Cristina Rodríguez-Rodríguez

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.

N-Aryl-substituted 3-(β-D-glucopyranosyloxy)-2-methyl-4(1H)-pyridinones as agents for Alzheimer's therapy

Molecules designed to sequester, redistribute and/or remove metal ions are attractive therapeutic agents in neurodegenerative diseases such as Alzheimers disease. The multifactorial nature of the condition and the generally poor target specificity associated with metal ion-binding therapy has led to the development of multifunctional 3-hydroxy-4-(1H)-pyridinone pro-ligands. The excellent qualities of the basic 3-hydroxy-4-pyridinone framework as a low toxicity metal chelator and an antioxidant, as well as its antibacterial and analgesic properties among other functions, inspired us to functionalize it with a framework derived from thioflavin-T, the well-known traditional dye used as a marker to detect amyloid deposits in tissue sections. Thus 2-methyl-3-hydroxy-1-(4-dimethylaminophenyl)-4(1H)-pyridinone (HL1), 2-methyl-3-hydroxy-1-(4-methylaminophenyl)-4(1H)-pyridinone (HL2), 1-(4-aminophenyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (HL3), 1-(6-benzothiazolyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (HL4), 1-(2-benzothiazolyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (HL5) and 2-methyl-3-hydroxy-1-[4-(4-bromophenyl)-2-thiazolyl]-4(1H)-pyridinone (HL6) were obtained. Glycosylation, as well as incorporation of structures mimicking those of known amyloid imaging agents, may target drug action to the site of interest, the metal-overloaded amyloid plaques in the Alzheimers brain. The pro-ligands were assessed for their antioxidant activity, cytotoxicity and ability to interfere with metal ion-induced amyloid peptide aggregation to screen promising lead compounds. Finally, in a brain uptake study with a radiolabeled glucoconjugate pyridinone, 3-(β-D-glucopyranosyloxy)-1-[4-(4-[125I]iodophenyl)-2-thiazolyl]-2-methyl-4(1H)-pyridinone ([125I]-GL7) was shown to cross the blood–brain barrier using an in situ rat brain perfusion technique.

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.

Ab Initio Design of Chelating Ligands Relevant to Alzheimer’s Disease: Influence of Metalloaromaticity

Evidence supporting the role of metal ions in Alzheimers disease (AD) has rendered metal ion chelation as a promising therapeutic treatment. The rational design of efficient chelating ligands requires, however, a good knowledge of the electronic and molecular structure of the complexes formed. In the present work, the coordinative properties of a set of chelating ligands toward Cu(II) have been analyzed by means of DFT(B3LYP) calculations. Special attention has been paid to the aromatic behavior of the metalated rings of the complex and its influence on the chelating ability of the ligand. Ligands considered have identical metal binding sites (through N/O coordination) and only differ on the kind and size of the aromatic moieties. Results indicate that there is a good correlation between the stability constants (log β(2)) and the degree of metalloaromaticity determined through the I(NG) and HOMA indices; that is, the higher the metalloaromaticity, the larger the log β(2) value. MOs and aromaticity descriptors confirm that present complexes exhibit Möbius metalloaromaticity. Detailed analysis of the nature of the Cu(II)-ligand bonding, performed through an energy decomposition analysis, indicates that ligands with less aromatic moieties have the negative charge more localized in the metalated ring, thus increasing their σ-donor character and the metalloaromaticity of the complexes they form.

In silico strategies for the selection of chelating compounds with potential application in metal-promoted neurodegenerative diseases

The development of new strategies to find commercial molecules with promising biochemical features is a main target in the field of biomedicine chemistry. In this work we present an in silico-based protocol that allows identifying commercial compounds with suitable metal coordinating and pharmacokinetic properties to act as metal-ion chelators in metal-promoted neurodegenerative diseases (MpND). Selection of the chelating ligands is done by combining quantum chemical calculations with the search of commercial compounds on different databases via virtual screening. Starting from different designed molecular frameworks, which mainly constitute the binding site, the virtual screening on databases facilitates the identification of different commercial molecules that enclose such scaffolds and, by imposing a set of chemical and pharmacokinetic filters, obey some drug-like requirements mandatory to deal with MpND. The quantum mechanical calculations are useful to gauge the chelating properties of the selected candidate molecules by determining the structure of metal complexes and evaluating their stability constants. With the proposed strategy, commercial compounds containing N and S donor atoms in the binding sites and capable to cross the BBB have been identified and their chelating properties analyzed.

Insights on the Binding of Thioflavin Derivative Markers to Amyloid-Like Fibril Models from Quantum Chemical Calculations

Thioflavin-T (ThT) is one of the most widely used dyes for staining and identifying amyloid fibrils, which share a common parallel in register β-sheet structure. Unfortunately, ThT is a charged molecule, which limits its ability to cross the blood brain barrier and its use as an efficient dye for in vivo detection of amyloid fibrils. For this reason, several uncharged ThT derivatives have been designed and their binding properties to Aβ fibrils studied by fluorescence assays. However, there are still many unknowns on the binding mechanism and the role of noncovalent interactions on the affinity of these ligands toward β-sheet structures. The present contribution analyzes the binding of ThT (1) and neutral ThT derivatives (2-7) to a β-sheet model by means of quantum chemical B3LYP-D calculations and including solvent effects with the continuum CPCM method. Results show that, in all cases, ligand binding is mainly driven by dispersion interactions. In addition, ligands with -NH groups display hydrogen bond interactions with CO groups of the peptide strand, increasing the intrinsic affinity toward the β-sheet surface. Solvent effects notably reduce the affinity of charged ThT, as compared to neutral systems, due to its larger solvation energy. As a result, neutral derivatives display significantly higher affinities than ThT in solution, in agreement with experimental observations. Analysis of the hydrogen bonding network of the β-sheet structure indicates that stacking interactions upon ligand binding induce a shortening of interstrand hydrogen bonding, suggesting a strengthening of the β-sheet.

Synthesis, characterization, and cytotoxicity studies of Cu(II), Zn(II), and Fe(III) complexes of N-derivatized 3-hydroxy-4-pyridiones

The deleterious role of metal ions in Alzheimers disease has inspired the study of various metal chelators. We previously showed the synthesis and in vitro activity of several bidentate hydroxypyridinone compounds, including 3-hydroxy-2-methyl-1-phenyl-4(1H)-pyridinone (1), 1-(4-aminophenyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (2), and 1-(2-benzothiazolyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (3). While the focus has been on the Cu(II) ion, the other biorelevant metals, Zn(II) and Fe(III) have been largely neglected. Herein, we report the synthesis of Zn(II) and Fe(III) complexes of ligands 1, 2, and 3, and their characterization by infrared (IR) spectroscopy, high resolution mass spectrometry (HR-MS), elemental analysis, and NMR, where applicable. Solid state structures of Zn(1)2, Fe(1)3, and Cu(3)2 are analyzed with X-ray crystallography. The cytotoxicity of pro-ligands 1, 2, and 3, and the three metal complexes of 2 are examined in a neuronal cell line to determine the effect of metal chelation on toxicity of the compounds.

3-Hydroxy-4-pyridinone derivatives designed for fluorescence studies to determine interaction with amyloid protein as well as cell permeability.

Finding a cure for Alzheimers disease is an urgent goal. Multifunctional metal binders are used to elucidate its pathological features and investigated as potential therapeutics. The use of physicochemical and TD-DFT calculations constituted successful strategy in the design of 1-(4-(benzo[d]oxazol-2-yl)phenyl)-3-hydroxy-2-methylpyridin-4(1H)-one (HL21) and 1-(4-(benzo[d]thiazol-2-yl)phenyl)-3-hydroxy-2-methylpyridin-4(1H)-one (HL22). We report the synthesis and full characterization of these compounds, including X-ray crystallography. Using fluorescent signal as the readout, it was determined that HL22 interacts with amyloid-beta protein fibrils, and permeates into bEnd.3 cells used as a mimic of the blood-brain barrier. This provides the first example of direct investigation of our hydroxypyridinone compounds within a biological setting.

Multi-isotope SPECT imaging of the 225Ac decay chain: feasibility studies

Effective use of the [Formula: see text] decay chain in targeted internal radioimmunotherapy requires the retention of both [Formula: see text] and progeny isotopes at the target site. Imaging-based pharmacokinetic tests of these pharmaceuticals must therefore separately yet simultaneously image multiple isotopes that may not be colocalized despite being part of the same decay chain. This work presents feasibility studies demonstrating the ability of a microSPECT/CT scanner equipped with a high energy collimator to simultaneously image two components of the [Formula: see text] decay chain: [Formula: see text] (218 keV) and [Formula: see text] (440 keV). Image quality phantoms were used to assess the performance of two collimators for simultaneous [Formula: see text] and [Formula: see text] imaging in terms of contrast and noise. A hotrod resolution phantom containing clusters of thin rods with diameters ranging between 0.85 and 1.70 mm was used to assess resolution. To demonstrate ability to simultaneously image dynamic [Formula: see text] and [Formula: see text] activity distributions, a phantom containing a [Formula: see text] generator from [Formula: see text] was imaged. These tests were performed with two collimators, a high-energy ultra-high resolution (HEUHR) collimator and an ultra-high sensitivity (UHS) collimator. Values consistent with activity concentrations determined independently via gamma spectroscopy were observed in high activity regions of the images. In hotrod phantom images, the HEUHR collimator resolved all rods for both [Formula: see text] and [Formula: see text] images. With the UHS collimator, no rods were resolvable in [Formula: see text] images and only rods  ⩾1.3 mm were resolved in [Formula: see text] images. After eluting the [Formula: see text] generator, images accurately visualized the reestablishment of transient equilibrium of the [Formula: see text] decay chain. The feasibility of evaluating the pharmacokinetics of the [Formula: see text] decay chain in vivo has been demonstrated. This presented method requires the use of a high-performance high-energy collimator.

188Re image performance assessment using small animal multi-pinhole SPECT/PET/CT system

PURPOSE The goal of this study was to investigate the performance of a pre-clinical SPECT/PET/CT system for 188Re imaging. METHODS Phantom experiments were performed aiming to assess the characteristics of two multi-pinhole collimators: ultra-high resolution collimator (UHRC) and high-energy ultra high resolution collimator (HE-URHC) for imaging 188Re. The spatial resolution, image contrast and contrast-to-noise ratio (CNR) were investigated using micro-Jaszczak phantoms. Additionally, the quantification accuracy of 188Re images was evaluated using two custom-designed phantoms. The 188Re images were compared to those obtained with 99mTc (gold standard); the acquired energy spectra were analyzed and Monte-Carlo simulations of the UHRC were performed. To verify our findings, a C57BL/6-mouse was injected with 188Re-microspheres and scanned with both collimators. RESULTS The spatial resolution achieved in 188Re images was comparable to that of 99mTc. Acquisitions using HE-UHRC yielded 188Re images with higher contrast and CNR than UHRC. Studies of quantitative accuracy of 188Re images resulted in <10% errors for both collimators when the activity was calculated within a small VOI around the object of interest. Similar quantification accuracy was achieved for 99mTc. However, 188Re images showed much higher levels of noise in the background. Monte-Carlo simulations showed that 188Re imaging with UHRC is severely affected by down-scattered photons from high-energy emissions. The mouse images showed similar biodistribution of 188Re-microspheres for both collimators. CONCLUSIONS VECTor/CT provided 188Re images quantitatively accurate and with quality comparable to 99mTc. However, due to large penetration of UHRC by high-energy photons, the use of the HE-UHRC for imaging 188Re in VECTor/CT is recommended.