Pilar González-Duarte

Design, selection, and characterization of thioflavin-based intercalation compounds with metal chelating properties for application in Alzheimer's disease.

Metal chelation is considered a rational therapeutic approach for interdicting Alzheimers amyloid pathogenesis. At present, enhancing the targeting and efficacy of metal-ion chelating agents through ligand design is a main strategy in the development of the next generation of metal chelators. Inspired by the traditional dye Thioflavin-T, we have designed new multifunctional molecules that contain both amyloid binding and metal chelating properties. In silico techniques have enabled us to identify commercial compounds that enclose the designed molecular framework (M1), include potential antioxidant properties, facilitate the formation of iodine-labeled derivatives, and can be permeable through the blood-brain barrier. Iodination reactions of the selected compounds, 2-(2-hydroxyphenyl)benzoxazole (HBX), 2-(2-hydroxyphenyl)benzothiazole (HBT), and 2-(2-aminophenyl)-1H-benzimidazole (BM), have led to the corresponding iodinated derivatives HBXI, HBTI, and BMI, which have been characterized by X-ray diffraction. The chelating properties of the latter compounds toward Cu(II) and Zn(II) have been examined in the solid phase and in solution. The acidity constants of HBXI, HBTI, and BMI and the formation constants of the corresponding ML and ML2 complexes [M = Cu(II), Zn(II)] have been determined by UV-vis pH titrations. The calculated values for the overall formation constants for the ML2 complexes indicate the suitability of the HBXI, HBTI, and BMI ligands for sequestering Cu(II) and Zn(II) metal ions present in freshly prepared solutions of beta-amyloid (Abeta) peptide. This was confirmed by Abeta aggregation studies showing that these compounds are able to arrest the metal-promoted increase in amyloid fibril buildup. The fluorescence features of HBX, HBT, BM, and the corresponding iodinated derivatives, together with fluorescence microscopy studies on two types of pregrown fibrils, have shown that HBX and HBT compounds could behave as potential markers for the presence of amyloid fibrils, whereas HBXI and HBTI may be especially suitable for radioisotopic detection of Abeta deposits. Taken together, the results reported in this work show the potential of new multifunctional thioflavin-based chelating agents as Alzheimers disease therapeutics.

Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis

A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.

RECOMBINANT SYNTHESIS OF MOUSE ZN3-BETA AND ZN4-ALPHA METALLOTHIONEIN 1 DOMAINS AND CHARACTERIZATION OF THEIR CADMIUM(II) BINDING CAPACITY

Abstract. Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn4-αMT and Zn3-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn3-βMT with CdCl2 results in the formation of Cd3-βMT. The addition of excess Cd(II) leads to Cd4-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd9-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd3-βMT species is stable in the presence of this strong metal-chelating agent.

A new insight into the Ag+ and Cu+ binding sites in the metallothionein β domain

Abstract The copper( I ) and silver( I ) binding properties of the β fragment of recombinant mouse metallothionein 1 have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn 3 -βMT at pH 7 or apo-βMT at pH 3 with Cu + leads to the formation of species having the same stoichiometry and structure: Cu 6 -βMT, Cu 7 -βMT and Cu 10 -βMT. In the first stage of the titration of Zn 3 -βMT with Cu + at pH 7 one additional species of formula Cu 4 Zn 1 -βMT was detected. In contrast, the titration of Zn 3 -βMT at pH 7.5 and of apo-βMT at pH 2.5 with Ag + proceeds through different reaction pathways, affording Zn x Ag 3 -βMT, Ag 6 -βMT and Ag 9 -βMT or Ag 3 -βMT, Ag 6 -βMT and Ag 9 -βMT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag 6 -βMT and Ag 9 -βMT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.

Synthesis, characterisation and magnetic properties of cobalt(II) complexes with picolinic acid derivatives: the crystal and molecular structures of [Co(MeC5H3NCOO)2(H2O)2] and [CoCl2(C5H4NCOOPri)2]

The reaction of Co(AcO) 2 with 6-methyl-2-pyridinecarboxylic acid, and that of CoCl 2 with the isopropyl ester of 2-pyridinecarboxylic acid, both with a 1:2 metal to ligand molar ratio, afford optimum yields for the synthesis of cis -[Co(MeC 5 H 3 NCOO) 2 (H 2 O) 2 ] and cis -[CoCl 2 (C 5 H 4 NCOOPr i ) 2 ], respectively. According to X-ray crystallography they both consist of mononuclear discrete molecules with octahedral coordination around Co(II). The ESR powder spectra of the related Zn phases doped with 0.25% Co(II) show the strong anisotropy of the g and A tensors. The magnetic behaviour of the solid complexes is characteristic of Co(II) systems with important spin–orbit coupling and weak antiferromagnetic interactions. Electronic UV–Vis and magnetic data indicate that these complexes, as well as the already known trans -[Co(C 5 H 4 NCOO) 2 (H 2 O) 2 ], have a high-spin octahedral geometry in solution.

Crystal structure of thioflavin-T and its binding to amyloid fibrils: insights at the molecular level

Combining X-ray data on thioflavin-T and theoretical calculations on its binding to a peptide model for Abeta(1-42) fibrils gives evidence of main stabilizing interactions, which influence the dihedral angle between the two moieties of thioflavin-T and thereby its fluorescence properties; these results shed new light on possible strategies for the design of dyes to bind amyloid fibrils more specifically.

Monitoring of the metal displacement from the recombinant mouse liver metallothionein Zn7-complex by capillary zone electrophoresis with electrospray MS detection

A solution of a Zn-complex of recombinant mouse MT-1 isoform (Zn(7)-MT-1) was prepared and titrated with Cd(2+) ions. A method based on the coupling of capillary zone electrophoresis (CZE) with electrospray MS detection was developed for the analysis of the stoichiometry of the species formed during the titration. The method offered the possibility of the on-line removal of up to 100 mM Tris or phosphate buffer solutions that would otherwise suppress the electrospray signal. By allowing the determination of the metal stoichiometry of the complex species present in solution the method was shown to be complementary to circular dichroism and UV-VIS spectrophotometry conventionally used for similar studies. The titration of the Zn(7)-MT complex with Cd(II) showed the sequential displacement of the Zn by Cd. The unusually high stability of the Cd(6)Zn(1)-MT species was observed which suggests a structural role of the remaining Zn(II) ion.

MTO: the second member of a Drosophila dual copper-thionein system.

Drosophila MTO metal binding features were analyzed for comparison with MTN, the paralogous Drosophila metallothionein, and to classify MTO as either zinc‐ or copper‐thionein. This was achieved by a combination of in vivo, in vitro and in silico methodologies. All the results unambiguously classified MTO as a second Drosophila copper‐thionein, putting Drosophila forward as the only metazoan in which any zinc‐thionein has still to be reported. Interestingly, experimental data only showed minor differences in the coordinative behavior of both MTs, but provided a characteristic spectroscopic fingerprint, revealing the possible binding of chloride anions in certain metal‐MTO aggregates.

Drosophila MTN: a metazoan copper-thionein related to fungal forms

Two Drosophila metallothioneins (MT) have been reported: MTN, a 40 residue peptide including 10 Cys, and MTO, a 43 residue peptide including 12 Cys. However, neither functional nor evolutionary analyses for either of the Drosophila MT are available. Here, heterologous expression of Mtn in Escherichia coli is reported. The metal binding abilities of the Cu‐ and Zn‐MTN complexes conformed in vivo, as well as the features of the Cd‐ and Cu‐aggregates produced by metal replacement in vitro, have been determined by atomic emission spectrometry, circular dichroism and electrospray ionization mass spectrometry. Primary structure relationships with other MT have been examined. The results indicate a close resemblance of MTN to fungal copper‐thioneins.

Zinc(II) is required for the in vivo and in vitro folding of mouse copper metallothionein in two domains

We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu,Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)α(Cu4Zn1)β-MT, (Cu3Zn2)α(Cu4Zn1)β-MT and (Cu4Zn1)α(Cu6)β-MT. The cooperative formation of (Cu3Zn2)α(Cu4Zn1)β-MT from (Zn4)α(Cu4Zn1)β-MT indicates that the preference of Cu(I) for binding to the β domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent α and β fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)α(Cu4Zn1)β-MT], while that of αMT has rendered a mixture of Cu4Zn1-αMT (40%), Cu5Zn1-αMT (20%) and Cu7-αMT (40%). In the case of βMT, a mixture of Cu6-βMT (25%) and Cu7-βMT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-βMT from native sources may not be adequate.