Inhibition of the lymphoid tyrosine phosphatase: The effect of zinc(II) ions and chelating ligand fragments on enzymatic activity

A 96-member chelator fragment library (CFL-1.1) was screened to identify inhibitors of the lymphoid tyrosine phosphatase in the absence and presence of zinc acetate. Fragments that inhibit LYP activity more potently in the presence of zinc, fragments that rescue LYP activity in the presence of inhibitory concentrations of zinc, and fragments that inhibit LYP activity independent of zinc concentration were identified. Of these, 1,2-dihydroxynaphthalene was the most potent inhibitor with an IC₅₀ value of 2.52 ± 0.06 μM after 2 h of incubation. LYP inhibition by 1,2-dihydroxynaphthalene was very similar to inhibition by 1,2-naphthoquinone (IC₅₀ = 1.10 ± 0.03 µM), indicating that the oxidized quinone species is likely the active inhibitor. The inhibition was time-dependent, consistent with covalent modification of the enzyme.     

Anti-leukemia Activity of 7-hydroxy-2-substituted-methyl-5 H -oxazolo[3,2- a] pyrimidin-5-one Derivatives

The synthesis of new 7-hydroxy-2-substituted-methyl-5 H -oxazolo[3,2- a] pyrimidin-5-ones derivatives, designed as structural bicyclic analogues of the iron chelator deferiprone, is described. They were tested for their ability to inhibit proliferation in human Bcr-Abl + leukemia cells.     

A simple,cheap but reliable method for evaluation of zinc chelating properties

BackgroundZinc is an essential trace element. Both its lack and excess are associated with pathological states. The former is more common and can ensue from the excessive treatment with clinically used iron/copper chelators.Aim and methodThe aim of this work was to prepare a reliable, rapid and cheap method for the screening of zinc chelation. Spectrophotometric assessment using a known zinc indicator dithizone was selected.ResultsInitial screening performed by comparison of spectra of dithizone and its complex with zinc suggested 530 and 570 nm as suitable wavelengths for determination of zinc at pH 4.5 while 540 and 590 nm for pH 5.5–7.5. Additional research showed the lower wavelengths to be more suitable. The sensitivity of the method was always bellow 1 μM with good linearity relationship between absorbance and zinc concentration. The method suitability was confirmed by use of two known zinc chelators, ethylenediaminetetraacetic acid (EDTA) and N,N,N′,N′-tetrakis(2-pyridylmethyl)-1,2-ethylenediamine (TPEN).ConclusionThis method represents a sufficiently precise method for zinc chelation screening usable at pathophysiologically relevant pH conditions. Such method can be employed for both screening of novel zinc chelators and for testing affinity of other metal chelators for zinc.     

Chelator-mediated iron efflux from reticulocytes

The mechanism by which bipyridine and phenanthroline types of iron chelator inhibit iron uptake from transferrin and iron efflux mediated by pyridoxal isonicotinoyl hydrazone was investigated using rabbit reticulocytes with the aim of providing more information on the normal process of iron uptake by developing erythroid cells. It was shown that the chelators block cellular uptake by chelating the iron immediately after release from transferrin while it is still in the membrane fraction of the cells. The iron-chelator is then released from the cells by a process which is very similar to that of transferrin release with respect to kinetics and sensitivity to incubation temperature and the effects of metabolic inhibitors and other chemical reagents. These results are compatible with the conclusion that both transferrin and the iron-chelators in the cells are mainly present in endocytotic vesicles and are released from the cells by exocytosis. The chelators were also shown to block the pyridoxal isonicotinoyl hydrazone-mediated efflux of iron from cells which had taken up iron in the presence of isoniazid, an inhibitor of haem synthesis, by chelating the iron in the cytosol and the mitochondria. In this case, the iron-chelator complexes were not released from the cells. Measurement of the diethyl ether/water partition coefficients of bipyridine and 1,10-phenanthroline and their iron complexes gave much higher values for the free chelators, supporting the concept that the chelators trap the iron intracellularly because of differences in the lipid solubility and, hence, membrane permeability to the free chelators and their iron complexes.     

The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals

Tannic acid (TA) has well-described antimutagenic and antioxidant activities. The antioxidant activity of TA has been previously attributed to its capacity to form a complex with iron ions, interfering with the Fenton reaction [Biochim. Biophys. Acta 1472, 1999, 142]. In this work, we observed that TA inhibits, in the micromolar range, in vitro Cu(II) plus ascorbate-mediated hydroxyl radical (*OH) formation (determined as 2-deoxyribose degradation) and oxygen uptake, as well as copper-mediated ascorbate oxidation and ascorbate radical formation (quantified in EPR studies). The effect of TA against 2-deoxyribose degradation was three orders of magnitude higher than classic *OH scavengers, but was similar to several other metal chelators. Moreover, the inhibitory effectiveness of TA, by the four techniques used herein, was inversely proportional to the Cu(II) concentration in the media. These results and the observation of copper-induced changes in the UV spectra of TA are indications that the antioxidant activity of TA relates to its copper chelating ability. Thus, copper ions complexed to TA are less capable of inducing ascorbate oxidation, inhibiting the sequence of reactions that lead to 2-deoxyribose degradation. On the other hand, the efficiency of TA against 2-deoxyribose degradation declined considerably with increasing concentrations of the *OH detector molecule, 2-deoxyribose, suggesting that the copper-TA complex also possesses an *OH trapping activity.     

Lipophilic ionophore complexes as superoxide dismutase mimetics

A wide range of metal ion complexes exhibit superoxide dismutase like activities as detected by inhibition of nitroblue tetrazolium reduction. Mn(II) and Cu(II) complexes of EDTA, EHPG, and EGTA exhibit SOD like activities commensurate with many of the purpose-built SOD mimics. Here, we report analogous lipophilic chelators that localise metal ions (Cu(II), Mn(II), and Fe(III)) in the lipid membranes and lipoproteins to protect them from superoxide mediated oxidative damage. Spectroscopic titrations and Jobs method confirm that both 1:1 and 2:1 metal ion monensin complexes form. The cupric complexes are the most active exhibiting IC(50) values of 0.09 and 0.18 microM for 2Cu(II)-monensin and Cu(II)-monensin, respectively, for superoxide destruction. In addition, the IC(50) value for Mn(II)-monensin is 0.31 microM. In conclusion, Mn(II) and Cu(II) complexes of the ionophore monensin exhibit considerable superoxide scavenging activities and represent a novel class of catalytic antioxidants for the protection of lipid structures.     

Iron homeostasis and eye disease

Background

Iron is necessary for life, but excess iron can be toxic to tissues. Iron is thought to damage tissues primarily by generating oxygen free radicals through the Fenton reaction.

Methods

We present an overview of the evidence supporting iron's potential contribution to a broad range of eye disease using an anatomical approach.

Results

Iron can be visualized in the cornea as iron lines in the normal aging cornea as well as in diseases like keratoconus and pterygium. In the lens, we present the evidence for the role of oxidative damage in cataractogenesis. Also, we review the evidence that iron may play a role in the pathogenesis of the retinal disease age-related macular degeneration. Although currently there is no direct link between excess iron and development of optic neuropathies, ferrous iron's ability to form highly reactive oxygen species may play a role in optic nerve pathology. Lastly, we discuss recent advances in prevention and therapeutics for eye disease with antioxidants and iron chelators.

General significance

Iron homeostasis is important for ocular health.     

Mathematical calculations of iron complex stoichiometry by direct UV–Vis spectrophotometry

The effects of iron-chelating agents on miscellaneous pathologies are currently largely tested. Due to various indications, different properties for chelators are required. A stoichiometry of the complex in relation to pH is one of the crucial factors. Moreover, the published data on the stoichiometry, especially concerning flavonoids, are equivocal.In this study, a new complementary approach was employed for the determination of stoichiometry in 10 iron-chelating agents, including clinically used drugs, by UV–Vis spectrophotometry at relevant pH conditions and compared with the standard Job’s method.This study showed that the simple approach based on absorbance at the wavelength of complex absorption maximum was sufficient when the difference between absorption maximum of substance and complex was high. However, in majority of substances this difference was much lower (9–73 nm). The novel complementary approach was able to determine the stoichiometry in all tested cases. The major benefit of this method compared to the standard Job’s approach seems to be its capability to reveal a reaction stoichiometry in chelators with moderate affinity to iron.In conclusion, using this complementary method may explain several previous contradictory data and lead to a better understanding of the underlying mechanisms of chelator’s action.     

Receptor-Mediated Tumor Targeting with Radiopeptides. Part 1. General Concepts and Methods: Applications to Somatostatin Receptor-Expressing Tumors

Radiolabeled peptides have become important tools in nuclear oncology, both as diagnostics and more recently also as therapeutics. They represent a distinct sector of the molecular targeting approach, which in many areas of therapy will implement the old “magic bullet” concept by specifically directing the therapeutic agent to the site of action. In this three-part review, we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part I summarizes the general concepts and methods of targeting, the selection of radioisotopes, chelators, and the criteria of peptide ligand development. Then, the >400 studies on the application to somatostatin/somatostatin-release inhibiting factor receptor-mediated tumor localization and treatment will be reviewed, demonstrating that peptide radiopharmaceuticals have gained an important position in clinical medicine.     

SO2907, a putative TonB-dependent receptor, is involved in dissimilatory iron reduction by Shewanella oneidensis strain MR-1

Shewanella oneidensis strain MR-1 utilizes soluble and insoluble ferric ions as terminal electron acceptors during anaerobic respiration. The components of respiratory metabolism are localized in the membrane fractions which include the outer membrane and cytoplasmic membrane. Many of the biological components that interact with the various iron forms are proposed to be localized in these membrane fractions. To identify the iron-binding proteins acting either as an iron transporter or as a terminal iron reductase, we used metal-catalyzed oxidation reactions. This system catalyzed the oxidation of amino acids in close proximity to the iron binding site. The carbonyl groups formed from this oxidation can then be labeled with fluoresceinamine (FLNH(2)). The peptide harboring the FLNH(2) can then be proteolytically digested, purified by HPLC and then identified by MALDI-TOF tandem MS. A predominant peptide was identified to be part of SO2907 that encodes a putative TonB-dependent receptor. Compared with wild type (wt), the so2907 gene deletion (ΔSO2907) mutant has impaired ability to reduce soluble Fe(III), but retains the same ability to respire oxygen or fumarate as the wt. The ΔSO2907 mutant was also impacted in reduction of insoluble iron. Iron binding assays using isothermal titration calorimetry and fluorescence tryptophan quenching demonstrated that a truncated form of heterologous-expressed SO2907 that contains the Fe(III) binding site, is capable of binding soluble Fe(III) forms with K(d) of approximate 50 μm. To the best of our knowledge, this is the first report of the physiological role of SO2907 in Fe(III) reduction by MR-1.