Design of ß-amyloid aggregation inhibitors from a predicted structural motif.

Drug design studies targeting one of the primary toxic agents in Alzheimer's disease, soluble oligomers of amyloid ß-protein (Aß), have been complicated by the rapid, heterogeneous aggregation of Aß and the resulting difficulty to structurally characterize the peptide. To address this, we have developed [Nle(35), D-Pro(37)]Aß(42), a substituted peptide inspired from molecular dynamics simulations which forms structures stable enough to be analyzed by NMR. We report herein that [Nle(35), D-Pro(37)]Aß(42) stabilizes the trimer and prevents mature fibril and ß-sheet formation. Further, [Nle(35), D-Pro(37)]Aß(42) interacts with WT Aß(42) and reduces aggregation levels and fibril formation in mixtures. Using ligand-based drug design based on [Nle(35), D-Pro(37)]Aß(42), a lead compound was identified with effects on inhibition similar to the peptide. The ability of [Nle(35), D-Pro(37)]Aß(42) and the compound to inhibit the aggregation of Aß(42) provides a novel tool to study the structure of Aß oligomers. More broadly, our data demonstrate how molecular dynamics simulation can guide experiment for further research into AD.

A molecular dynamics study of the interaction of D-peptide amyloid inhibitors with their target sequence reveals a potential inhibitory pharmacophore conformation.

The self-assembly of soluble proteins and peptides into beta-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short D-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent beta-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.

New strategy for the generation of specific D-peptide amyloid inhibitors.

The conversion of a soluble protein into beta-sheet-rich oligomeric structures and further fiber formation are critical steps in the pathogenesis of the group of human diseases known as amyloidoses. Drugs that interfere with this process may thus be able to prevent and/or cure these diseases. Recent results have shown that short amino acid stretches can provide most of the driving force needed to trigger amyloid formation of a protein. These evidence suggest that compounds that specifically bind to peptides synthesized upon the sequence of such amyloidogenic protein stretches might also be able to inhibit amyloid formation of the corresponding full-length protein and, likely, amyloid-induced cytotoxicity as well. Here we present a general strategy to obtain d-peptides that specifically interact with protein amyloid stretches. The screening of a d-peptide combinatorial library for inhibitors of an amyloidogenic peptide designed de novo has allowed us to extract a set of empirical rules for the design of d-peptide inhibitors of any six-residue amyloidogenic stretch. d-peptides generated on these bases prevent amyloid formation and disassemble preformed fibrils of different amyloid hexapeptides identified in human amyloid proteins. In addition, they are also specific for their target sequence. The d-peptide designed here for the Alzheimer's Abeta(1-42) peptide not only inhibits and disassembles amyloid material but also reduces Abeta(1-42) amyloid-induced cytotoxicity in cell culture.

Amyloid toxicity is independent of polypeptide sequence, length and chirality.

By using an amyloid sequence pattern, here we have identified putative six-residue amyloidogenic stretches in several relevant amyloid proteins. Hexapeptides synthesized on the bases of the sequence stretches matching the pattern have been shown to form amyloid fibrils in vitro. As larger pathological peptides such as A beta(1-42) do, these short amyloid peptides form heterogeneous mixtures of small aggregates that induce cell death in PC12 cells and primary hippocampal neurons. Toxic mixtures of small aggregates from these hexapeptides bind to cell membranes and can be further internalized, as also observed for natural amyloid proteins. In neurons, toxic aggregates obtained from the full length A beta(1-42) amyloid peptide or their amyloid stretch A beta(16-21) peptide preferentially localize in synapses, leading to the re-organization of the underlying actin cytoskeleton. This process does not involve stereospecific interactions between membrane and toxic species as D-sequences are as toxic as L ones, suggesting that is not receptor mediated. Based on these results, we propose here that regardless of polypeptide sequence, length and amino acid chirality, amyloid prefibrillar aggregates exert their cytotoxic effect through a common cell death mechanism related to a particular quaternary structure. The degree of toxicity of these species seems to depend, however, on cell membrane composition.

Hacking the code of amyloid formation: the amyloid stretch hypothesis.

Many research efforts in the last years have been directed towards understanding the factors determining protein misfolding and amyloid formation. Protein stability and amino acid composition have been identified as the two major factors in vitro. The research of our group has been focused on understanding the relationship between amino acid sequence and amyloid formation. Our approach has been the design of simple model systems that reproduce the biophysical properties of natural amyloids. An amyloid sequence pattern was extracted that can be used to detect amyloidogenic hexapeptide stretches in proteins. We have added evidence supporting that these amyloidogenic stretches can trigger amyloid formation by nonamyloidogenic proteins. Some experimental results in other amyloid proteins will be analyzed under the conclusions obtained in these studies. Our conclusions together with evidences from other groups suggest that amyloid formation is the result of the interplay between a decrease of protein stability, and the presence of highly amyloidogenic regions in proteins. As many of these results have been obtained in vitro, the challenge for the next years will be to demonstrate their validity in in vivo systems.

Peptide model systems for amyloid fiber formation: design strategies and validation methods.

The rational understanding of the factors involved in the formation of amyloid deposits in tissue is fundamental to the identification of novel therapeutic strategies to prevent or cure pathological conditions such as Alzheimer's and Parkinson's disease or spongiform encephalopathies. Given the complexity of the molecular events driving protein self-association, a frequent strategy in the field has consisted of designing simplified model systems that facilitate the analysis of the elements that predispose polypeptides toward amyloid formation. In fact, these systems have provided very valuable knowledge on the determinants underlying structural transitions to the polymeric beta-sheet state present in amyloid fibers and more disordered aggregates. In this chapter, we will describe different approaches to obtain and design model systems for amyloidogenesis, as well as the methodologies that are typically used to validate them. We will also show how some of the general principles obtained from these studies can be applied for de novo design purposes and for the sequence-based identification of amyloidogenic stretches in proteins.

The amyloid stretch hypothesis: recruiting proteins toward the dark side.

A detailed understanding of the molecular events underlying the conversion and self-association of normally soluble proteins into amyloid fibrils is fundamental to the identification of therapeutic strategies to prevent or cure amyloid-related disorders. Recent investigations indicate that amyloid fibril formation is not just a general property of the polypeptide backbone depending on external factors, but that it is strongly modulated by amino acid side chains. Here, we propose and address the validation of the premise that the amyloidogenicity of a protein is indeed localized in short protein stretches (amyloid stretch hypothesis). We demonstrate that the conversion of a soluble nonamyloidogenic protein into an amyloidogenic prone molecule can be triggered by a nondestabilizing six-residue amyloidogenic insertion in a particular structural environment. Interestingly enough, although the inserted amyloid sequences clearly cause the process, the protease-resistant core of the fiber also includes short adjacent sequences from the otherwise soluble globular domain. Thus, short amyloid stretches accessible for intermolecular interactions trigger the self-assembly reaction and pull the rest of the protein into the fibrillar aggregate. The reliable identification of such amyloidogenic stretches in proteins opens the possibility of using them as targets for the inhibition of the amyloid fibril formation process.

Design of model systems for amyloid formation: lessons for prediction and inhibition.

The determination of the physico-chemical principles underlying amyloid deposition is fundamental to the identification of therapeutic strategies to prevent or cure amyloid-related disorders. Given the complexity of the molecular events involved in protein self-association, researchers have designed simplified systems that facilitate the discovery of factors that predispose polypeptides to amyloid formation and aggregation. These systems have provided valuable knowledge about the determinants underlying the structural transitions to the polymeric beta-sheet state present in amyloid fibers and in more disordered aggregates. The integration of this knowledge is crucial to the identification of the regions responsible for the amyloidogenic and aggregating behavior of a given protein. The reliable discovery of amyloid-promoting fragments in proteins should have a great impact on the development of anti-amyloid agents. Also, methods that identify aggregation-prone motifs have a broad range of biotechnological applications, such as the improvement of the solubility of recombinant proteins for pharmaceutical and industrial purposes, and peptide-based biomaterial engineering.