RESUMO
Aberrant aggregates of amyloid-ß (Aß) and tau protein (tau), called amyloid, are related to the etiology of Alzheimer disease (AD). Reducing amyloid levels in AD patients is a potentially effective approach to the treatment of AD. The selective degradation of amyloids via small molecule-catalyzed photooxygenation in vivo is a leading approach; however, moderate catalyst activity and the side effects of scalp injury are problematic in prior studies using AD model mice. Here, leuco ethyl violet (LEV) is identified as a highly active, amyloid-selective, and blood-brain barrier (BBB)-permeable photooxygenation catalyst that circumvents all of these problems. LEV is a redox-sensitive, self-activating prodrug catalyst; self-oxidation of LEV through a hydrogen atom transfer process under photoirradiation produces catalytically active ethyl violet (EV) in the presence of amyloid. LEV effectively oxygenates human Aß and tau, suggesting the feasibility for applications in humans. Furthermore, a concept of using a hydrogen atom as a caging group of a reactive catalyst functional in vivo is postulated. The minimal size of the hydrogen caging group is especially useful for catalyst delivery to the brain through BBB.
Assuntos
Doença de Alzheimer , Pró-Fármacos , Animais , Pró-Fármacos/farmacologia , Camundongos , Doença de Alzheimer/metabolismo , Catálise , Modelos Animais de Doenças , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Humanos , Proteínas tau/metabolismo , Proteínas tau/químicaRESUMO
Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, catalysis driven by amyloid-substrate complex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH2 (NL6), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe-Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.
Assuntos
Amiloide , Catálise , Amiloide/química , Amiloide/metabolismo , Aminas/química , Aminas/metabolismo , Ligação de Hidrogênio , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Concentração de Íons de Hidrogênio , HumanosRESUMO
Amyloids, proteinous aggregates with ß-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat ß-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model. In this study, we further explored the residue-specific properties of ThT-binding to the flat ß-sheet by introducing systematic mutations. We found that site-specific mutations at the ThT-binding channel enhanced affinity. We also evaluated the binding of a ThT-based photocatalyst, which showed the photooxygenation activity on the amyloid fibril upon light radiation. Upon binding of the photocatalyst to the PSAM variant, singlet oxygen-generating activity was observed. The results of this study expand our understanding of the detailed binding mechanism of amyloid-specific molecules.
Assuntos
Benzotiazóis , Benzotiazóis/química , Catálise , Ligação Proteica , Conformação Proteica em Folha beta , Amiloide/química , Mutação , Oxigênio Singlete/química , Oxigênio Singlete/metabolismo , Corantes Fluorescentes/químicaRESUMO
N/C-terminal protected amyloidogenic peptides are valuable biomaterials. Optimization of the protective structures at both termini is, however, synthetically laborious because a linear sequence of solid-phase peptide synthesis protocol (on-resin peptide assembly/peptide removal from resin/high-performance liquid chromatography purification) is required for the peptides each time the protective group is modified. In this study, we demonstrate a modular synthetic strategy for the purpose of rapidly deriving the N/C-terminal structures of amyloidogenic peptides. The precursor sequences that can be easily synthesized due to a non-amyloidogenic property were stocked as the synthetic intermediates. Condensation of the intermediates with N/C-terminal units in a liquid phase followed by high-performance liquid chromatography purification gave the desired peptides P1-P8. The amyloidogenic peptides that have various N/C-terminal protective structures were therefore synthesized in a labor-effective manner. This method is suggested to be useful for synthesizing amyloidogenic peptides possessing divergent protective structures at the N/C-terminus.
Assuntos
Materiais Biocompatíveis , Peptídeos , Peptídeos/química , Cromatografia Líquida de Alta Pressão , Técnicas de Síntese em Fase SólidaRESUMO
Aggregation of α-synuclein (α-syn) into amyloid is the pathological hallmark of several neurodegenerative disorders, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. It is widely accepted that α-syn aggregation is associated with neurodegeneration, although the mechanisms are not yet fully understood. Therefore, the inhibition of α-syn aggregation is a potential therapeutic approach against these diseases. This study used the photocatalyst for α-syn photo-oxygenation, which selectively adds oxygen atoms to fibrils. Our findings demonstrate that photo-oxygenation using this photocatalyst successfully inhibits α-syn aggregation, particularly by reducing its seeding ability. Notably, we also discovered that photo-oxygenation of the histidine at the 50th residue in α-syn aggregates is responsible for the inhibitory effect. These findings indicate that photo-oxygenation of the histidine residue in α-syn is a potential therapeutic strategy for synucleinopathies.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , Histidina/análise , Doença de Parkinson/terapia , Doença de Parkinson/patologia , Corpos de Lewy/patologia , Fenômenos Fisiológicos RespiratóriosRESUMO
Catalytic photo-oxygenation of tau amyloid is a potential therapeutic approach to tauopathies, including Alzheimer disease (AD). However, tau is a complex target containing great molecular size and heterogeneous isoforms/proteoforms. Although catalytic photo-oxygenation has been confirmed when using catalyst 1 and recombinant tau pretreated with heparin, its effects on tau from human patients have not yet been clarified. In this study, focusing on the histidine residues being oxygenated, we have constructed two assay systems capable of quantitatively evaluating the catalytic activity when used on human patient tau: (1) fluorescence labeling at oxygenated histidine sites and (2) LC-MS/MS analysis of histidine-containing fragments. Using these assays, we identified 2 as a promising catalyst for oxygenation of human tau. In addition, our results suggest that aggregated tau induced by heparin is different from actual AD patient tau in developing effective photo-oxygenation catalysts.
Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Doença de Alzheimer/metabolismo , Proteínas tau/metabolismo , Cromatografia Líquida , Histidina , Espectrometria de Massas em Tandem , Tauopatias/metabolismoRESUMO
We developed catalyst 11 to promote selective photo-oxygenation of α-synuclein amyloid and attenuate its aggregation. Catalyst 11 effectively oxygenated both small and large aggregates. The oxygenated α-synuclein exhibited lower seeding activity than intact α-synuclein. This study corroborates the feasibility of catalytic photo-oxygenation as an anti-synucleinopathy strategy.
Assuntos
Amiloide , alfa-SinucleínaRESUMO
Alzheimer's disease (AD) is characterized by the aggregation and deposition of 2 amyloid proteins: amyloid ß peptide (Aß) and tau protein. Immunotherapies using anti-Aß antibodies to promote the clearance of aggregated Aß have recently been highlighted as a promising disease-modifying approach against AD. However, immunotherapy has still some problems, such as low efficiency of delivery into the brain and high costs. We have developed the "amyloid selective photo-oxygenation technology" as a comparable to immunotherapy for amyloids. The photo-oxygenation can artificially attach the oxygen atoms to specific amino acids in amyloid proteins using photocatalyst and light irradiation. We revealed that in vivo photo-oxygenation for living AD model mice reduced the aggregated Aß in the brain. Moreover, we also showed that microglia were responsible for this promoted clearance of photo-oxygenated Aß from the brain. These results indicated that our photo-oxygenation technology has the potential as a disease-modifying therapy against AD to promote the degradation of amyloids, resulting in being comparable to immunotherapy. Here, we introduce our technology and its effects in vivo that we showed previously in Ozawa et al., Brain, 2021, as well as a further improvement towards non-invasive in vivo photo-oxygenation described in another publication Nagashima et al., Sci. Adv., 2021, as expanded discussion.
RESUMO
Alzheimer disease (AD) is associated with the aggregation of two amyloid proteins: tau and amyloid-ß (Aß). The results of immunotherapies have shown that enhancing the clearance and suppressing the aggregation of these two proteins are effective therapeutic strategies for AD. We have developed photocatalysts that attach oxygen atoms to Aß and tau aggregates via light irradiation. Photo-oxygenation of these amyloid aggregates reduced their neurotoxicity by suppressing their aggregation both in vitro and in vivo. Furthermore, photo-oxygenation enhanced the clearance of Aß in the brain and microglial cells. Here, we describe the effects of photo-oxygenation on tau and Aß aggregation, and the potential of photo-oxygenation as a therapeutic strategy for AD, acting via microglial clearance.
RESUMO
Knoevenagel condensation, an olefin-forming reaction from active methyl/methylene-containing compounds and aldehydes, is a fundamental and useful synthetic method. Benzothiazoles are, however, out of the scope of Knoevenagel condensation. Here, we report that Knoevenagel condensation between aldehydes and 2-methyl-thiazolo[4,5-b]pyrazines (MeTPy), a fused ring structure comprising pyrazine and thiazole, proceeded smoothly, despite minor structural differences from benzothiazoles. This finding will be useful for short synthesis of MeTPy-containing functional molecules, such as a tau probe analog 1.
Assuntos
Aldeídos/química , Alcenos/síntese química , Pirazinas/química , Alcenos/química , Estrutura MolecularRESUMO
Misfolded proteins produce aberrant fibrillar aggregates, called amyloids, which contain cross-ß-sheet higher order structures. The species generated in the aggregation process (i.e., oligomers, protofibrils, and fibrils) are cytotoxic and can cause various diseases. Interfering with the amyloid formation of proteins could be a drug development target for treating diseases caused by aberrant protein aggregation. In this review, we introduce a variety of chemical catalysts that oxygenate amyloid proteins under light irradiation using molecular oxygen as the oxygen atom donor (i.e., photooxygenation catalysts). Catalytic photooxygenation strongly inhibits the aggregation of amyloid proteins due to covalent installation of hydrophilic oxygen atoms and attenuates the neurotoxicity of the amyloid proteins. Recent in vivo studies in disease model animals using photooxygenation catalysts showed promising therapeutic effects, such as memory improvement and lifespan extension. Moreover, photooxygenation catalysts with new modes of action, including interference with the propagation of amyloid core seeds and enhancement in the metabolic clearance of amyloids in the brain, have begun to be identified. Manipulation of catalytic photooxygenation with secured amyloid selectivity is indispensable for minimizing the side effects in clinical application. Here we describe several strategies for designing catalysts that selectively photooxygenate amyloids without reacting with other non-amyloid biomolecules.
Assuntos
Proteínas Amiloidogênicas/química , Oxigênio/química , Processos Fotoquímicos , Animais , Catálise , HumanosRESUMO
Amyloid formation and the deposition of the amyloid-ß peptide are hallmarks of Alzheimer's disease pathogenesis. Immunotherapies using anti-amyloid-ß antibodies have been highlighted as a promising approach for the prevention and treatment of Alzheimer's disease by enhancing microglial clearance of amyloid-ß peptide. However, the efficiency of antibody delivery into the brain is limited, and therefore an alternative strategy to facilitate the clearance of brain amyloid is needed. We previously developed an artificial photo-oxygenation system using a low molecular weight catalytic compound. The photocatalyst specifically attached oxygen atoms to amyloids upon irradiation with light, and successfully reduced the neurotoxicity of aggregated amyloid-ß via inhibition of amyloid formation. However, the therapeutic effect and mode of actions of the photo-oxygenation system in vivo remained unclear. In this study, we demonstrate that photo-oxygenation facilitates the clearance of aggregated amyloid-ß from the brains of living Alzheimer's disease model mice, and enhances the microglial degradation of amyloid-ß peptide. These results suggest that photo-oxygenation may represent a novel anti-amyloid-ß strategy in Alzheimer's disease, which is compatible with immunotherapy.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Compostos de Boro/farmacologia , Encéfalo/efeitos dos fármacos , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Microglia/metabolismo , Fototerapia/métodos , Agregados Proteicos/efeitos dos fármacosRESUMO
Protein degradation induced by small molecules by recruiting endogenous protein degradation systems, such as ubiquitin-proteasome systems, to disease-related proteins is an emerging concept to inhibit the function of undruggable proteins. Protein targets without reliable ligands and/or existing outside the cells where ubiquitin-proteasome systems do not exist, however, are beyond the scope of currently available protein degradation strategies. Here, we disclose photooxygenation catalyst 7 that permeates the blood-brain barrier and selectively and directly degrades an extracellular Alzheimer's disease-related undruggable protein, amyloid-ß protein (Aß). Key was the identification of a compact but orange color visible light-activatable chemical catalyst whose activity can be switched on/off according to its molecular mobility, thereby ensuring high selectivity for aggregated Aß. Chemical catalyst-promoted protein degradation can be applied universally for attenuating extracellular amyloids and various pathogenic proteins and is thus a new entry to induced protein degradation strategies.
RESUMO
Many types of amyloidoses are pathologically characterized by the deposition of amyloid, which is comprised of fibrils formed by abnormally aggregated proteins, in various peripheral tissues and the central nervous system (CNS). Neurodegenerative disorders, such as Alzheimer disease (AD), Parkinson disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS), are well-known CNS amyloidoses that are characterized by amyloid deposition both inside and outside of cells. The amyloidogenic proteins of each disease have distinct primary sequences, and they normally function as soluble proteins. However, these proteins all aggregate and form amyloid with a common intermolecular tertiary structure, namely, a cross-ß-sheet structure, finally leading to the onset of each disease. Therefore, inhibition of the aggregation of amyloid proteins or efficient clearance of the already formed amyloids are thought to be promising therapeutic strategies against amyloidoses.
Assuntos
Doença de Alzheimer , Amiloidose , Demência Frontotemporal , Doença de Parkinson , Amiloide , Amiloidose/terapia , HumanosRESUMO
The oligomer species of amyloid-ß peptide (Aß) may be relevant to the development of Alzheimer's disease. Isolating specific oligomer species of Aß (i.e., dimers, trimers, tetramers, etc.), however, is difficult due to the transient, labile property of the oligomers. Here, we improved the resistance to intertrimer mis-aggregation of chemically tethered Aß25-35 trimers by introducing charged structures to the cyclic peptide tether that is covalently attached to the Aß chain. The resistance to aggregation of the chemically tethered trimers positively correlated with the number of negative charges at the tether. Thus, a chemical trimer possessing three malonic acids at the tether exhibited high resistance because of the attenuated self-association by anionic repulsion. In addition, the malonic acid trimer possessed amyloidogenic properties such as cross-ß-sheet structures, seeding activity, and cytotoxicity. This is the first study demonstrating that chemical modifications at the non-Aß component enhance the resistance to aggregation of chemically tethered Aß oligomers, by which the structural integrity of Aß is maintained. Biological/biophysical evaluations of the intertrimer aggregation-resistant trimer may offer new, useful insights into the pathological functions of Aß oligomers.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Doença de Alzheimer/tratamento farmacológico , Humanos , Fragmentos de Peptídeos , Peptídeos CíclicosRESUMO
A combination of time-resolved optical spectroscopy and nanoscale imaging has been used to study the complex binding to amyloids of a photocatalyst that selectively photo-oxygenates pathogenic aggregates, as well as the consequences of its irradiation. Correlative atomic force microscopy (AFM) and fluorescence microscopy reveals topography-dependent binding of the dye to model ß-lactoglobulin fibers, which may also explain the observed difference in their response to photodegradation. We provide direct evidence of the photosensitization of singlet oxygen by the photocatalyst bound to amyloid fibers by direct detection of its NIR phosphorescence. The effect of singlet oxygen at the molecular level brings about nanoscale morphological changes that can be observed with AFM at the single-fiber level. We also find differential response of two α-synuclein mutants to photodamage, which can be rationalized by the presence of amino acids susceptible to photo-oxygenation. Overall, our results help to unravel some of the complexity associated with highly heterogeneous amyloid populations and contribute to the development of improved phototherapeutic strategies for amyloid-related disorders.
Assuntos
Amiloide/química , Luz/efeitos adversos , Microscopia de Força Atômica/métodos , alfa-Sinucleína/química , Benzotiazóis/química , Lactoglobulinas/química , Microscopia de Fluorescência/métodos , Espécies Reativas de Oxigênio/química , Oxigênio Singlete/química , Espectroscopia de Luz Próxima ao Infravermelho/métodosRESUMO
The photophysical properties of newly identified B,B-fluoro-perfluoroalkyl BODIPYs (2 and 3), which possess a fluoro group and a trifluoromethyl or pentafluoroethyl group at the boron center, were investigated. B,B-Fluoro-perfluoroalkyl BODIPYs 2 and 3 exhibited better photophysical/chemical properties than B,B-difluoro-BODIPY 1, as follows: (1) higher photostability both in methanol solvent and in a live cell environment, (2) higher stability against acid degradation, and (3) improved fluorescence signal-to-noise ratios in a cell system. These favorable properties of B,B-fluoro-perfluoroalkyl BODIPYs are likely due to the highly electron-withdrawing nature of the perfluoroalkyl groups on the boron atom, which reduces the reactivity to 1O2 and strengthens the complexation of the dipyrromethene ligands to the boron atom. Thus, B,B-fluoro perfluoroalkyl BODIPYs may be useful functional molecules for various applications.
RESUMO
Tau amyloid formation and deposition are responsible for the onset of Alzheimer's disease. In particular, the seeding activity of the tau protein plays an important role in the spreading of tau pathology via its propagation in the human brain. Here we demonstrate that catalytic photo-oxygenation markedly suppresses tau seeding activity, resulting in the inhibition of amyloid formation, both in vitro and in cultured cells.
Assuntos
Oxigênio/metabolismo , Processos Fotoquímicos , Proteínas tau/antagonistas & inibidores , Proteínas tau/biossíntese , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas Amiloidogênicas/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Catálise , Reação de Cicloadição , Células HEK293 , HumanosRESUMO
A characteristic feature of higher-order structures of amyloid ß peptide (Aß) aggregates observed in Alzheimer disease is the salt-bridge between the side-chains of Asp23 (carboxylate) and Lys28 (ammonium). We synthesized an [Met35(O)]Aß42 possessing a covalently bound lactam tether as an Asp23/Lys28 salt-bridge surrogate (compound 3). The lactam tether of 3 markedly promoted the formation of stable protofibril-like species that exhibited amyloidogenic properties such as a cross-ß-sheet structure and cytotoxicity. This finding is consistent with reports that the Asp23/Lys28 salt-bridge of Aß42 is transiently formed in aggregation intermediates.
Assuntos
Peptídeos beta-Amiloides/química , Lactamas/química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/toxicidade , Animais , Lactamas/toxicidade , Células PC12 , Fragmentos de Peptídeos/toxicidade , Conformação Proteica em Folha beta , Engenharia de Proteínas , Multimerização Proteica , RatosRESUMO
We developed a palladium-catalyzed C-H N-heteroarylation of N-protected-2,5-disubstituted imidazoles at the C4-position using N-heteroaryl halides as a coupling partner. Intensive reaction condition screening led us to identify fluorinated bathophenanthroline 7 as the optimum ligand for the palladium catalyst. This reaction will enhance lead optimization of drug candidates by facilitating the synthesis of heterobiaryl compounds containing an imidazole ring.