RESUMEN
Amyloid ß-protein (Aß42) aggregates have been demonstrated to induce cognitive decline and neurodegeneration in Alzheimer's disease (AD). Thus, functional food ingredients that inhibit Aß42 aggregation are valuable for AD prevention. Although several food ingredients have been studied for their anti-aggregation activity, information on their bioavailability in the brain, incorporated forms, and relevance to AD etiology is limited. Here, we first detected the sulfate- and glucuronic-acid-conjugated forms of green perilla-derived chalcone (1) and taxifolin (2), which inhibit Aß42 aggregation, in the brain, small intestine, and plasma of mice (1 and 2 were administered orally) using ultra-performance liquid chromatography-tandem mass spectrometry. We observed that the conjugated metabolites (sulfate (4) and glucuronide (5)) of 1 prevented the fibrillization and oligomerization of Aß42. These findings imply that the conjugated metabolites of 1 can prove beneficial for AD treatment.
Asunto(s)
Enfermedad de Alzheimer , Chalconas , Ingredientes Alimentarios , Ratones , Animales , Péptidos beta-Amiloides/metabolismo , Flavonoides , Espectrometría de Masas en Tándem , Cromatografía Liquida , Enfermedad de Alzheimer/metabolismo , Sulfatos , Fragmentos de Péptidos/químicaRESUMEN
Oligomers of amyloid ß (Aß) represent an early aggregative form that causes neurotoxicity in the pathogenesis of Alzheimer's disease (AD). Thus, preventing Aß aggregation is important for preventing AD. Despite intensive studies on dietary compounds with anti-aggregation properties, some identified compounds are susceptible to autoxidation and/or hydration upon incubation in water, leaving unanswered issues regarding which active structures in metastable compounds are actually responsible for the inhibition of Aß aggregation. In this study, we observed the site-specific inhibition of 42-mer Aß (Aß42) oligomerization by the green perilla-derived chalcone 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC), which was converted to its decomposed flavonoids (dDDC, 1-3) via nucleophilic aromatic substitution with water molecules. DDC suppressed Aß42 fibrillization and slowed the transformation of the ß-sheet structure, which is rich in Aß42 aggregates. To validate the contribution of dDDC to the inhibitory effects of DDC on Aß42 aggregation, we synthesized 1-3 and identified 3, a catechol-type flavonoid, as one of the active forms of DDC. 1H-15N SOFAST-HMQC NMR revealed that 1-3 as well as DDC could interact with residues between His13 and Leu17, which were near the intermolecular ß-sheet (Gln15-Ala21). The nucleation in Aß42 aggregates involves the rate-limiting formation of low-molecular-weight oligomers. The formation of a Schiff base with dDDC at Lys16 and Lys28 in the dimer through autoxidation of dDDC was associated with the suppression of Aß42 nucleation. Of note, in two AD mouse models using immunoaffinity purification-mass spectrometry, adduct formation between dDDC and brain Aß was observed in a similar manner as reported in vitro. The present findings unraveled the lysine-targeting inhibitory mechanism of metastable dietary ingredients regarding Aß oligomerization.
RESUMEN
Aggregation of amyloid ß42 (Aß42) is one of the hallmarks of Alzheimer's disease (AD). Inhibition of Aß42 aggregation is thus a promising approach for AD therapy. Kampo medicine has been widely used to combat dementias such as AD. Crude drug known as Shoyaku is an ingredient of Kampo that could have potential as a natural source of novel drugs. However, given that a mixture of compounds, rather than singular compounds, could contribute to the biological functions of crude drug, there are very limited studies on the structure and mechanism of each constituent in crude drug which may have anti-Aß42 aggregation properties. Herein we provide an efficient method, using LC-MS combined with principal component analysis (PCA), to search for activity-dependent compounds that inhibit Aß42 aggregation from 46 crude drug extracts originating from 18 plants. Only 5 extracts (Kakou, Kayou, Gusetsu, Rensu, and Renbou) from lotus demonstrated differentially inhibitory activities depending on the part of the plant from which they are derived (e.g. petiole, leaf, root node, stamen, and receptacle, respectively). To compare the anti-aggregative properties of compounds of active crude drug with those of inactive crude drug, these extracts were subjected to LC-MS measurement, followed by PCA. From 12 candidate compounds identified from the analysis, glucuronized and glucosidized quercetin, as well as 6 flavonoids (datiscetin, kaempferol, morin, robinetin, quercetin, and myricitrin), including catechol or flatness moiety suppressed Aß42 aggregation, whereas curcumol, a sesquiterpene, did not. In conclusion, this study offers a new activity-differential methodology to identify bioactive natural products in crude drugs that inhibit Aß42 aggregation and that could be applied to future AD therapies.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Medicamentos Herbarios Chinos/farmacología , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Análisis de Componente Principal , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/aislamiento & purificación , Humanos , Espectrometría de Masas , Medicina Kampo , Estructura Molecular , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales/química , Agregado de Proteínas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Aggregation of amyloid ß42 (Aß42) is one of the hallmarks of Alzheimer's disease (AD). The mechanism of Aß42 aggregation mainly consists of two phases, nucleation and elongation (including plateau region as a saturation phase). During the nucleation phase, the monomer gradually forms toxic oligomers. During the elongation phase, each nucleus acts as a template and associates with monomers to initiate less toxic fibrillization. We previously proposed a method of classifying compounds into nine groups based on their ability to modulate the nucleation and/or elongation phases. An orcein derivative (O4), which is a phenoxazine dye isolated from the lichen Roccella tinctoria and containing a 2,5-cyclohexadienone moiety, was reported to convert oligomers into relatively inert fibrils, resulting in the reduction of the neurotoxicity of Aß42. Focusing on O4 in the pursuit of anti-AD drugs, we herein screened 480 natural products including NPDepo (RIKEN) for the compounds that delayed the nucleation phase and promoted the elongation phase. The signal intensities for Aß42 treated with each of the 15 compounds that met these criteria were lowered in dot blotting using antioligomer antibody, and the fibril formation of Aß42 in the presence of these compounds was observed in transmission electron microscopy. Among the 15 compounds, 12 compounds (80%) reduced the toxicity of Aß42 against mouse neuroblastoma Neuro-2a cells. Some of these anticytotoxic compounds contain 2-pyrone and 4-pyrone that interacted with Aß42, maybe by shifting the equilibrium of Aß from toxic oligomer into inert fibrils.
Asunto(s)
Enfermedad de Alzheimer , Productos Biológicos , Enfermedad de Alzheimer/tratamiento farmacológico , Amiloide , Péptidos beta-Amiloides , Animales , Ascomicetos , Ratones , Fragmentos de PéptidosRESUMEN
Protein persulfidation plays a role in redox signaling as an anti-oxidant. Dimers of amyloid ß42 (Aß42), which induces oxidative stress-associated neurotoxicity as a causative agent of Alzheimer's disease (AD), are minimum units of oligomers in AD pathology. Met35 can be susceptible to persulfidation through its substitution to homoCys residue under the condition of oxidative stress. In order to verify whether persulfidation has an effect in AD, herein we report a chemical approach by synthesizing disulfide dimers of Aß42 and their evaluation of biochemical properties. A homoCys-disulfide dimer model at position 35 of Aß42 formed a partial ß-sheet structure, but its neurotoxicity was much weaker than that of the corresponding monomer. In contrast, the congener with an alkyl linker generated ß-sheet-rich 8-16-mer oligomers with potent neurotoxicity. The length of protofibrils generated from the homoCys-disulfide dimer model was shorter than that of its congener with an alkyl linker. Therefore, the current data do not support the involvement of Aß42 persulfidation in Alzheimer's disease.
RESUMEN
Here, we report the first synthesis of quasi-stable trimer models of full-length Aß40 with a toxic conformation using a 1,3,5-phenyltris-l-alanyl linker at position 34, 36, or 38. The only trimer to exhibit weak neurotoxicity against SH-SY5Y cells was the one which was linked at position 38. This suggests that such a propeller-type trimer model is not prone to forming oligomers with potent neurotoxicity, which is in contrast with its corresponding dimer model.
RESUMEN
Herein we report that a preferable inhibition of the nucleation phase of Aß42, related to the formation of toxic oligomers, by triterpenoids from medicinal herbs originates from a salt bridge of their carboxy groups with Lys16 and 28 in Aß42. Such a direct interaction targeting the monomer, dimer, and trimer suppressed further oligomerization. In contrast, the corresponding congeners without carboxy groups failed to do so.
Asunto(s)
Péptidos beta-Amiloides/química , Ácidos Carboxílicos/farmacología , Fármacos Neuroprotectores/farmacología , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacología , Fragmentos de Péptidos/química , Antraquinonas/química , Antraquinonas/farmacología , Ácidos Carboxílicos/química , Línea Celular Tumoral , Humanos , Lisina/química , Fármacos Neuroprotectores/química , Ácido Oleanólico/química , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/farmacología , Multimerización de Proteína , Triterpenos/química , Triterpenos/farmacologíaRESUMEN
(R)-Apomorphine (1) has the potential to reduce the accumulation of amyloid ß-protein (Aß42), a causative agent of Alzheimer's disease (AD). Although the inhibition of Aß42 aggregation by 1 is ascribable to the antioxidative effect of its phenol moiety, its inhibitory mechanism at the molecular level remains to be fully elucidated. LC-MS and UV analyses revealed that 1 is autoxidized during incubation to produce an unstable o-quinone form (2), which formed a Michael adduct with Lys 16 and 28 of Aß42. A further autoxidized form of 1 (3) with o-quinone and phenanthrene moieties suppressed Aß42 aggregation comparable to 1, whereas treating 1 with a reductant, tris(2-carboxyethyl)phosphine diminished its inhibitory activity. 1H-15N SOFAST-HMQC NMR studies suggested that 1 interacts with Arg5, His13,14, Gln15, and Lys16 of the Aß42 monomer. These regions form intermolecular ß-sheets in Aß42 aggregates. Since 3 did not perturb the chemical shift of monomeric Aß42, we performed aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aß42 to investigate whether 3 associates with Aß42 oligomers. Compounds 1 and 3 delayed the onset of the oligomer-driven nucleation phase. Despite their cytotoxicity, they did not exacerbate Aß42-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. These results demonstrate that extension of the conjugated system in 1 by autoxidation can promote its planarity, which is required for intercalation into the ß-sheet of Aß42 nuclei, thereby suppressing further aggregation.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Apomorfina/farmacología , Péptidos beta-Amiloides/metabolismo , Apomorfina/síntesis química , Apomorfina/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Agregado de Proteínas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The prevention of amyloid aggregation is promising for the treatment of age-related diseases such as Alzheimer's (AD) and type 2 diabetes (T2D). Ten antioxidant flavonoids isolated from the medicinal halophyte Tamarix gallica were tested for their amyloid aggregation inhibition potential. Glucuronosylated flavonoids show relatively strong inhibitory activity of Amyloid ß (Aß) and human islet amyloid polypeptide (hIAPP) aggregation compared to their aglycone analogs. Structure-activity relationship of the flavonoids suggests that the catechol moiety is important for amyloid aggregation inhibition, while the methylation of the carboxyl group in the glucuronide moiety and of the hydroxyl group in the aglycone flavonoids decreased it.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Antioxidantes/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Flavonoides/farmacología , Tamaricaceae , Amiloide/antagonistas & inhibidores , Antioxidantes/aislamiento & purificación , Flavonoides/aislamiento & purificación , Humanos , Componentes Aéreos de las Plantas , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Agregación Patológica de Proteínas/metabolismoRESUMEN
The formation of soluble oligomers of amyloid ß42 and 40 (Aß42, Aß40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aß42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the C-terminal region. However, in addition to Aß42, Aß40 dimers can also contribute to AD progression because of the more abundance of Aß40 monomer in biological fluids. Here, we describe the synthesis and characterization of three dimer models of the toxic-conformation constrained E22P-Aß40 using l,l-2,6-diaminopimeric acid (DAP) or l,l-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel ß-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aß40 dimer (1) and E22P-A30DAZ-Aß40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity toward SH-SY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aß40 dimer (3) formed ß-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility-mass spectrometry suggested that high molecular-weight oligomers (12-24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel ß-sheet, triggers the formation of toxic Aß oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Línea Celular , Dicroismo Circular , Humanos , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Neuronas/efectos de los fármacos , Neuronas/patologíaRESUMEN
Oligomers of the 42-mer amyloid-ß protein (Aß42), rather than fibrils, cause synaptic dysfunction in the pathology of Alzheimer's disease (AD). The nucleation phase in a nucleation-dependent aggregation model of Aß42 is related to the formation of oligomers. Uncaria rhynchophylla is one component of "Yokukansan", a Kampo medicine, which is widely used for treating AD symptoms. Previously, an extract of U. rhynchophylla was found to reduce the aggregation of Aß42, but its active principles have yet to be identified. In the present work, uncarinic acid C (3) was identified as an inhibitor of Aß42 aggregation that is present in U. rhynchophylla. Moreover, compound 3 acted as a specific inhibitor of the nucleation phase of Aß42 aggregation. Compound 3 was synthesized from saponin A (10), an abundant byproduct of rutin purified from Uncaria elliptica. Comprehensive structure-activity studies on 3 suggest that both a C-27 ferulate and a C-28 carboxylic acid group are required for its inhibitory activity. These findings may aid the development of oligomer-specific inhibitors for AD therapy.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Triterpenos/síntesis química , Triterpenos/farmacología , Uncaria/química , Supervivencia Celular , Medicamentos Herbarios Chinos , Humanos , Japón , Estructura Molecular , Neuronas/metabolismo , Fragmentos de Péptidos , Rifamicinas , Relación Estructura-Actividad , Triterpenos/químicaRESUMEN
Amyloid ß-protein (Aß42) oligomerization is an early event in Alzheimer's disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aß42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aß42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aß42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aß42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aß42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aß42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales/líquido cefalorraquídeo , Fragmentos de Péptidos/inmunología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/toxicidad , Animales , Anticuerpos Monoclonales/química , Estudios de Casos y Controles , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Masculino , Conformación Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/toxicidad , Ratas Wistar , Resonancia por Plasmón de SuperficieRESUMEN
The prevention of 42-mer amyloid ß-protein (Aß42) aggregation is promising for the treatment of Alzheimer's disease. We previously described the site-specific inhibitory mechanism for Aß42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aß42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aß42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The (1)H-(15)N SOFAST-HMQC NMR of Aß42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular ß-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aß42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aß42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aß42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aß42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aß42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aß42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aß42 aggregation.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Flavonoides/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/química , Relación Dosis-Respuesta a Droga , Flavonoides/síntesis química , Flavonoides/química , Estructura Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Relación Estructura-ActividadRESUMEN
Amyloid fibrils in senile plaque mainly consist of the 40-mer and 42-mer amyloid ß-proteins (Aß40 and Aß42). Although Aß42 plays more important role in the pathogenesis of Alzheimer's disease (AD), Aß40 could be involved in the progression of AD pathology because of its large amount. Recent studies revealed that variable sizes of Aß oligomers contributed to the neuronal death and cognitive dysfunction. However, how large oligomeric species are responsible for AD pathogenesis remains unclear. We previously proposed a toxic dimer model of Aß with turn structure at positions 22 and 23 using solid-state NMR and systematic proline replacement. Based on this model, we herein show the synthesis and biological activities of an E22P-Aß40 dimer at position 30, which was connected to l,l-2,6-diaminopimeric acid. The E22P-Aß40 dimer formed stable 6â¼8-mer oligomers without amyloid fibrils, but was not neurotoxic on human neuroblastoma cells. On the other hand, E22P-Aß40 generated high molecular-weight oligomers into fibrils, and showed the neurotoxicity. These results suggest that such kind of Aß40 dimer with a parallel ß-sheet might not be pathological.