Gregorio Valencia

Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative.

In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation.

Recent advances in the synthesis of complex N-glycopeptides

Abstract This review describes the recent advances in the development of efficient strategies, chemical, enzymatic and chemo-enzymatic, for the synthesis of complex N- glycopeptides. A selected number of illustrative examples will also be discussed.

O-glycosyl α-amino acids as building blocks for glycopeptide synthesis

Abstract A review of O -glycosyl α-amino acid structures representing most of the glycosylation sites of known O -glycoproteins found in nature, their preparation by chemical methods and application as building blocks for either solution or solid-phase synthesis of O -glycopeptides is presented.

Arylation of Phe and Tyr Side Chains of Unprotected Peptides by a Suzuki−Miyaura Reaction in Water

An efficient arylation in water of tyrosine and phenylalanine side chains from unprotected iodopeptides is accomplished by using Suzuki-Miyaura cross-coupling processes. The method is compatible with the hydrophilic and thermolabile nature of biologically active peptides. Also of interest, the arylated tyrosine peptides can be accessed in one-pot mode starting from native peptides.

Human transthyretin in complex with iododiflunisal: structural features associated with a potent amyloid inhibitor

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 N(zeta) 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, induced by iododiflunisal, increase the stability of the tetramer impairing the formation of amyloid fibrils. The crystal structure of this complex opens perspectives for the design of more specific and effective drugs for familial amyloidotic polyneuropathy patients.

Iodine Atoms: A New Molecular Feature for the Design of Potent Transthyretin Fibrillogenesis Inhibitors

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis.

Influence of solvent and water activity on kinetically controlled peptide synthesis

alpha-Chymotrypsin deposited on Celite was used to catalyse peptide synthesis reactions between N-protected amino acid esters and leucine amide in organic media with low water content. The influence of the solvent and the thermodynamic water activity on the reaction kinetics was studied. The substrate specificity in the reactions was shown to be a combination of the substrate specificity of the enzyme in aqueous media and the influence of the solvents. The magnitude of the solvent effects differed greatly depending on the substrates used. In hydrophobic solvents high reaction rates were observed and the competing hydrolysis of the ester substrate occurred to only a minor extent. Reactions occurred at water activities as low as 0.11, but the rate constants increased with increasing water activity and were about two orders of magnitude higher at the highest water activity tested (0.97).

Comparative in vitro and ex vivo activities of selected inhibitors of transthyretin aggregation: relevance in drug design.

Destabilization of the tetrameric fold of TTR (transthyretin) is important for aggregation of the protein which culminates in amyloid fibril formation. Many TTR mutations interfere with tetramer stability, increasing the amyloidogenic potential of the protein. The vast majority of proposed TTR fibrillogenesis inhibitors are based on in vitro assays with isolated protein, limiting their future use in clinical assays. In the present study we investigated TTR fibrillogenesis inhibitors using a cellular system that produces TTR intermediates/aggregates in the medium. Plasmids carrying wild-type TTR, V30M or L55P cDNA were transfected into a rat Schwannoma cell line and TTR aggregates were investigated in the medium using a dot-blot filter assay followed by immunodetection. Results showed that, in 24 h, TTR L55P forms aggregates in the medium, whereas, up to 72 h, wild-type TTR and V30M do not. A series of 12 different compounds, described in the literature as in vitro TTR fibrillogenesis inhibitors, were tested for their ability to inhibit L55P aggregate formation; in this system, 2-[(3,5-dichlorophenyl) amino] benzoic acid, benzoxazole, 4-(3,5-difluorophenyl) benzoic acid and tri-iodophenol were the most effective inhibitors, as compared with the reference iododiflunisal, previously shown by ex vivo and in vitro procedures to stabilize TTR and inhibit fibrillogenesis. Among these drugs, 2-[(3,5-dichlorophenyl) amino] benzoic acid and tri-iodophenol stabilized TTR from heterozygotic carriers of V30M in the same ex vivo conditions as those used previously for iododiflunisal. The novel cellular-based test herein proposed for TTR fibrillogenesis inhibitor screens avoids not only lengthy and cumbersome large-scale protein isolation steps but also artefacts associated with most current in vitro first-line screening methods, such as those associated with acidic conditions and the absence of serum proteins.

Optimization and kinetic studies of the enzymatic synthesis of Ac-Phe-Leu-NH2 in reversed micelles

Abstract Optimization and kinetic studies of the α-chymotrypsin-catalysed synthesis of Ac-Phe-Leu-NH 2 were performed in reversed micelles. The micellar reaction system included n -octane, either 1-hexanol or 1-octanol as cosurfactants, CTAB, and Tris-HCl buffer. Following the response surface methodology, in a first set of experiments the combined effects on the reaction yields of the following experimental factors were examined: W 0 , pH, ionic strength, temperature, percentage of cosurfactant, and surfactant concentration. The reaction yields were mainly influenced by three parameters: W 0 , pH, and, mostly, percentage of consurfactant. In addition, a 2 2 factorial design was devised to further study the influence of W 0 and pH on the reaction performance. It was found that there is not a well-defined maximum, since there is a range of conditions that have optimal response. These optimal conditions always rendered peptide yields as high as 85%. In a parallel set of experiments, it was also shown that the substitution of 1-hexanol by 1-octanol led, in these reaction systems, to slightly lower reaction yields; therefore, the structure and the polarity of the cosurfactant were shown to be factors greatly determining the reaction yield.

Isatin derivatives, a novel class of transthyretin fibrillogenesis inhibitors

The isatin core structure was found to be a novel chemical scaffold in transthyretin (TTR) fibrillogenesis inhibitor design. Among the series of isatin analogues prepared and tested, the nitro compound 1,3-dihydro-3-[(4-nitrophenyl)imino]-2H-indol-2-one (2r) is as potent as triiodophenol, which is one of the most active known TTR inhibitors. The E/Z stereochemistry of these molecules in solution, elucidated by (1)H NMR, does not influence their biological activity. The compounds do not bind to the native tetrameric TTR suggesting that their inhibitory action is independent of the protein binding and stabilization.