The iron(III) coordinating properties of citrate and α-hydroxycarboxylate containing siderophores.

The iron(III) binding properties of citrate and rhizoferrin, a citrate containing siderophore, are compared. Citrate forms many oligonuclear complexes, whereas rhizoferrin forms a single mononuclear complex. The α-hydroxycarboxylate functional group, which is present in both citrate, and rhizoferrin, has a high affinity and selectivity for iron(III) under most biological conditions. The nature of the toxic form of iron found in the blood of patients suffering from many haemoglobinopathies and haemochromatosis is identified as a mixture of iron(III)citrate complexes. The significance of the presence of this iron pool to patients suffering from systemic iron overload is discussed. The wide utilisation of the α-hydroxycarboxylate functional group in siderophore structures is described, as is their photo-induced decarboxylation leading to the release of iron(II) ions. The importance of this facile dissociation to algal iron uptake is discussed.

The Colorimetric Detection of the Hydroxyl Radical.

An aromatic substrate for hydroxylation by hydroxyl radicals (•OH) was investigated. The probe, N,N'-(5-nitro-1,3-phenylene)-bis-glutaramide, and its hydroxylated product do not bind either iron(III) or iron(II), and so they do not interfere with the Fenton reaction. A spectrophotometric assay based on the hydroxylation of the substrate was developed. The synthesis and purification methods of this probe from previously published methodologies were improved upon, as well as the analytical procedure for monitoring the Fenton reaction through its use, enabling univocal and sensitive •OH detection. The assay was utilised to demonstrate that the iron(III) complexes of long-chain fatty acids lack Fenton activity under biological conditions.

Antioxidant and anti-tyrosinase activity of a novel stilbene analogue as an anti-browning agent.

BACKGROUND: Tyrosinase inhibitors find potential application in food, cosmetic and medicinal products, but most of the identified tyrosinase inhibitors are not suitable for practical use because of safety regulations or other problems. For the purpose of development of novel tyrosinase inhibitors that meet the requirement for practical application, a novel stilbene analogue (SA) was designed. RESULTS: SA was found to possess a potent inhibitory effect against both mono- and diphenolase activities of mushroom tyrosinase, with IC50 values of 1.56 and 7.15 µmol L-1 , respectively. Compared with a natural tyrosinase inhibitor - kojic acid - the anti-tyrosinase effect of SA was significantly improved. Analysis of inhibition kinetics indicated that SA was a reversible and competitive-noncompetitive mixed-type inhibitor. SA was also found to possess more potent antioxidant activities (DPPH, superoxide anion radical and hydroxyl radical scavenging ability) than those of kojic acid. Cell viability studies revealed that SA was non-toxic to two cell lines. Furthermore, an anti-browning test demonstrated that SA effectively delayed the blackening of shrimp. CONCLUSION: SA has potential as an anti-browning agent in foods. © 2021 Society of Chemical Industry.

Intravenous iron preparations transiently generate non-transferrin-bound iron from two proposed pathways.

Intravenous iron-carbohydrate complex preparations (IVIPs) are non-interchangeable pro-drugs: their pharmacokinetics (PK) varies determined by semi-crystalline iron core and carbohydrate shell structures, influences pharmacodynamics (PD) and thus efficacy and safety. Examining PK/PD relationships of 3 IVIPs we identify a two-pathway model of transient NTBI generation following single dose administration. 28 hypoferremic non-anemic patients randomized to 200mg iron as ferric carboxymaltose (Fe-carboxymaltose), iron sucrose (Fe-sucrose), iron isomaltoside 1000 (Fe-isomaltoside-1000), n=8/arm, or placebo, n=4, on a 2-week PK/PD study, had samples analysed for total serum iron, IVIP-iron, transferrin-bound iron (TBI) by HPLC-ICP-MS, transferrin saturation (TSAT), serum ferritin (s-Ferritin) by standard methods, non-TBI (NTBI) and hepcidin as published before. IVIP-dependent increases in these parameters returned to baseline in 48-150h, except for s-Ferritin and TSAT. NTBI was low with Fe-isomaltoside-1000 (0.13µM at 8h), rapidly increased with Fe-sucrose (0.8µM at 2h, 1.25µM at 4h), and delayed for Fe-carboxymaltose (0.57µM at 24h). NTBI AUCs were 7-fold greater for Fe-carboxymaltose and Fe-sucrose than for Fe-isomaltoside-1000. Hepcidin peak time varied, but not AUC or mean levels. s-Ferritin levels and AUC were highest for Fe-carboxymaltose and greater than placebo for all IVIPs. We propose 2 mechanisms for the observed NTBI kinetics: rapid and delayed NTBI appearance consistent with direct (circulating IVIP-to-plasma) and indirect (IVIP-to-macrophage-to-plasma) iron release based on IVIP plasma half-life and s-Ferritin dynamics. IVIPs generate different, broadly stability- and PK-dependent, NTBI and s-Ferritin signatures, which may influence iron bioavailability, efficacy and safety. Longer-term studies should link NTBI exposure to subsequent safety and efficacy parameters and potential clinical consequences.

Dipeptide inhibitors of the prostate specific membrane antigen (PSMA): A comparison of urea and thiourea derivatives.

Glutamate carboxypeptidase II (GCP(II)), also known as the prostate-specific membrane antigen (PSMA), is a transmembrane zinc(II) metalloenzyme overexpressed in prostate cancer. Inhibitors of this receptor are used to target molecular imaging agents and molecular radiotherapy agents to prostate cancer and if the affinity of inhibitors for GCP(II)/PSMA could be improved, targeting might also improve. Compounds containing the dipeptide OH-Lys-C(O)-Glu-OH (compound 3), incorporating a urea motif, have high affinity for GCP(II)/PSMA. We hypothesized that substituting the zinc-coordinating urea group for a thiourea group, thus incorporating a sulfur atom, could facilitate stronger binding to zinc(II) within the active site, and thus improve affinity for GCP(II)/PSMA. A structurally analogous urea and thiourea pair (HO-Glu-C(O)-Glu-OH - compound 5 and HO-Glu-C(S)-Glu-OH - compound 6) were synthesized and the inhibitory concentration (IC50) of each compound measured with a cell-based assay, allowing us to refute the hypothesis: the thiourea analogue showed 100-fold weaker binding to PSMA than the urea analogue.

Glutathione and the intracellular labile heme pool.

One candidate for the cytosolic labile iron pool is iron(II)glutathione. There is also a widely held opinion that an equivalent cytosolic labile heme pool exists and that this pool is important for the intracellular transfer of heme. Here we describe a study designed to characterise conjugates that form between heme and glutathione. In contrast to hydrated iron(II), heme reacts with glutathione, under aerobic conditions, to form the stable hematin-glutathione complex, which contains iron(III). Thus, glutathione is clearly not the cytosolic ligand for heme, indeed we demonstrate that the rate of heme degradation is enhanced in the presence of glutathione. We suggest that the concentration of heme in the cytosol is extremely low and that intracellular heme transfer occurs via intracellular membrane structures. Should any heme inadvertently escape into the cytosol, it would be rapidly conjugated to glutathione thereby protecting the cell from the toxic effects of heme.

Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling.

Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.

Hydroxypyridinone Journey into Metal Chelation.

Hydroxypyridinones (HOPOs) form outstanding building blocks for the development of a variety of agents in the field of metal chelation. The pyridinone ring is easily synthesized and readily converted into tetradentate, hexadentate, and octadentate chelators. There is considerable potential for the control of the stereochemistry of the resulting metal complex and hence the properties of these multidentate molecules. Their ability to rapidly bind hard metals in aqueous media has facilitated the development of efficient applications in both biological and medical contexts. In this Review, an in-depth analysis of the synthetic methodologies for HOPO-based ligands is presented, as well as the many aspects to achieve optimal biological activity. Recent advances and current challenges for the future application of HOPO structures are outlined. The present flourishing development of drug candidates and diagnostic agents based on this chemical scaffold opens access to many new applications in analytical, environmental, and clinical science.

Functionality study of chalcone-hydroxypyridinone hybrids as tyrosinase inhibitors and influence on anti-tyrosinase activity.

In an attempt to synthesise new tyrosinase inhibitors, we designed and synthesised a series of chalcone-hydroxypyridinone hybrids as potential tyrosinase inhibitors adopting strategic modifications of kojic acid. All the newly synthesised compounds were characterised by NMR and mass spectrometry. Initial screening of the target compounds demonstrated that compounds 1a, 1d, and 1n had relatively strong inhibitory activities against tyrosinase monophenolase, with IC50 values of 3.07 ± 0.85, 2.25 ± 0.8 and 2.75 ± 1.19 µM, respectively. The inhibitory activity against monophenolase was 6- to 8-fold higher than that of kojic acid. Compounds 1a, 1d, and 1n also showed inhibition of diphenolase, with IC50 values of 17.05 ± 0.07, 11.70 ± 0.03 and 19.3 ± 0.28 µM, respectively. The inhibition kinetics of diphenolase indicates that compounds 1a and 1d induce reversible inhibition on tyrosinase. Finally, we found that copper coordination should be one of the important inhibitory mechanism of these compounds in tyrosinase.

A chemically characterized ethanolic extract of Thai Perilla frutescens (L.) Britton fruits (nutlets) reduces oxidative stress and lipid peroxidation in human hepatoma (HuH7) cells.

Perilla frutescens is cultivated in East Asian countries including Thailand, and the nutlets (single-seeded fruits) are used as traditional and medicinal food. Perilla nutlets extracted by ethyl acetate (EA), 80% ethanol (Eth), and hot water (HW) sequentially were chemically characterized using high-resolution accurate liquid chromatography-mass spectrometry with the main compounds detected assigned as rosmarinic acid and derivatives of the flavones apigenin and luteolin, with the more diverse chemical composition observed with the Eth extract. All extracts showed dose-dependent free-radical scavenging activity, with the Eth extract the most potent (IC50  = 3.43 mg/ml for ABTS• scavenging and 0.27 mg/ml for DPPH• scavenging). The Eth extract also inhibited AAPH-induced hemolysis (IC50  = 0.07 mg/ml) more potently than did the HW (IC50  = 0.38 mg/ml) and EA extracts (IC50  = 1.63 mg/ml). An MTT test revealed all the extracts were noncytotoxic at concentrations up to 200 µg/ml. Only the Eth and EA extracts showed protective effects against the generation of reactive oxygen species and lipid peroxidation in FeCl3 -induced HuH7 cells in a dose-dependent manner. Our findings suggest the Eth extract of Thai perilla nutlets, containing rosmarinic acid and flavones and their derivatives, may have potential to provide protection against oxidative stress in hepatic disorders.

Design of Bifunctional Dendritic 5-Aminolevulinic Acid and Hydroxypyridinone Conjugates for Photodynamic Therapy.

Iron chelators have recently attracted interest in the field of photodynamic therapy (PDT) owing to their role in enhancement of intracellular protoporphyrin IX (PpIX) generation induced by 5-aminolevulinic acid (ALA) via the biosynthetic heme cycle. Although ALA is widely used in PDT, cellular uptake of ALA is limited by its hydrophilicity. In order to improve ALA delivery and enhance the PpIX production, several dendrimers incorporating both ALA and 3-hydroxy-4-pyridinone (HPO) were synthesized. The ability of the dendrimers to enter cells and be metabolized to the PpIX photosensitizer was studied in several human cancer cell lines. The dendrimers were found to be significantly more efficient than ALA alone in PpIX production. The higher intracellular PpIX levels showed a clear correlation with enhanced cellular phototoxicity following light exposure. Dendritic derivatives are therefore capable of efficiently delivering both ALA and HPO, which act synergistically to amplify in vitro PpIX levels and enhance PDT efficacy.

Ion-Pairing with Spermine Targets Theophylline To the Lungs via the Polyamine Transport System.

Certain xenobiotics, such as paraquat, are sequestered into the lungs from the systemic circulation by the polyamine transporter system (PTS). The aim of this study was to investigate whether ion-pairing a drug (theophylline) with a PTS substrate (spermine) provides a means of using this active transport mechanism to target drug delivery to the lungs. Fourier transform infrared spectroscopy showed that two of the amine groups of spermine interact with C-N7 and C6═O of theophylline, leaving two free amines to interact with the PTS. In A549 cells, which possess a functional PTS (spermidine Km and Vmax, 0.6 ± 0.3 µM and 1.8 ± 0.3 pmol·min-1 per 105 cells, respectively), uptake of the theophylline-spermine ion-pair was increased 1.8-fold compared to free theophylline at 37 °C, but not at 4 °C. In an isolated perfused rat lung model (IPL) a 3.6-fold increase in lung theophylline concentration was observed after vascular administration of the ion-pair compared to free theophylline. Theophylline was cleared from the IPL with similar kinetics irrespective of whether it was delivered as the free drug or an ion-pair, although lung levels remained elevated after washout following delivery as an ion-pair. In vitro simulation of the theophylline-spermine break down demonstrated that a drop in pH from 9.6 to 7.4, such as that undergone by the ion-pair in biological matrices, induces rapid and almost complete dissociation of the ion-paired species. However, infusion of the ion-pair formulations via the vasculature provides almost immediate delivery to the pulmonary capillary bed permitting PTS-mediated active sequestering of ion-paired theophylline into the lungs.

Synthesis, iron binding and antimicrobial properties of hexadentate 3-hydroxypyridinones-terminated dendrimers.

Macromolecular chelators have potential applications in the medical area, for instance, in treatment of iron overload-related disorders and in the treatment of external infections. In this investigation, several novel iron(III)-selective hydroxypyridinone hexadentate-terminated first and second generation dendrimeric chelators were synthesized using a convergent strategy. Their iron chelating ability was demonstrated by UV/Visible spectrometry and high resolution mass spectrometry (HRMS). The iron binding affinities were also investigated by the competition with a fluorescent iron chelator CP691. The result indicated that these dendrimers possesses a high affinity for iron with a very high pFe3+ value, which is close to that of an isolated hexadentate unit. These dendrimeric chelators were found to exhibit inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria.

Hydroxypyridinone Chelators: From Iron Scavenging to Radiopharmaceuticals for PET Imaging with Gallium-68.

Derivatives of 3,4-hydroxypyridinones have been extensively studied for in vivo Fe3+ sequestration. Deferiprone, a 1,2-dimethyl-3,4-hydroxypyridinone, is now routinely used for clinical treatment of iron overload disease. Hexadentate tris(3,4-hydroxypyridinone) ligands (THP) complex Fe3+ at very low iron concentrations, and their high affinities for oxophilic trivalent metal ions have led to their development for new applications as bifunctional chelators for the positron emitting radiometal, 68Ga3+, which is clinically used for molecular imaging in positron emission tomography (PET). THP-peptide bioconjugates rapidly and quantitatively complex 68Ga3+ at ambient temperature, neutral pH and micromolar concentrations of ligand, making them amenable to kit-based radiosynthesis of 68Ga PET radiopharmaceuticals. 68Ga-labelled THP-peptides accumulate at target tissue in vivo, and are excreted largely via a renal pathway, providing high quality PET images.

Copper(II) binding properties of hepcidin.

Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu(II) and Ni(II) through the amino terminal copper-nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu(II) with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu(II) than that of native hepcidin. The log K 1 value of hepcidin for Cu(II) was determined as 7.7. Cu(II) binds to albumin more tightly than hepcidin (log K 1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu(II) present in blood will be bound to albumin. It is estimated that the concentration of Cu(II)-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin.

Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders.

Non-transferrin-bound iron and its labile (redox active) plasma iron component are thought to be potentially toxic forms of iron originally identified in the serum of patients with iron overload. We compared ten worldwide leading assays (6 for non-transferrin-bound iron and 4 for labile plasma iron) as part of an international inter-laboratory study. Serum samples from 60 patients with four different iron-overload disorders in various treatment phases were coded and sent in duplicate for analysis to five different laboratories worldwide. Some laboratories provided multiple assays. Overall, highest assay levels were observed for patients with untreated hereditary hemochromatosis and ß-thalassemia intermedia, patients with transfusion-dependent myelodysplastic syndromes and patients with transfusion-dependent and chelated ß-thalassemia major. Absolute levels differed considerably between assays and were lower for labile plasma iron than for non-transferrin-bound iron. Four assays also reported negative values. Assays were reproducible with high between-sample and low within-sample variation. Assays correlated and correlations were highest within the group of non-transferrin-bound iron assays and within that of labile plasma iron assays. Increased transferrin saturation, but not ferritin, was a good indicator of the presence of forms of circulating non-transferrin-bound iron. The possibility of using non-transferrin-bound iron and labile plasma iron measures as clinical indicators of overt iron overload and/or of treatment efficacy would largely depend on the rigorous validation and standardization of assays.

Design and synthesis of novel hydroxypyridinone derivatives as potential tyrosinase inhibitors.

Two groups of novel hydroxypyridinone derivatives 6(a-e) and 12(a-c), were designed as potential tyrosinase inhibitors, and synthesized using kojic acid as a starting material. The tyrosinase inhibitory activity of these two groups was demonstrated to be potent, especially compounds 6e and 12a, whose IC50 values for monophenolase activity were 1.95µM and 2.79µM, respectively. Both of these values are lower than that of kojic acid (IC50=12.50µM). Compounds 6e and 12a were investigated for the inhibitory effect on diphenolase activity. The results showed that the inhibitory mechanism of these two compounds was reversible and that the inhibitory type was a competitive-uncompetitive mixed-type. The values of IC50 of 6e and 12a on the diphenolase activity of tyrosinase were determined to be 8.97µM and 26.20µM, respectively. The inhibitory constants (KI and KIS) of 6e were determined as 17.17µM and 22.09µM, respectively; and the KI and KIS values of 12a were 34.41µM and 79.02µM, respectively. Compound 6e showed a greater ability to reduce copper and a stronger copper chelating ability than kojic acid.