Novel diaroylhydrazine ligands as iron chelators: coordination chemistry and biological activity

The search for orally effective drugs for the treatment of iron overload disorders is an important goal in improving the health of patients suffering diseases such as beta-thalassemia major. Herein, we report the syntheses and characterization of some new members of a series of N-aroyl-N'-picolinoyl hydrazine chelators (the H2IPH analogs). Both 1:1 and 1:2 Fe-III:L complexes were isolated and the crystal structures of Fe(HPPH)Cl-2, Fe(4BBPH)Cl-2, Fe(HAPH)(APH) and Fe(H3BBPH)(3BBPH) were determined (H2PPH=N,N'-bis-picolinoyl hydrazine; H(2)APH=N-4-aminobenzoyl-N'-picolinoyl hydrazine, H(2)3BBPH=N-3-bromobenzoyl-N'-picolinoylhydrazine and H(2)4BBPH=N-(4-bromobenzoyl)-N'-(picolinoyl)hydrazine). In each case, a tridentate N,N,O coordination mode of each chelator with Fe was observed. The Fe-III complexes of these ligands have been synthesized and their structural, spectroscopic and electrochemical characterization are reported. Five of these new chelators, namely H2BPH (N-(benzoyl)-N'-(picolinoyl)hydrazine), H2TPH (N-(2-thienyl)-N'-(picolinoyl)-hydrazine), H2PPH, H(2)3BBPH and H(2)4BBPH, showed high efficacy at mobilizing Fe-59 from cells and inhibiting Fe-59 uptake from the serum Fe transport protein, transferrin (Tf). Indeed, their activity was much greater than that found for the chelator in current clinical use, desferrioxamine (DFO), and similar to that observed for the orally active chelator, pyridoxal isonicotinoyl hydrazone (H2PIH). The ability of the chelators to inhibit Fe-59 uptake could not be accounted for by direct chelation of Fe-59-Tf. The most effective chelators also showed low antiproliferative activity which was similar to or less than that observed with DFO, which is important in terms of their potential use as agents to treat Fe-overload disease.

A recombinant diheme SoxAX cytochrome - Implications for the relationship between EPR signals and modified heme-ligands

The multiheme SoxAX proteins are notable for their unusual heme ligation (His/Cys-persulfide in the SoxA subunit) and the complexity of their EPR spectra. The diheme SoxAX protein from Starkeya novella has been expressed using Rhodobacter capsulatus as a host expression system. rSoxAX was correctly formed in the periplasm of the host and contained heme c in similar amounts as the native SoxAX. ESI-MS showed that the full length rSoxA, in spite of never having undergone catalytic turnover, existed in several forms, with the two major forms having masses of 28 687 +/- 4 and 28 718 +/- 4 Da. The latter form exceeds the expected mass of rSoxA by 31 4 Da, a mass close to that of a sulfur atom and indicating that a fraction of the recombinant protein contains a cysteine persulfide modification. EPR spectra of rSoxAX contained all four heme-dependent EPR signals (LS1a, LS1b, LS2, LS3) found in the native SoxAX proteins isolated from bacteria grown under sulfur chemolithotrophic conditions. Exposure of the recombinant SoxAX to different sulfur compounds lead to changes in the SoxA mass profile as determined by ESI while maintaining a fully oxidized SoxAX visible spectrum. Thiosulfate, the proposed SoxAX substrate, did not cause any mass changes while after exposure to dimethylsulfoxide a + 112 +/- 4 Da form of SoxA became dominant in the mass spectrum. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and anti-viral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

Isomeric distribution and catalyzed isomerization of cobalt(III) complexes with pentadentate macrocyclic ligands. Importance of hydrogen bonding

We have investigated the isomeric distribution and rearrangement of complexes of the type [CoXLn](2+,3+) (where X = Cl-, OH-, H2O, and L-n represents a pentadentate 13-, 14-, and 15-membered tetraaza or diaza-dithia (N-4 or N2S2) macrocycle bearing a pendant primary amine). The preparative procedures for chloro complexes produced almost exclusively kinetically preferred cis isomers (where the pendant primary amine is cis to the chloro ligand) that can be separated by careful cation-exchange chromatography. For L-13 and L-14 the so-called cis-V isomer is isolated as the kinetic product, and for L-15 the cis-VI form (an N-based diastereomer) is the preferred, while for the L-14(S) complex both cis-V and trans-I forms are obtained. All these complexes rearrange to form stable trans isomers in which the pendent primary amine is trans to the monodentate aqua or hydroxo ligand, depending on pH and the workup procedure. In total 11 different complexes have been studied. From these, two different trans isomers of [CoCIL14S](2+) have been characterized crystallographically for the first time in addition to a new structure of cis-V-[CoCIL14S](2+); all were isolated as their chloride perchlorate salts. Two additional isomers have been identified and characterized by NMR as reaction intermediates. The remaining seven forms correspond to the complexes already known, produced in preparative procedures. The kinetic, thermal, and baric activation parameters for all the isomerization reactions have been determined and involve large activation enthalpies and positive volumes of activation. Activation entropies indicate a very important degree of hydrogen bonding in the reactivity of the complexes, confirmed by density functional theory studies on the stability of the different isomeric forms. The isomerization processes are not simple and even some unstable intermediates have been detected and characterized as part of the above-mentioned 11 forms of the complexes. A common reaction mechanism for the isomerization reactions has been proposed for all the complexes derived from the observed kinetic and solution behavior.

Prediction of HLA-DQ3.2 beta ligands: evidence of multiple registers in class II binding peptides

Motivation: While processing of MHC class II antigens for presentation to helper T-cells is essential for normal immune response, it is also implicated in the pathogenesis of autoimmune disorders and hypersensitivity reactions. Sequence-based computational techniques for predicting HLA-DQ binding peptides have encountered limited success, with few prediction techniques developed using three-dimensional models. Methods: We describe a structure-based prediction model for modeling peptide-DQ3.2 beta complexes. We have developed a rapid and accurate protocol for docking candidate peptides into the DQ3.2 beta receptor and a scoring function to discriminate binders from the background. The scoring function was rigorously trained, tested and validated using experimentally verified DQ3.2 beta binding and non-binding peptides obtained from biochemical and functional studies. Results: Our model predicts DQ3.2 beta binding peptides with high accuracy [area under the receiver operating characteristic (ROC) curve A(ROC) > 0.90], compared with experimental data. We investigated the binding patterns of DQ3.2 beta peptides and illustrate that several registers exist within a candidate binding peptide. Further analysis reveals that peptides with multiple registers occur predominantly for high-affinity binders.

Cobalt complexes of tripodal hexadentate ligands: Electrochemically driven rearrangements

The Co-III complexes of the hexadentate tripodal ligands HOsen (3-(2'-aminoethylamino)-2,2-bis((2 ''-aminoethylamino) methyl) propan-1-ol) and HOten (3-(2'-aminoethylthia)-2,2-bis((2 ''-aminoethylthia) methyl) propan-1-ol) have been synthesized and fully characterized. The crystal structures of [Co(HOsen)]Cl-3 center dot H2O and [Co(HOten)](ClO4)Cl-2 are reported and in both cases the ligands coordinate as tripodal hexadentate N-6 and N3S3 donors, respectively. Cyclic voltammetry of the N3S3 coordinated complex [Co(HOten)](3+) is complicated and electrode dependent. On a Pt working electrode an irreversible Co-III/II couple ( formal potential - 157 mV versus Ag-AgCl) is seen, which is indicative of dissociation of the divalent complex formed at the electrode. The free HOten released by the dissociation of [Co(HOten)](2+) can be recaptured by Hg as shown by cyclic voltammetry experiments on a static Hg drop electrode ( or in the presence of Hg2+ ions), which leads to the formation of an electroactive Hg-II complex of the N3S3 ligand (formal potential + 60 mV versus Ag-AgCl). This behaviour is in contrast to the facile and totally reversible voltammetry of the hexaamine complex [Co(HOsen)](3+) ( formal potential (Co-III/II) - 519 mV versus Ag-AgCl), which is uncomplicated by any coupled chemical reactions. Akinetic and thermodynamic analysis of the [Co(HOten)](2+)/[Hg(HOten)](2+) system is presented on the basis of digital simulation of the experimental voltammetric data.

Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. in the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional H-1 NMR spectral studies: only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands, As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.

Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 Angstrom resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 Angstrom resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 .when the nucleotide product is formed. The 2.6 Angstrom resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by similar to 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex. (C) 1998 Academic Press.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn2+. The EPR spectrum of Mn2+-activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5 (+/- 0.1) Angstrom. In the X-ray absorption spectrum of Mn2+-activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k less than or equal to 12 Angstrom(-1) by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15 Angstrom, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5 Angstrom, is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn2+-activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.

Crystal and molecular structure of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and its iron(III) complex: an iron chelator with anti-tumour activity

Previous studies have demonstrated that 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and several other aroylhydrazone chelators possess anti-neoplastic activity due to their ability to bind intracellular iron. In this study we have examined the structure and properties of NIH and its Fe-III complex in order to obtain further insight into its anti-tumour activity. Two tridentate NIH ligands deprotonate upon coordination to Fe-III in a meridional fashion to form a distorted octahedral, high-spin complex. Solution electrochemistry of [Fe(NIH-H)(2)](+) shows that the trivalent oxidation state is dominant over a wide potential range and that the Fe-II analogue is not a stable form of this complex. The fact that [Fe(NIH-H)(2)](+) cannot-cycle between the Fe-II and Fe-III states suggests that the production of toxic free- radical species, e.g. OH. or O2(.-),is not part of this ligand's cytotoxic action. This suggestion is supported by cell culture experiments demonstrating that the addition of Fe-III to NIH prevents its anti-proliferative effect. The chemistry of this chelator and its Fe-III complex are discussed in the context of understanding its anti-tumour activity.