Structural Characterization and Inhibition of Toxic Amyloid-β Oligomeric Intermediates

Abstract

Misfolding of amyloid-beta (Aβ) has been implicated in Alzheimer’s disease (AD). Recent studies have shown that early oligomeric intermediates formed in the misfolding pathway are the main toxic species as they kill neuronal cells. Therefore, the development of compounds to inhibit Aβ aggregation—and thus to avoid the formation of toxic oligomeric intermediates—is urgently needed as they could be potential drugs to treat AD (1). Although this has been well realized by the scientific community and many research groups have been working on developing such compounds, the main limitation has been the lack of biophysical techniques that can provide atomic-level structural insights into the misfolding process. Fortunately, NMR techniques can be utilized to obtain such high-resolution information on amyloid peptides and proteins as demonstrated by recent studies. In this context, the study by Raditsis et al. (2) demonstrates the value of solution NMR techniques in understanding the molecular basis for inhibition of oligomerization Aβ1−42 (and also a peptide fragment Aβ12−28) by the iron-transport glycoprotein transferrin that is present in plasma and cerebrospinal fluid (2). Specifically, using off-resonance relaxation and saturation transfer difference NMR experiments, they have shown that transferrin preferentially binds to Aβ oligomers and inhibits the aggregation of monomers into large oligomers. The similarity between the mechanisms of Aβ self-association inhibition by transferrin and another plasma protein human serum albumin (3) is intriguing, and indicates that the Aβ-oligomer-binding model proposed in this study could be a general strategy shared by multiple plasma proteins. In fact, to fully understand the Aβ toxicity, it is essential to solve the three-dimensional structures of early oligomers as they provide insights into molecular interactions that drive the misfolding and aggregation of Aβ. Although NMR is one of the most commonly used biophysical tools in structural biology, its poor sensitivity severely limits structural investigation of fast aggregating amyloid proteins. Increasing the amount of peptide/protein or temperature to enhance NMR sensitivity is not desirable, for it increases the rate of amyloid aggregation. In addition, the presence of heterogeneous species—monomers, oligomers, and protofibers—significantly lower the spectral resolution though only the fast-tumbling small size species are detected in solution NMR measurements. Despite these limitations, a recent NMR study determined the high-resolution structure of Aβ1-40 from solution that could be a nucleus in the aggregation process (Fig. 1 A). Remarkably, the NMR structure reveals that the hydrophobic H13–D23 region of Aβ1-40 forms a 310 helix and the unstructured hydrophobic residues of the N- and C-termini collapse against the central 310 helix (4). Though previous studies have detected the formation of a transient, on-pathway α-helical intermediate, to our knowledge this is the first experimentally determined high-resolution structure of Aβ1-40 (5). This structure is increasingly utilized in the development of compounds to inhibit the aggregation of Aβ peptides. Figure 1 (A) A partially folded structure of Aβ1-40 (PDB:2LFM) in solution determined by NMR spectroscopy (4). (B) NMR investigations for identification of interactions of Aβ with proteins, metal ions, and/or small molecules (6–9). Because large oligomers are not detected in solution NMR experiments, NMR signals detected from monomers are used to characterize Aβ-ligand and Aβ-protein interactions (6–9). Such studies have been used to develop compounds that can inhibit Aβ aggregation as well as avoid metal-induced aggregation by modulating the interaction between the metal (e.g., Cu(II) and Zn(II)) and the peptide (6,7,9) (Fig. 1 B). In fact, simple high-throughput two-dimensional NMR experiments such as NOESY, HSQC, TROSY-HSQC, or SOFAST-HMQC are increasingly utilized to: 1. Identify direct interactions (structures) of compounds with Aβ at a molecular level, which leads to a better design or optimization of molecules to efficiently suppress the formation of toxic Aβ species; and 2. Screen a chemical library against Aβ aggregation in a high-throughput manner. Recent studies have demonstrated the feasibility of real-time measurement of the aggregation of Aβ and IAPP using 19F NMR (10,11). These studies have revealed the formation of several different types of small-size Aβ oligomers, the nonexistence of appreciable nonfibrillar islet amyloid polypeptide protein intermediates, and the effect of an amyloid inhibitor epigallocatechin gallate (10,11). Due to both fundamental and translation aspects, drug discovery for multiple targets in human diseases including the misfolding proteins (amyloids) has been assisted and will continue to greatly benefit by the development and applications of NMR spectroscopy. In this context, the results reported by Raditsis et al. (2) are exciting and further demonstrate the power of simple NMR experiments to characterize protein-Aβ interactions. At the same time, it should also be realized that the investigation of the Aβ-membrane interaction and the roles of membrane components are essential to understanding the mechanism of Aβ toxicity in AD (12).