Iodine Atoms: A New Molecular Feature for the Design of Potent Transthyretin Fibrillogenesis Inhibitors (original) (raw)
Related papers
Biochemical Journal, 2005
Ex vivo and in vitro studies have revealed the remarkable amyloid inhibitory potency and specificity of iododiflunisal in relation to transthyretin [Almeida, Macedo, Cardoso, Alves, Valencia, Arsequell, Planas and Saraiva (2004) Biochem. J. 381, 351–356], a protein implicated in familial amyloidotic polyneuropathy. In the present paper, the crystal structure of transthyretin complexed with this diflunisal derivative is reported, which enables a detailed analysis of the protein–ligand interactions. Iododiflunisal binds very deep in the hormone-binding channel. The iodine substituent is tightly anchored into a pocket of the binding site and the fluorine atoms provide extra hydrophobic contacts with the protein. The carboxylate substituent is involved in an electrostatic interaction with the Nζ of a lysine residue. Moreover, ligand-induced conformational alterations in the side chain of some residues result in the formation of new intersubunit hydrogen bonds. All these new interactions...
Chemical Communications, 2010
All reagents were purchased from Aldrich, the commercial ThT(Cl) was recrystallized in acetonitrile. The slow addition of an aqueous saturated solution of KI (5 ml) to an aqueous solution (20 ml) of (ThT)Cl (0.600 g, 1.88 mmol) resulted in the formation of a yellow solid that was filtered off, washed with cold water (10 ml) and dried in vacuo. MS(+ESI-MS) showed the presence of ThT + exclusively. m/z (relative intensity): 283.1 ([ThT] + , 100). A saturated CHCl 3 solution of this solid was allowed to evaporate slowly at room temperature under open atmosphere. Reddish crystals, (ThT) 2 I 4 •2CHCl 3 (1), were obtained after 24 h and yellow crystals, (ThT)I•CHCl 3 (2), after ca. one week, both types of crystals being suitable for X-ray diffraction. 2. X-ray diffraction Single-crystal X-ray diffraction analyses were performed on a Bruker SMART-APEX CCD area-detector diffractometer at room temperature with graphite-monochromated MoKα radiation. Lorentz-polarization and absorption corrections were applied using Bruker SAINT [1] and SADABS [2] software. Structures were solved by direct methods and refined by full-matrix least-squares on F 2 for all reflections using SHELXTL. [3] In 1, the chloroform molecule is highly disordered. Four geometrically constrained CHCl 3 groups have been considered and refined with isotropic displacement parameters. The rest of nonhydrogen atoms in the structures 1 and 2 were refined with anisotropic displacement parameters. Hydrogen atoms were included with riding model constraints and isotropic displacement parameters 1.2 times U eq value of the corresponding carbons. The two crystal structures have been deposited at the Cambridge Crystallographic Data Centre under the following deposition numbers: (ThT) 2 I 4 •2CHCl 3 (CCDC 728298) and (ThT)I•CHCl 3 (CCDC 728299). 3. Computational details 3.1. DFT calculations Gas phase calculations for (ThT) + have been carried out at the B3LYP [4, 5] level of theory with the 6-31++G(d,p) basis set. In the case of ThT + interacting with a model of the β-sheet structure optimizations have been performed at the B3LYP-D level; that is, adding an empirical correction for dispersion of the form-C 6 •R-6 (s 6 =1.05) [6] to the B3LYP energy. In this case, and due to the size of the system, we used the 6-31G(d) basis set for geometry optimizations and the 6-31+G(d,p) basis for single point energy calculations. This procedure was calibrated for one of the minimum located (ϕ = 21) and found to be very
PLoS ONE, 2009
Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of b-aminoxypropionic acids (compounds 5-21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR:inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp 2 -hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22-32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding. (JCS) . These authors contributed equally to this work.
Journal of Enzyme Inhibition and Medicinal Chemistry
Transthyretin (TTR) is a 54 kDa homotetrameric protein that transports thyroxine (T4) and retinol (vitamin A), through its association with retinol binding protein (RBP). Under unknown conditions, it aggregates to form fibrils associated with TTR amyloidosis. Ligands able to inhibit fibril formation have been studied by X-ray crystallography. The use of polyethylene glycol (PEG) instead of ammonium sulphate or citrate has been evaluated as an alternative to obtain new TTR complexes with (R)-3-(9-fluoren-9-ylideneaminooxy)-2-methyl-N-(methylsulfonyl) propionamide (48R(1)) and 2-(9H-fluoren-9-ylideneaminooxy) acetic acid (ES8(2)). The previously described fluorenyl based inhibitors (S)-3-((9H-fluoren-9-ylideneamino)oxy)-2-methylpropanoic acid (6BD) and 3-((9H-fluoren-9-ylideneamino)oxy)propanoic acid (7BD) have been re-evaluated with the changed crystallization method. The new TTR complexes with compounds of the same family show that the 9-fluorenyl motif can occupy alternative hydrophobic binding sites. This augments the potential use of this scaffold to yield a large variety of differently substituted mono-aryl compounds able to inhibit TTR fibril formation.
Journal of Enzyme Inhibition and Medicinal Chemistry, 2016
Transthyretin (TTR), a b-sheet-rich tetrameric protein, in equilibrium with an unstable amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils, is associated with the onset of neurodegenerative diseases, such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. One of the therapeutic strategies is to use small molecules to stabilize the TTR tetramer and thus curb amyloid fibril formation. Here, we report the synthesis, the in vitro evaluation of several halogen substituted 9-fluorenyl-and di-benzophenon-based ligands and their three-dimensional crystallographic analysis in complex with TTR. The synthesized compounds bind TTR and stabilize the tetramer with different potency. Of these compounds, 2c is the best inhibitor. The dual binding mode prevalent in the absence of substitutions on the fluorenyl ring, is disfavored by (2,7-dichlorofluoren-9-ylideneaminooxy)-acetic acid (1b), (2,7-dibromo-fluoren-9-ylideneaminooxy)-acetic acid (1c) and (E/Z)-((3,4-dichloro-phenyl)-methyleneaminooxy)-acetic acid (2c), all with halogen substitutions.
Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor
Nature chemistry, 2012
Amyloid formation has been implicated in the pathology of over 20 human diseases, but the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labelling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin (the peptide responsible for islet amyloid formation in type 2 diabetes) with a known inhibitor (rat amylin). Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 h after mixing, rat amylin blocks the N-terminal β-sheet instead. At 24 h after mixing, rat amylin blocks neither β-sheet and forms its own β-sheet, most probably on the outside of the human fibrils. This is striking, because rat amylin is natively disordered and not previously known to form amyloid β-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.
Fiber Diffraction As a Screen for Amyloid Inhibitors
Current Alzheimer Research, 2008
Targeting the initial formation of amyloid assemblies is a preferred approach to therapeutic intervention in amyloidoses, which include such diseases as Alzheimer's, Parkinson's, Huntington's, etc., as the early-stage, oligomers that form before the development of -conformation-rich fibers are thought to be toxic. X-ray patterns from amyloid assemblies always show two common intensity maxima: one at 4.7 Å corresponding to the hydrogen-bonding spacing between the -chains, and the other at ~10 Å corresponding to the spacing between -pleated sheets. We report here the application of fiber x-ray diffraction to monitor these structural indicators of amyloid fiber assembly in the presence of small, aromatic molecules, some of which have been assessed by other techniques as being inhibitory. The compounds included butylated hydroxytoluene, chloramphenicol, cotinine, curcumin, diphenylalanine (FF), ethyl 3-aminobenzoate methane sulfonate, hexachlorophene, melatonin, methylpyrrolidine, morin, nicotine, phenolphthalaine, PTI-00703 ® (Cat's claw), pyridine, quinine, sulfadiazine, tannic acid, tetracaine, tetrachlorosalicylanilide, and tetracycline. Their effects on the aggregation of A 1-40, A 11-25, A 12-28, A 17-28, A 16-22, and A 16-22[methylated] analogues were characterized in terms of the integral widths and integrated intensities of the two characteristic reflections.
Biophysical Journal, 1993
To elucidate the relation between amyloid fibril formation in Alzheimer disease and the primary structure of the fl/A4 protein, which is the major component of the amyloid, we have been investigating the ability of peptides sharing sequences with ,B/A4 to form fibrils in vitro. In previous studies we focused on the macroscopic morphology of the assemblies formed by synthetic peptides corresponding in sequence to different regions of this protein. In the present study we analyze the x-ray diffraction patterns obtained from these assemblies.
Acta crystallographica. Section D, Biological crystallography, 2015
The analysis of structural data obtained by X-ray crystallography benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF. A core laboratory featuring various spectrometers, named the Cryobench, is now in its third version and houses portable devices that can be directly mounted on beamlines. This paper reports the current status of the Cryobench, which is now located on the MAD beamline ID29 and is thus called the ID29S-Cryobench (where S stands for `spectroscopy'). It also reviews the diverse experiments that can be performed at the Cryobench, highlighting the various scientific questions that can be addressed.