Comparative Study of Inhibition at Multiple Stages of Amyloid-β Self-Assembly Provides Mechanistic Insight

Abstract

The "amyloid cascade hypothesis," linking self-assembly of the amyloid-␤ protein (A␤) to the pathogenesis of Alzheimer's disease, has led to the emergence of inhibition of A␤ self-assembly as a prime therapeutic strategy for this currently unpreventable and devastating disease. The complexity of A␤ selfassembly, which involves multiple reaction intermediates related by nonlinear and interconnected nucleation and growth mechanisms, provides multiple points for inhibitor intervention. Although a number of small-molecule inhibitors of A␤ selfassembly have been identified, little insight has been garnered concerning the point at which these inhibitors intervene within the A␤ assembly process. In the current study, a julolidine derivative is identified as an inhibitor of A␤ self-assembly. To gain insight into the mechanistic action of this inhibitor, the inhibition of fibril formation from monomeric protein is assessed quantitatively and compared with the inhibition of two distinct mechanisms of growth for soluble A␤ aggregation intermediates. This compound is observed to significantly inhibit soluble aggregate growth by lateral association while having little effect on soluble aggregate elongation via monomer addition. In addition, inhibition of soluble A␤ aggregate association exhibits an IC 50 with a somewhat lower stoichiometric ratio than the IC 50 determined for inhibition of fibril formation from monomeric A␤. This quantitative comparison of inhibition within multiple A␤ self-assembly assays suggests that this compound binds the lateral surface of on-pathway intermediates exhibiting a range of sizes to prevent their association with other aggregates, which is required for further assembly into mature fibrils.Alzheimer's disease (AD) is currently the most common type of dementia, affecting an estimated 5.2 million Americans (Alzheimer's Association, 2008). As the life expectancy in the United States and other postindustrialized nations increases, AD presents a burgeoning epidemic. AD initially affects short-term memory and progresses to include pervasive cognitive and emotional dysfunction. These manifested symptoms are hypothesized to result from a cascade of events initiated by the self-assembly of monomeric amyloid-␤ protein (A␤), leading first to the formation of soluble aggregates and later progressing into larger insoluble fibrils, which ultimately deposit in the extracellular space of the brain parenchyma. This "amyloid cascade hypothesis" is supported by experimental evidence (Walsh and Selkoe, 2007), including genetic correlations, transgenic animal models, and cell culture studies, and has established the inhibition of A␤ self-assembly as a prime therapeutic strategy in the fight against AD.Several small molecules that inhibit the in vitro formation of amyloid fibrils from monomeric A␤ have been identified (Findeis, 2000;Hamaguchi et al., 2006;LeVine, 2007). Studies using quantitative measures of inhibition assembled from light scattering measurements, thioflavin T (ThT) fluorescence, or immun...

Biochemistry of Amyloid -Protein and Amyloid Deposits in Alzheimer Disease

“…The structure of the carboxyl terminus may involve novel metastable conformations (b-hairpin at 35 -37), but these remain to be confirmed by crystallographic methods . Synthetic oligomers are also being used in attempts to discover small molecules which are able to target these specific assemblies (Davis and Berkowitz 2009a;Davis et al 2009;Feng et al 2009;Liu et al 2009a;Nerelius et al 2009;Pitt et al 2009;Riviere et al 2009;Smith et al 2009;Sun et al 2009;Yamin et al 2009;Hawkes et al 2010;Ladiwala et al 2010). …”

Inhibition of amyloid-β aggregation by coumarin analogs can be manipulated by functionalization of the aromatic center

“…These results demonstrate the strong potential of functionalized coumarin structures to attenuate Ab aggregation. Coumarin derivatives have been shown to also intervene with other processes associated with symptomatic treatment and progression of AD, including slowing neurotransmitter breakdown via inhibition of AChE, 19,29,30 reducing the release of Ab from APP via inhibition of b-secretase activity 30,31 or modulation of c-secretase activity, 32 and serving as a free radical scavenger to protect cells against Ab-induced toxicity. 33 The additional action of coumarin analogs as Ab aggregation inhibitors further establishes these structures as potential multi-target AD therapeutics.…”

“…Other aromatic small molecules, however, do not impact this measurement technique. 29 To ensure that the coumarin analogs to be assessed as potential Ab aggregation inhibitors (Scheme 1) fell within the latter category, the ability of each molecule to interfere with aggregate detection via thioflavin T fluorescence was evaluated. These measurements were acquired when coumarin based compounds were present at a fivefold molar excess over Ab, the stoichiometric ratio present in samples used to monitor Ab aggregation reactions.…”

“…29 Briefly, 20 lM Ab 1-40 monomer was combined with 0 lM (control) or 0.2-100 lM coumarin analog in the presence of 40 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2.5% (v/v) DMSO. Initial experimentation utilized a fivefold molar excess of coumarin analog over Ab 1-40 monomer to give a robust effect for both weak and strong inhibitors and facilitate comparisons of inhibitory capabilities among coumarin analogs.…”

Strategies for the Inhibition of Protein Aggregation in Human Diseases

“…Nevertheless, small inhibitors have been reported to disrupt soluble oligomers and to interfere with formed larger structures. More specifically, small compounds have been tested as inhibitors of a-syn aggregation (e.g., polyphenols, phenothiazines, porphyrins, polyene macrolides, curcumins, and Congo red and its derivatives [145,146] ), polyQ aggregation for HD treatment (e.g., benzothiazole [147] ), tau protein (e.g., anthracyclines such as daunorubicin, aminothienopyridazines, phenothiazines such as methylene blue, Nphenylamines, phenylthiazolyl-hydrazides, polyphenols, porphyrins, quinazolines, rhodanines, cyanine dyes, [148,149] and compound N774 [150,151] ), prion proteins (e.g., porphyrins and phthalocyanines, [152] lysosomotropic antimalarial compounds [153] ), and Ab peptide [154] (e.g., curcumin derivatives, [155] N-phenylanthranilic acid analogues, [156] quinacrine conjugates, [157] bis-styrylpyridine and bis-styrylbenzene derivatives, [158] tricyclic pyrones, [159] julolidine aldehyde [160] ). Detailed discussion of single compounds is beyond the aim of this review, but it must be emphasized that compounds claiming anti-aggregating action have in most cases been evaluated for their overall effect on the aggregation process without any detailed investigation of the specific pre-fibrillar species targeted by the inhibitor.…”