The mapping of active sites of amyloid‐targeting compounds through the utilization of immobilized fragments of amyloid hexamers

Abstract Background As amyloid‐β (Aβ) aggregates are considered as the biomarkers and key factors in the pathology of Alzheimer’s disease, there has been extensive investigation into Aβ‐targeting compounds for the development of diagnostics and drug discovery related to the disorder. However, the polymorphic and heterogenous nature of Aβ aggregates impedes the structural understanding of their structure. Consequently it is a major challenge to develop new diagnostic and therapeutic development of AD and to study the mechanism of Aβ‐targeting compounds. Method Utilization of solid‐phase peptide synthesis, 37 Aβ hexameric Aβ fragments were synthesized. These fragments were immobilized on the 96‐well microplane to develop a mapping plate for investigating the active sites of Aβ‐targeting compounds. To confirm the mapping potential of our plate, previously reported fluorescent Aβ‐imaging agents (CRANAD‐28, bis‐ANS), Aβ aggregation inhibitors (curcumin, scyllo‐inositol), and Aβ aggregate dissociators (necrostatin‐1, sunitinib) were treated. Result Using the mapping plate, we confirmed that CRANAD‐28 interacted with three domains of Aβ HHQKLVFF (Aβ 13‐20 ), IIGLMV (Aβ 31‐36 ), and GGVVIA (Aβ 37‐42 ), and bis‐ANS were bound to the predominant parts of Aβ sequences except N‐terminal regions. We further determined that curcumin inhibited three distinct fragmental regions of Aβ (Aβ 14‐21 , Aβ 29‐35 , and Aβ 37‐42 ) and scyllo‐inositol targeted VHHQKLVFF (Aβ 12‐20 ), VGSNKGAIIGL (Aβ 24‐39 ), and GGVVIA (Aβ 37‐42 ) sites. Lastly, we confirmed that both necrostatin‐1 and sunitinib strongly dissociated GLMVGGVVIA (Aβ 33‐42 ), the C‐terminal hydrophobic residue. Conclusion This mapping plate can be employed to understand the active sites of Aβ‐targeting compounds, even in the lack of a comprehensive structural understanding of Aβ aggregates. Moreover, the insights gained from identifying targeting sites using this mapping plate can contribute to the rational approach in the development of diagnostic and therapeutic strategies for Alzheimer’s disease (AD).