other iron formulations. Similar properties of increased iron absorption were also shown by lipophilic iron complexes of 8-hydroxyquinoline, tropolone, 2-hydroxy-4-methoxypyridine-1-oxide, and related analogues. The interactions of the KHP iron complexes with natural chelators, drugs, metal ions, proteins, and other molecules appear to affect the pharmacological and metabolic effects of both iron and the KHP chelators. A new era in the treatment of IDA and other possible clinical applications, such as theranostic and anticancer formulations and metal radiotracers in diagnostic medicine, are envisaged from the introduction of maltol, KHP, and similar lipophilic chelators.

Deferoxamine B: A Natural, Excellent and Versatile Metal Chelator.

Deferoxamine B is an outstanding molecule which has been widely studied in the past decade for its ability to bind iron and many other metal ions. The versatility of this metal chelator makes it suitable for a number of medicinal and analytical applications, from the well-known iron chelation therapy to the most recent use in sensor devices. The three bidentate hydroxamic functional groups of deferoxamine B are the centerpiece of its metal binding ability, which allows the formation of stable complexes with many transition, lanthanoid and actinoid metal ions. In addition to the ferric ion, in fact, more than 20 different metal complexes of deferoxamine b have been characterized in terms of their chemical speciation in solution. In addition, the availability of a terminal amino group, most often not involved in complexation, opens the way to deferoxamine B modification and functionalization. This review aims to collect and summarize the available data concerning the complex-formation equilibria in solutions of deferoxamine B with different metal ions. A general overview of the progress of its applications over the past decade is also discussed, including the treatment of iron overload-associated diseases, its clinical use against cancer and neurodegenerative disorders and its role as a diagnostic tool.

In vitro studies of deferasirox derivatives as potential organelle-targeting traceable anti-cancer therapeutics.

We report here strategic functionalization of the FDA approved chelator deferasirox (1) in an effort to produce organelle-targeting iron chelators with enhanced activity against A549 lung cancer cells. Derivative 8 was found to have improved antiproliferative activity relative to 1. Fluorescent cell imaging revealed that compound 8 preferentially localises within the lysosome.

Recent Advances in Iron Chelation and Gallium-Based Therapies for Antibiotic Resistant Bacterial Infections.

Iron is essential for multiple bacterial processes and is thus required for host colonization and infection. The antimicrobial activity of multiple iron chelators and gallium-based therapies against different bacterial species has been characterized in preclinical studies. In this review, we provide a synthesis of studies characterizing the antimicrobial activity of the major classes of iron chelators (hydroxamates, aminocarboxylates and hydroxypyridinones) and gallium compounds. Special emphasis is placed on recent in-vitro and in-vivo studies with the novel iron chelator DIBI. Limitations associated with iron chelation and gallium-based therapies are presented, with emphasis on limitations of preclinical models, lack of understanding regarding mechanisms of action, and potential host toxicity. Collectively, these studies demonstrate potential for iron chelators and gallium to be used as antimicrobial agents, particularly in combination with existing antibiotics. Additional studies are needed in order to characterize the activity of these compounds under physiologic conditions and address potential limitations associated with their clinical use as antimicrobial agents.

In vitro identification and characterisation of iron chelating catechol-containing natural products and derivatives.

Iron is an essential component for multiple biological processes. Its regulation within the body is thus tightly controlled. Dysregulation of iron levels within the body can result in several disorders associated with either excess iron accumulation, including haemochromatosis and thalassaemia, or iron deficiency. In cases of excess body iron, therapy involves depleting body iron levels either by venesection, typically for haemochromatosis, or using iron chelators for thalassemia. However, the current chelation options for people with iron overload are limited, with only three iron chelators approved for clinical use. This presents an opportunity for improved therapeutics to be identified and developed. The aim of this study was to examine multiple compounds from within the Davis open access natural product-based library (512 compounds) for their ability to chelate iron. In silico analysis of this library initially identified nine catechol-containing compounds and two closely related compounds. These compounds were subsequently screened using an in vitro DNA breakage assay and their ability to chelate biological iron was also examined in an iron-loaded hepatocyte cellular assay. Toxicity was assessed in hepatocyte and breast cancer cell lines. One compound, RAD362 [N-(3-aminopropyl)-3,4-dihydroxybenzamide] was able to protect against DNA damage, likely through the prevention of free radicals generated via the Fenton reaction; RAD362 treatment resulted in decreased ferritin protein levels in iron-loaded hepatocytes. Lastly, RAD362 resulted in significantly less cell death than the commonly used iron chelator deferoxamine. This is the first study to identify compound RAD362 as an iron chelator and potential therapeutic.

Preparation, characterization and in vitro stability of iron-chelating peptides from mung beans.

Mung bean protein was enzymatically hydrolyzed with either alcalase, neutral protease, or papain. The mung bean protein hydrolysates (MPH) showed good ability to chelate ferrous ions, and the chelates had high stability in vitro. The hydrolysates prepared by alcalase showed the highest degree of hydrolysis and the highest ferrous chelating rate. Single factor tests showed that the pH and the material ratio had significant effects on ferrous chelating rates. The optimal MPH to FeCl2·4H2O material ratio was 8:1 (w/w) and the optimal pH of the reaction was 7.0, which yielded a chelating rate of 96.19 ± 0.94%. The fraction 3 with the highest ferrous chelating activity up to 61.25 ± 1.02 µg/mg was obtained from MPH by affinity chromatography. Meanwhile, the MPH-Fe complex had higher digestive stability than just MPH in both in vitro and acid-alkali tolerance assays. The characterization results showed that ferrous ions mainly combined with the amino, carboxyl, imidazole and other chelating active groups in mung bean peptides to form peptide-iron chelates. Scanning electron microscopy (SEM) analysis showed that mung bean peptide chelated ferrous ions to form polymer particles. These results provided insight into ways to develop functional foods such as iron-fortified cereals.