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Pathophysiological shifts in the cerebral levels of sphingolipids in Alzheimer's disease (AD) patients suggest a link between sphingolipid metabolism and the disease pathology. Sphingosine ( SP ), a structural backbone of sphingolipids, is an amphiphilic molecule that is able to undergo aggregation into micelles and micellar aggregates. Considering its structural properties and cellular localization, we hypothesized that SP potentially interacts with amyloid-β (Aβ) and metal ions that are found as pathological components in AD-affected brains, with manifesting its reactivity towards metal-free Aβ and metal-bound Aβ (metal-Aβ). Herein, we report, for the first time , that SP is capable of interacting with both Aβ and metal ions and consequently affects the aggregation of metal-free Aβ and metal-Aβ. Moreover, incubation of SP with Aβ in the absence and presence of metal ions results in the aggravation of toxicity induced by metal-free Aβ and metal-Aβ in living cells. As the simplest acyl derivatives of SP , N -acetylsphingosine and 3- O -acetylsphingosine also influence metal-free Aβ and metal-Aβ aggregation to different degrees, compared to SP . Such slight structural modifications of SP neutralize its ability to exacerbate the cytotoxicity triggered by metal-free Aβ and metal-Aβ. Notably, the reactivity of SP and the acetylsphingosines towards metal-free Aβ and metal-Aβ is determined to be dependent on their formation of micelles and micellar aggregates. Our overall studies demonstrate that SP and its derivatives could directly interact with pathological factors in AD and modify their pathogenic properties at concentrations below and above critical aggregation concentrations., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Aggregation of amyloidogenic peptides could cause the onset and progression of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These amyloidogenic peptides can coordinate to metal ions, including Zn(ii), which can subsequently affect the peptides' aggregation and toxicity, leading to neurodegeneration. Unfortunately, the detection of metal-amyloidogenic peptide complexation has been very challenging. Herein, we report the development and utilization of a probe ( A-1 ) capable of monitoring metal-amyloid-β (Aβ) complexation based on Förster resonance energy transfer (FRET). Our probe, A-1 , is composed of Aβ 1-21 grafted with a pair of FRET donor and acceptor capable of providing a FRET signal upon Zn(ii) binding even at nanomolar concentrations. The FRET intensity of A-1 increases upon Zn(ii) binding and decreases when Zn(ii)-bound A-1 aggregates. Moreover, as the FRET intensity of Zn(ii)-added A-1 is drastically changed when their interaction is disrupted, A-1 can be used for screening a chemical library to determine effective inhibitors against metal-Aβ interaction. Eight natural products (out of 145 compounds; >80% inhibition) were identified as such inhibitors in vitro , and six of them could reduce Zn(ii)-Aβ-induced toxicity in living cells, suggesting structural moieties useful for inhibitor design. Overall, we demonstrate the design of a FRET-based probe for investigating metal-amyloidogenic peptide complexation as well as the feasibility of screening inhibitors against metal-bound amyloidogenic peptides, providing effective and efficient methods for understanding their pathology and finding therapeutic candidates against neurodegenerative disorders.

Metal ion homeostasis in conjunction with amyloid-β (Aβ) aggregation in the brain has been implicated in Alzheimer's disease (AD) pathogenesis. To uncover the interplay between metal ions and Aβ peptides, synthetic, multifunctional small molecules have been employed to modulate Aβ aggregation in vitro . Naturally occurring flavonoids have emerged as a valuable class of compounds for this purpose due to their ability to modulate both metal-free and metal-induced Aβ aggregation. Although, flavonoids have shown anti-amyloidogenic effects, the structural moieties of flavonoids responsible for such reactivity have not been fully identified. In order to understand the structure-interaction-reactivity relationship within the flavonoid family for metal-free and metal-associated Aβ, we designed, synthesized, and characterized a set of isoflavone derivatives, aminoisoflavones ( 1 - 4 ), that displayed reactivity ( i.e. , modulation of Aβ aggregation) in vitro . NMR studies revealed a potential binding site for aminoisoflavones between the N-terminal loop and central helix on prefibrillar Aβ different from the non-specific binding observed for other flavonoids. The absence or presence of the catechol group differentiated the binding affinities and enthalpy/entropy balance between aminoisoflavones and Aβ. Furthermore, having a catechol group influenced the binding mode with fibrillar Aβ. Inclusion of additional substituents moderately tuned the impact of aminoisoflavones on Aβ aggregation. Overall, through these studies, we obtained valuable insights on the requirements for parity among metal chelation, intermolecular interactions, and substituent variation for Aβ interaction.