Real‐Time BODIPY‐Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1–42 Peptide

Abstract

Misfolding and aggregation of amyloid b1-42 peptide( A b1-42) play ac entral role in the pathogenesis of Alzheimer's disease (AD). Ta rgeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents ap romising therapeutic strategy to reduce the toxicity associated with Ab1-42. Currently,t he thioflavin T( ThT) assay is the only established spectrofluorometric methodt os creen aggregation inhibitors. The successo ft he ThT assay is that it can detect Ab1-42 aggregates with high b-sheet content,s uch as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately,b yu sing the ThT assay,t he detection of inhibitors of early soluble oligomers that present al ow b-sheet character is challenging. Herein, an ew,f acile, and robust boron-dipyrromethene( BODIPY) real-time assay suitable for 96-well plate format, which allows screening of compounds as selectivei nhibitors of the formation of Ab1-42 oligomers, is reported.T hese inhibitors decrease the cellular toxicityo fA b1-42, although they fail in the ThT assay.T he findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is ac onvenient methodt o screen and discover new candidate compoundst hat slow down or stop the pathological earlyo ligomerization process and are active in the cellular assay.T herefore, it is as uitable complementary screening method of the current ThT assay.Alzheimer's disease (AD) is ad evastating neurodegenerative disease that leads to progressive cognitive decline, functional impairment, and loss of independence. [1] The number of people worldwide suffering from AD is expected to reach 75 million by 2030, [2] butn oc ausative treatment exists for AD.The search for small molecules that inhibitt he aggregation of the 42-residue amyloid b protein fragment (Ab1-42; Figure 1A) is still ongoing to find at herapy for AD. Ab1-42 spontaneously self-associates into soluble oligomers and insolublea ggregates, such as protofibrilsa nd fibrils with high b-sheetc ontent.I th as been recognized that small and soluble Ab1-42 oligomers are particularly cytotoxic. [3][4][5][6][7] Thus, therapeutics trategies that intervene in the early oligomerization process, rather than in the later fibril formation step, have recently attracted attention. [8] However,d espite its therapeutic significance,t he screening of potentiali nhibitors of the early oligomerization process is still challenging. The thioflavin T( ThT) fluorescencea ssayi su sed routinelytodetermine the influence of compounds on amyloid aggregation kinetics. [9,10] However,T hT only exhibits as ubstantial fluorescencei ncreaseu pon binding to b-sheet-rich amyloid protofilaments and fibrils, but has low sensitivity to soluble early-stage oligomers. [11][12][13][14] Molecules that do not show any apparenti nhibition of amyloid aggregation,a ccording to the ThT assay,a re usually not considered for any subsequentt est...

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

“…These probes interact with high sensitivity with Aβ-peptide fibrils, showing an increment in their fluorescence emission, and some of them, such as CRANAD probes, suffer a blue-shift of the maximum of the emission upon binding. Recently, a Triazole-BODIPY-based probe was shown to detect, in a time-dependent manner, the first stages of Aβ-peptide oligomerization before fibril formation [ 104 ]. Additionally, the same BODIPY derivative identified the presence of oligomers of the toxic p31–43 peptide related to celiac disease [ 49 ].…”

“…CD is widely used to evaluate the formation of amyloid fibrils at different experimental conditions such as time, changes in pH, and presence of different molecules, among others. The Aβ-peptide [ 104 ], the Ac-Phe-Phe-Cys-NH 2 (Ac-FFC-NH 2 ) amyloid peptide model [ 61 ], and proteins such as alpha-synuclein [ 152 ] and β2-microglobulin [ 153 ] have been shown to change their conformation from random coil to β-sheet structure over time during the aggregation process, which is compatible with the formation of fibrils. Other systems, such as the rationally designed DN1 peptide, have been shown to self-aggregate into fibrils in a concentration-dependent manner [ 154 ].…”

“…In 0 (black), 0.5 (red), 1 (violet) 3 (blue) and 7 h (green). This image was extracted and re-created from Tonali et al [ 104 ]. ( B ) Spectra showing the structural transformation of the 33-mer gliadin peptide at 613 µM depending the temperature, at −5 °C (red) corresponds to PPII structure and at 37 °C (black) to the β-sheet signature.…”

The Positive Side of the Alzheimer’s Disease Amyloid Cross-Interactions: The Case of the Aβ 1-42 Peptide with Tau, TTR, CysC, and ApoA1

“…Aggregates of toxic Aβ in the form of soluble Aβ oligomers, intraneuronal Aβ, and amyloid plaques injure the synapses and ultimately cause neurodegeneration and dementia [6,7]. One of the strategies adopted to stop or reverse the progression of the disease is to modulate or inhibit the aggregation process of Aβ, by various mechanisms: stabilization of its native state, destabilization of its incorrectly folded state [8], bypass of the on-pathway oligomer formation, inhibition of the fibril elongation and disaggregation of the already formed amyloid aggregates [9][10][11][12].…”

“…Instead, the second class is characterized by peptides which are identified from libraries, that may or may not show sequence similarly to wild type, and these are termed as randomly generated peptides. Other approaches have been exploited in the field of peptidomimetics, such as synthetic peptide derivates-β sheet breakers and β peptide hairpins [9][10][11][12].…”