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Amphiphilic Distyrylbenzene Derivatives as Potential Therapeutic and Imaging Agents for Soluble and Insoluble Amyloid β Aggregates in Alzheimer's Disease.

ABSTRACT: Alzheimer's Disease (AD) is the most common neurodegenerative disease, and efficient therapeutic and early diagnostic agents for AD are still lacking. Herein, we report the development of a novel amphiphilic compound, LS-4, generated by linking a hydrophobic amyloid-binding distyrylbenzene fragment with a hydrophilic triazamacrocycle, which dramatically increases the binding affinity toward various amyloid β (Aβ) peptide aggregates, especially for soluble Aβ oligomers. Moreover, upon the administration of LS-4 to 5xFAD mice, fluorescence imaging of LS-4-treated brain sections reveals that LS-4 can penetrate the blood-brain barrier and bind to the Aβ oligomers in vivo. In addition, the treatment of 5xFAD mice with LS-4 reduces the amount of both amyloid plaques and associated phosphorylated tau aggregates vs the vehicle-treated 5xFAD mice, while microglia activation is also reduced. Molecular dynamics simulations corroborate the observation that introducing a hydrophilic moiety into the molecular structure of LS-4 can enhance the electrostatic interactions with the polar residues of the Aβ species. Finally, exploiting the Cu²⁺-chelating property of the triazamacrocycle, we performed a series of imaging and biodistribution studies that show the ⁶⁴Cu-LS-4 complex binds to the amyloid plaques and can accumulate to a significantly larger extent in the 5xFAD mouse brains vs the wild-type controls. Overall, these results illustrate that the novel strategy, to employ an amphiphilic molecule containing a hydrophilic moiety attached to a hydrophobic amyloid-binding fragment, can increase the binding affinity for both soluble and insoluble Aβ aggregates and can thus be used to detect and regulate various Aβ species in AD.

SUBMITTER: Sun L

PROVIDER: S-EPMC8762579 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The specific amyloid-beta (Aβ) species or other amyloid-precursor protein cleavage products that are most directly related to human neurodegeneration and clinical dementia of the Alzheimer's type have not yet been directly identified. Without a clear understanding of the most relevant species, it is difficult to determine whether therapeutic candidates successfully engaged the correct target(s). Here, we review some of the controversies regarding soluble Aβ aggregates (also termed oligomers, dimers, trimers, Aβ*56, amylospheroids, etc.) and propose experiments designed to move forward towards new therapeutic approaches. Specifically, we review the increasing evidence for the relevance of non-canonical forms of Aβ, the much more potent toxicity attributable to native species than to synthetic Aβ, and the evidence implicating the ratio of soluble Aβ aggregates to plaques in differentiating demented patients from non-demented high Aβ plaque pathology controls. To move forward, we propose four related directions. 1) Narrowing the focus to species derived from human Alzheimer's disease (AD) brain tissue, as opposed to synthetic Aβ, cell culture-derived species, or species primarily present in animal models. 2) Careful study of differences between patients with dementia of the Alzheimer's type vs. non-demented controls with high Aβ plaque pathology. This will involve testing the hypothesis that, under some circumstances, plaques may buffer soluble toxic species, but later release them into the surrounding milieu. 3) Investigations of other protein constituents of soluble Aβ aggregates in addition to Aβ itself. Our initial data based on chemical cleavage experiments indicate that other proteins do appear to be part of the human brain soluble Aβ aggregates. 4) Multimodal experimental assessments of toxicity, including longer term effects on synapse loss, related deleterious cellular responses, and degeneration in human-derived neuron-like cells. Overall, the goal is to identify specific Aβ species, other amyloid precursor protein cleavage products, or other key proteins in aggregates present in human AD brains, less abundant in non-demented high pathology control brains, and robustly toxic in a wide variety of relevant assays. These species themselves, the enzymatic or cellular processes involved in their production, and their routes of clearance would be highly relevant therapeutic targets for dementia of the Alzheimer's type.

Project description:BackgroundAmyloid-β (Aβ) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer's disease (AD). A number of monoclonal antibodies have entered clinical trials for AD. Some of them have failed due to the lack of efficacy or side-effects, two antibodies are currently in phase 3, and one has been approved by FDA. The soluble intermediate aggregated species of Aβ, termed oligomers and protofibrils, are believed to be key pathogenic forms, responsible for synaptic and neuronal degeneration in AD. Therefore, antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest.MethodsWe designed and recombinantly produced a hexavalent antibody based on mAb158, an Aβ protofibril-selective antibody. The humanized version of mAb158, lecanemab (BAN2401), is currently in phase 3 clinical trials for the treatment of AD. The new designs involved recombinantly fusing single-chain fragment variables to the N-terminal ends of mAb158 antibody. Real-time interaction analysis with LigandTracer and surface plasmon resonance were used to evaluate the kinetic binding properties of the generated antibodies to Aβ protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aβ aggregates of different sizes. Finally, the ability of the antibodies to protect cells from Aβ-induced effects was evaluated by MTT assay.ResultsUsing real-time interaction analysis with LigandTracer, the hexavalent design promoted a 40-times enhanced binding with avidity to protofibrils, and most of the added binding strength was attributed to the reduced rate of dissociation. Furthermore, ELISA experiments demonstrated that the hexavalent design also had strong binding to small oligomers, while retaining weak and intermediate binding to monomers and insoluble fibrils. The hexavalent antibody also reduced cell death induced by a mixture of soluble Aβ aggregates.ConclusionWe provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aβ aggregates. This approach should be general and work for any aggregated protein or repetitive target.

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Amphiphilic Distyrylbenzene Derivatives as Potential Therapeutic and Imaging Agents for Soluble and Insoluble Amyloid β Aggregates in Alzheimer's Disease.

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