Benzo[d]imidazole-pyrrolo[1,2-a]pyrazine Hybrids Ameliorate Amyloid Aggregates in the Brain of Alzheimer Transgenic Mice.

Abnormal assembly of amyloid ß (Aß) in the brain is implicated in Alzheimer's disease (AD) and is associated with cognitive impairments. Since Aß accumulation occurs in advance of the onset of clinical symptoms, identifying preventable drug candidates regulating Aß accumulation is regarded as a promising approach in AD therapeutic. Herein, we synthesized eight Yonsei Institute of pharmaceutical sciences Alzheimer's Drug (YIAD) compounds based on 5-benzyl-6-phenylbenzo[4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazine structures. Subsequently, YIAD-0203 and YIAD-0205 were selected as effective candidates via thioflavin T assays and gel electrophoresis. The potential therapeutic effect of YIAD-0203 and YIAD-0205 on Aß aggregates was investigated through an AD transgenic mouse model with five familial AD mutations (5XFAD) by oral gavage. Significant amounts of Aß plaque and oligomer reduction were observed in the hippocampus region of both 4.3-month-old (early stage of AD) and 6.0-month-old (mid stage of AD) YIAD-0205-treated 5XFAD mice brains when compared to the nontreated brains. The ability of YIAD-0205 to ameliorate Aß aggregates in the early and mid stages of AD progression supports the notion that YIAD-0205 could be utilized as a reliable scaffold for the development of preventive AD drug candidates.

4-Acyl-3,4-dihydropyrrolo[1,2-a]pyrazine Derivative Rescued the Hippocampal-Dependent Cognitive Decline of 5XFAD Transgenic Mice by Dissociating Soluble and Insoluble Aß Aggregates.

Cerebral amyloid-ß (Aß) deposition is a representative hallmark of Alzheimer's disease (AD). Development of Aß-clearing small molecules could be an advantageous therapeutic strategy for Aß clearance considering the advantages in terms of side effects, cost-effectiveness, stability, and oral bioavailability. Here, we report an Aß-dissociating small molecule, YIAD-0121, a derivative of 4-acyl-3,4-dihydropyrrolo[1,2-a]pyrazine. Through a series of anti-Aß screening assays, YIAD-0121 was identified to inhibit Aß aggregation and dissociate preformed Aß fibrils in vitro. Furthermore, the administration of YIAD-0121 in 5XFAD transgenic AD mice inhibited the increase of cerebral Aß aggregation and progression of hippocampus-dependent cognitive decline, with ameliorated neuroinflammation.

Solid-phase synthesis and pathological evaluation of pyroglutamate amyloid-ß3-42 peptide.

Pyroglutamate amyloid-ß3-42 (AßpE3-42) is an N-terminally truncated and pyroglutamate-modified Aß peptide retaining highly hydrophobic, amyloidogenic, and neurotoxic properties. In Alzheimer's disease (AD) patients, AßpE3-42 peptides accumulate into oligomers and induce cellular toxicity and synaptic dysfunction. AßpE3-42 aggregates further seed the formation of amyloid plaques, which are the pathological hallmarks of AD. Given that AßpE3-42 peptides play critical roles in the development of neurodegeneration, a reliable and reproducible synthetic access to these peptides may support pathological and medicinal studies of AD. Here, we synthesized AßpE3-42 peptides through the microwave-assisted solid-phase peptide synthesis (SPPS). Utilizing thioflavin T fluorescence assay and dot blotting analysis with anti-amyloid oligomer antibody, the amyloidogenic activity of synthesized AßpE3-42 peptides was confirmed. We further observed the cytotoxicity of AßpE3-42 aggregates in cell viability test. To examine the cognitive deficits induced by synthetic AßpE3-42 peptides, AßpE3-42 oligomers were intracerebroventricularly injected into imprinting control region mice and Y-maze and Morris water maze tests were performed. We found that AßpE3-42 aggregates altered the expression level of postsynaptic density protein 95 in cortical lysates. Collectively, we produced AßpE3-42 peptides in the microwave-assisted SPPS and evaluated the amyloidogenic and pathological function of the synthesized peptides.

Immobilized Amyloid Hexamer Fragments to Map Active Sites of Amyloid-Targeting Chemicals.

As amyloid-ß (Aß) peptide is considered a biomarker and pathological culprit of Alzheimer's disease, Aß-targeting compounds have been investigated for diagnostics development and drug discovery of the disorder. Unlike amyloid plaque targeting agents, such as clinically available amyloid radiotracers intercalating into the ß-sheet structures of the aggregates, monomer and oligomer targeting chemicals are difficult to develop, as the transient and polymorphic nature of these peptides impedes their structural understanding. Here, we report a mapping approach to explore targeting residues of Aß-imaging probes and Aß-regulating drug candidates by utilizing a set of fragmented Aß hexamers immobilized on a 96-well microplate in combination with fluorescent full-length Aß for on-plate aggregation. To evaluate the mapping potential of the peptide plate, we tested previously reported fluorescent imaging agents (CRANAD-28, bis-ANS), aggregation inhibitors (curcumin, scyllo-inositol), and aggregate dissociators (necrostatin-1, sunitinib) targeting Aß. Our approach enabled mechanistic understanding of compounds targeting nonfibrillar Aß on an interacting sequence level.

A Therapeutic Nanovaccine that Generates Anti-Amyloid Antibodies and Amyloid-specific Regulatory T Cells for Alzheimer's Disease.

Alzheimer's disease (AD), the most common cause of dementia, is a complex condition characterized by multiple pathophysiological mechanisms including amyloid-ß (Aß) plaque accumulation and neuroinflammation in the brain. The current immunotherapy approaches, such as anti-Aß monoclonal antibody (mAb) therapy, Aß vaccines, and adoptive regulatory T (Treg) cell transfer, target a single pathophysiological mechanism, which may lead to unsatisfactory therapeutic efficacy. Furthermore, Aß vaccines often induce T helper 1 (Th1) cell-mediated inflammatory responses. Here, a nanovaccine composed of lipid nanoparticles loaded with Aß peptides and rapamycin is developed, which targets multiple pathophysiological mechanisms, exhibits the combined effects of anti-Aß antibody therapy and adoptive Aß-specific Treg cell transfer, and can overcome the limitations of current immunotherapy approaches for AD. The Nanovaccine effectively delivers rapamycin and Aß peptides to dendritic cells, produces both anti-Aß antibodies and Aß-specific Treg cells, removes Aß plaques in the brain, alleviates neuroinflammation, prevents Th1 cell-mediated excessive immune responses, and inhibits cognitive impairment in mice. The nanovaccine shows higher efficacy in cognitive recovery than an Aß vaccine. Unlike anti-Aß mAb therapy and adoptive Treg cell transfer, both of which require complicated and costly manufacturing processes, the nanovaccine is easy-to-prepare and cost-effective. The nanovaccines can represent a novel treatment option for AD.

Amyloid Against Amyloid: Dimeric Amyloid Fragment Ameliorates Cognitive Impairments by Direct Clearance of Oligomers and Plaques.

Amyloid-ß (Aß) in the form of neurotoxic aggregates is regarded as the main pathological initiator and key therapeutic target of Alzheimer's disease. However, anti-Aß drug development has been impeded by the lack of a target needed for structure-based drug design and low permeability of the blood-brain barrier (BBB). An attractive therapeutic strategy is the development of amyloid-based anti-Aß peptidomimetics that exploit the self-assembling nature of Aß and penetrate the BBB. Herein, we designed a dimeric peptide drug candidate based on the N-terminal fragment of Aß, DAB, found to cross the BBB and solubilize Aß oligomers and fibrils. Administration of DAB reduced amyloid burden in 5XFAD mice, and downregulated neuroinflammation and prevented memory impairment in the Y-maze test. Peptide mapping assays and molecular docking studies were utilized to elucidate DAB-Aß interaction. To further understand the active regions of DAB, we assessed the dissociative activity of DAB with sequence modifications.

Aryloxypropanolamine targets amyloid aggregates and reverses Alzheimer-like phenotypes in Alzheimer mouse models.

BACKGROUND: Aggregated amyloid-ß (Aß) is considered a pathogenic initiator of Alzheimer's disease (AD), in strong association with tau hyperphosphorylation, neuroinflammation, synaptic dysfunction, and cognitive decline. As the removal of amyloid burden from AD patient brains by antibodies has shown therapeutic potential, the development of small molecule drugs inducing chemical dissociation and clearance of Aß is compelling as a therapeutic strategy. In this study, we synthesized and screened aryloxypropanolamine derivatives and identified 1-(3-(2,4-di-tert-pentylphenoxy)-2-hydroxypropyl)pyrrolidin-1-ium chloride, YIAD002, as a strong dissociator of Aß aggregates. METHODS: The dissociative activity of aryloxypropanolamine derivatives against Aß aggregates were evaluated through in vitro assays. Immunohistochemical staining, immunoblot assays, and the Morris water maze were used to assess the anti-Alzheimer potential in YIAD002-treated 5XFAD and transgenic APP/PS1 mice. Target-ligand interaction mechanism was characterized via a combination of peptide mapping, fluorescence dissociation assays, and constrained docking simulations. RESULTS: Among 11 aryloxypropanolamine derivatives, YIAD002 exerted strongest dissociative activity against ß-sheet-rich Aß aggregates. Upon oral administration, YIAD002 substantially reduced amyloid burden and accordingly, improved cognitive performance in the Morris water maze and attenuated major pathological hallmarks of AD including tauopathy, neuroinflammation, and synaptic protein loss. Mechanism studies suggest that YIAD002 interferes with intermolecular ß-sheet fibrillation by directly interacting with KLVFFA and IGLMVG domains of Aß. In addition, YIAD002 was found to possess dissociative activity against aggregates of pyroglutamate-modified Aß and tau. CONCLUSIONS: Collectively, our results evince the potential of chemical-driven dissociation of Aß aggregates by aryloxypropanolamines as a therapeutic modality of the amyloid clearance approach.

Quinacrine directly dissociates amyloid plaques in the brain of 5XFAD transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia characterized by the abnormal accumulation of amyloid-ß (Aß) in the brain. Aß misfolding is associated with neuroinflammation and synaptic dysfunction, leading to learning and memory deficits. Therefore, Aß production and aggregation have been one of the most popular drug targets for AD. Failures of drug candidates regulating the aforementioned Aß cascade stimulated development of immunotherapy agents for clearance of accumulated Aß in the brain. Here, we report that quinacrine, a blood-brain barrier penetrating antimalarial chemical drug, dissociates Aß plaques in the brain of AD transgenic mice. When co-incubated with pre-formed Aß fibrils, quinacrine decreased thioflavin T-positive ß-sheets in vitro, on top of its inhibitory function on the fibril formation. We confirmed that quinacrine induced dissociation of high-molecular-weight Aß aggregates into low-molecular-weight species by dot blots in association with size cut-off filtrations. Quinacrine was then administered to adult 5XFAD transgenic mice via weekly intravenous injections for 6 weeks, and we found a significant reduction of Aß plaques and astrocytosis in their cortex and hippocampus. In western blots of quinacrine-administered mouse brains, amelioration of AD-related biomarkers, glial fibrillary acidic protein, postsynaptic protein 95, phosphorylated cAMP response element-binding protein, phosphorylated c-Jun N-terminal kinase were observed. Lastly, quinacrine-stimulated dissociation of misfolded aggregates induced recovery of synaptic function associated with Aß in excitatory post-synaptic current recordings of primary rat cortical neurons treated with Aß aggregates and quinacrine. Collectively, quinacrine can directly dissociate Aß fibrils and alleviate decreased synaptic functions.

Azetidine-Bearing Non-Ribosomal Peptides, Bonnevillamides D and E, Isolated from a Carrion Beetle-Associated Actinomycete.

Two new nonribosomal peptides, bonnevillamides D and E (1 and 2), have been discovered in Streptomyces sp. UTZ13 isolated from the carrion beetle, Nicrophorus concolor. Combinational analysis of the UV, MS, and NMR spectroscopic data revealed that their planar structures were comprised of dichlorinated linear peptides containing nonproteinogenic amino acid residues, such as 4-methylazetidinecarboxylic acid and 4-O-acetyl-5-methylproline. The configurations of bonnevillamides D and E (1 and 2) were determined based on ROESY correlations, the advanced Marfey's method, phenylglycine methyl ester derivatization, molecular modeling, and circular dichroism spectroscopy. The nonribosomal peptide synthetase biosynthetic pathway of bonnevillamides D and E has been proposed using bioinformatic analysis of the whole-genome sequence data of Streptomyces sp. UTZ13. Their biological activity toward the aggregation of amyloid-ß, which is one of the key pathogenic proteins in Alzheimer's disease, was evaluated using a thioflavin T assay and gel electrophoresis. Bonnevillamides D and E reversed the fibril formation by inducing the monomerization of amyloid-ß aggregates.