Soluble and toxic oligomers of amyloid beta (A beta) protein have been identified as the true neurotoxic species involved in Alzheimer's disease and considering them as targets to inhibit A beta aggregation might have a therapeutic value. We previously set up a CE method that enables the separation and quantification of transient oligomers of A beta protein-containing 42 amino acids (A beta(1-42)) along the pathway leading to fibrils and we now demonstrate how this method can be successfully applied to examine the in vitro inhibitory effects of small molecules on A beta oligomerization. To this end, we investigated mitoxantrone and pixantrone, two well-known anticancer drugs, as well as suramin and a suramin-like compound. By using CE, it is here shown how mitoxantrone and pixantrone either reduce or block A beta(1-42) oligomerization, while Thioflavin T spectrofluorimetric assay and transmission electron microscopy demonstrate how these two compounds also display antifibrillogenic activity. Interestingly, in vitro cell viability experiments indicated that pixantrone significantly reduces A beta(1-42) neurotoxicity.

Background: Identifying physiologically relevant binding partners of amyloid-β (Aβ) that modulate in vivo fibril formation may yield new insights into Alzheimer’s disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types.Objective:We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to Aβ42 and can modulate Aβ fibril formation.Methods:Specific interactions between LL-37 and Aβ (with Aβ in different aggregation states, assessed by capillary electrophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, Aβ42 alone, and LL-37/Aβ complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y).Results:SPRi shows binding specificity between LL-37 and Aβ, while TEM shows that LL-37 inhibits Aβ42 fibril formation, particularly Aβ’s ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents Aβ42 from adopting its typical β-type secondary structure. Microglia-mediated toxicities of LL-37 and Aβ42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides.Conclusion:Based on this body of evidence, we propose that LL-37 and Aβ42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

Converging analytical, biological, and in silico data explained the mechanism of action of 2a on Aβ1-42 oligomers formation and against Aβ-preformed fibrils. This evidence, combined with toxicity data, will orient the future design of safer analogues.

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In order to identify novel Alzheimer's modifying pharmacological tools, we developed bis-tacrines bearing a peptide moiety for specific interference with surface sites of human acetylcholinesterase (hAChE) binding amyloid-beta (Aβ). Accordingly, compounds 2a−c proved to be inhibitors of hAChE catalytic and noncatalytic functions, binding the catalytic and peripheral sites, interfering with Aβ aggregation and with the Aβ self-oligomerization process (2a). Compounds 2a−c in complex with TcAChE span the gorge with the bis-tacrine system, and the peptide moieties bulge outside the gorge in proximity of the peripheral site. These moieties are likely responsible for the observed reduction of hAChE-induced Aβ aggregation since they physically hamper Aβ binding to the enzyme surface. Moreover, 2a was able to significantly interfere with Aβ self-oligomerization, while 2b,c showed improved inhibition of hAChE-induced Aβ aggregation.

Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.