The formation of well-ordered fibrillar protein deposits is common to a large group of amyloidassociated disorders. This group consists of several major human diseases such as Alzheimer's disease, Parkinson's disease, prion diseases, and type II diabetes. Currently, there is no approved therapeutic agent directed towards the formation of fibrillar assemblies, which have been recently shown to have a key role in the cytotoxic nature of amyloidogenic proteins. One important approach in the development of therapeutic agents is the use of small molecules that specifically and efficiently inhibit the aggregation process. Several small polyphenol molecules have been demonstrated to remarkably inhibit the formation of fibrillar assemblies in vitro and their associated cytotoxicity. Yet, the inhibition mechanism was mostly attributed to the antioxidative properties of these polyphenol compounds. Based on several observations demonstrating that polyphenols are capable of inhibiting amyloid fibril formation in vitro, regardless of oxidative conditions, and in view of their structural similarities we suggest an additional mechanism of action. This mechanism is assuming structural constraints and specific aromatic interactions, which direct polyphenol inhibitors to the amyloidogenic core. This proposed mechanism is highly relevant for future de novo inhibitors' design as therapeutic agents for the treatment of amyloid-associated diseases.Key words: Aromatic Interactions, pi-pi interactions, polyphenols, protein misfolding, self-assembly. Received 4 October 2005, revised and accepted for publication 21 October 2005Amyloidogenic diseases are characterized by conformational changes that are followed by aggregation of proteins inside or outside cells. This self-assembly into well-ordered supramolecular assemblies and eventually deposits (also termed plaques in neurodegenerative diseases) is associated with major human diseases and has key medical importance. A partial list of more than 20 amyloid-related diseases includes Alzheimer's disease, Parkinson's disease, Huntington's disease, prion diseases, familial amyloidosis, and type II diabetes (1-13).In spite of the fact that various amyloid-forming proteins and polypeptides do not reveal any simple sequence homology, all amyloid assemblies share similar ultrastructural and physiochemical properties. All amyloid fibrils have a characteristic, elongated fibrillar morphology with a diameter of 5-15 nm, a b-sheet-rich structure (14-16), and typical X-ray fiber diffraction with a reflection of 4.6-4.8 in the meridian (17,18). Another well-known characteristic of all amyloid fibrils is their interaction with specific dyes such as Congo red and thioflavin T, which results in definite optical behavior such as birefringence and fluorescence, respectively (19,20).In this article, we will describe the up-to-date knowledge on the molecular interactions that lead to amyloid fibril formation and the therapeutic approaches to amyloidogenic diseases, and will focus on a small molecular family ...

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH 2 -DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH 2 -DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH 2 -FNKF-COOH tetrapeptide, the NH 2 -DFN-COOH tripeptide, or the NH 2 -DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.The process of amyloid fibril formation is associated with a large number of diseases of unrelated origin. A partial list includes Alzheimer's disease, Parkinson's disease, Type II diabetes, Prion diseases, Familial British dementia, and several familial amyloidosises (1-7). All of these diseases are characterized by the transformation of soluble proteins into aggregated fibrillar deposits in different organs and tissues. The pathological significance of amyloid fibril formation is not completely understood in all cases. One such example is aortic medial amyloid deposits that occur virtually in all individuals older than 60 years, but its medical consequence is not known yet (8).Although different amyloid-forming proteins do not share clear sequence homology, the fibrillar structures that are being formed have very similar physicochemical and ultrastructural characteristics as determined by transmission electron microscopy x-ray fiber diffraction, and other biophysical techniques (1-7). Furthermore, amyloid fibrils are being formed in vitro also by disease-unrelated proteins (9 -13). This finding suggests that amyloid fibrils may serve as a generic structural form of aggregated proteins (1-7). Nevertheless, despite the significant medical importance of the amyloid fibril formation process and its consideration as a universal structural form, the exact mechanism that leads to the self-assembly of polypeptides into ordered fibrils is not fully understood.Human calcitonin (hCT) 1 is a 32-amino acid polypeptide hor...

The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.

The formation of amyloid fibrils by the human islet amyloid polypeptide is associated with type II diabetes. While it was previously suggested that the formed fibrils are toxic to pancreatic beta-cells due to membrane permeation activity, more recent studies suggested that protofibrillar assemblies have significantly higher potency in permeating lipid bilayers. Here, we specifically studied the membrane interaction activity of soluble and insoluble islet amyloid polypeptide assemblies at high temporal resolution. A colorimetric analysis using lipid/polydiacetylene (PDA) biomimetic vesicles clearly demonstrated the transient formation of soluble assemblies that strongly interact with the lipid vesicles. A peak in the level of membrane binding of the soluble fraction, as reflected by the colorimetric assay, was observed after incubation for approximately 1 h, followed by a decrease in the level of membrane interaction of the assemblies. The transient nature of the membrane-active assemblies was independently confirmed by a fluorescence quenching assay. Ultrastructural analysis using transmission electron microscopy provided morphological evidence of prefibrillar assemblies, supported the transient existence of membrane interacting soluble species, and facilitated observation of the non-membrane-active filaments in the solution. Taken together, our results provide experimental evidence for the formation of transient soluble prefibrillar assemblies which are highly membrane-active. The implications of these observations are discussed in light of designed fibrillization inhibitors.