No abstract

The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. Fibril formation is accompanied by oligomeric species thought to be the primary pathogenic agents in many of these diseases. With the aim of identifying the structural determinants responsible for the toxicity of misfolded oligomers, we created 12 oligomeric variants from the N-terminal domain of the E. coli HypF protein (HypF-N) by replacing one or more charged amino acid residues with neutral apolar residues and allowing the mutated proteins to aggregate under two sets of conditions. The resulting oligomeric species have different degrees of cytotoxicity when added to the extracellular medium of the cells, as assessed by the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, apoptosis, and influx of Ca2+ into the cells. The structural properties of the oligomeric variants were characterized by evaluating their surface hydrophobicity with 8-anilinonaphthalene-1-sulfonate (ANS) binding and by measuring their size by means of turbidimetry as well as light scattering. We find that increases in the surface hydrophobicity of the oligomers following mutation can promote the formation of larger assemblies and that the overall toxicity correlates with a combination of both surface hydrophobicity and size, with the most toxic oligomers having high hydrophobicity and small size. These results have allowed the relationships between these three parameters to be studied simultaneously and quantitatively, and have enabled the generation of an equation that is able to rationalize and even predict toxicity of the oligomers resulting from their surface hydrophobicity and size.

The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer's disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a readout the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.Alzheimer's disease | amyloid-β peptide | protein misfolding | drug discovery | protein aggregation A lzheimer's disease (AD) is, to date, an incurable neurodegenerative disorder that imposes substantial social and economic costs worldwide (1). According to the amyloid hypothesis, the aggregation of the amyloid-β peptide (Aβ) initiates a cascade of molecular events leading eventually to neuronal death (2-11). Because the presence of abnormal Aβ metabolism can be detected 10-20 years before the onset of AD (12, 13), early interventions may be possible before widespread and irreversible neurodegeneration has occurred. Although targeting Aβ accumulation has been pursued as a major potential therapeutic strategy against AD (14-17), no compound selected for this purpose has yet entered clinical use (18,19).Although these failures have raised doubts about the amyloid hypothesis (20), they can also be attributed to an incomplete knowledge of the molecular mechanisms by which the compounds tested so far affect the nucleation and growth of Aβ aggregates. Indeed, it has been shown that inhibiting Aβ aggregation without a detailed understanding of the underlying microscopic processes could affect the toxicity in unexpected ways (21,22). For example, the inhibition of nucleation events may delay or decrease toxicity, whereas the inhibition of elongation may lead to an overall increase in toxicity (21,22). Therefore, effective therapeutic strategies must be aimed at targeting precise microscopic steps during the Aβ aggregation process (21,(23)(24)(25).We describe here the development of a systematic pipeline based on chemical kinetics to identify a pool of candidate molecules directed against the aggregation of the 42-residue form of Aβ (Aβ42), and to understand the key chemical features responsible for their inhibitory activity. Results and DiscussionA Quasi-Structure-Based Drug Discovery Strategy. We introduce first a quasi-structure-based drug discovery (QSBDD) strategy, which builds on the recent finding that the small molecule bexarotene delays primary nucleation in Aβ42 aggregation (22) (Fig. 1A). Bec...

Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-β peptide (Aβ42) are key pathogenic agents in Alzheimer’s disease (AD). To investigate the relationship between Aβ42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aβ42-induced toxicity. In addition to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small molecule to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.protein homeostasis | protein misfolding | protein aggregates | amyloid | extracellular chaperones