Thioflavin-T Assay Research Articles

Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons.

The role of α-synuclein (α-syn) pathology in Parkinson's disease (PD) is well established; however, effective therapies remain elusive. Two mechanisms central to PD neurodegeneration are the intracellular aggregation of misfolded α-syn and the uptake of α-syn aggregates into neurons. Cationic arginine-rich peptides (CARPs) are an emerging class of molecule with multiple neuroprotective mechanisms of action, including protein stabilisation. This study characterised both intracellular α-syn aggregation and α-syn uptake in cortical neurons in vitro. Thereafter, this study examined the therapeutic potential of the neuroprotective CARP, R18D (18-mer of D-arginine), to prevent the aforementioned PD pathogenic processes through a cell-free thioflavin-T (ThT) assay and in cortical neurons. To induce intracellular α-syn aggregation, rat primary cortical neurons were exposed to α-syn seed (0.14 μM) for 2 h to allow uptake of the protein, followed by R18D treatment (0.0625, 0.125, 0.25, 0.5 μM), and a subsequent measurement of α-syn aggregates 48 h later using a homogenous time-resolved fluorescence (HTRF) assay. To assess neuronal uptake, α-syn seeds were covalently labelled with an Alexa-Fluor 488 fluorescent tag, pre-incubated with R18D (0.125, 0.25, 0.5 μM), and then exposed to cortical neurons for 24 h and assessed via confocal microscopy. It was demonstrated that R18D significantly reduced both intracellular α-syn aggregation and α-syn seed uptake in neurons by 37.8% and 77.7%, respectively. Also, R18D reduced the aggregation of α-syn monomers in the cell-free assay. These findings highlight the therapeutic potential of R18D to inhibit key α-syn pathological processes and PD progression.

SMOC1 colocalizes with Alzheimer’s disease neuropathology and delays Aβ aggregation

SMOC1 has emerged as one of the most significant and consistent new biomarkers of early Alzheimer’s disease (AD). Recent studies show that SMOC1 is one of the earliest changing proteins in AD, with levels in the cerebrospinal fluid increasing many years before symptom onset. Despite this clear association with disease, little is known about the role of SMOC1 in AD or its function in the brain. Therefore, the aim of this study was to examine the distribution of SMOC1 in human AD brain tissue and to determine if SMOC1 influenced amyloid beta (Aβ) aggregation. The distribution of SMOC1 in human brain tissue was assessed in 3 brain regions (temporal cortex, hippocampus, and frontal cortex) using immunohistochemistry in a cohort of 73 cases encompassing advanced AD, mild cognitive impairment (MCI), preclinical AD, and cognitively normal controls. The Aβ- and phosphorylated tau-interaction with SMOC1 was assessed in control, MCI, and advanced AD human brain tissue using co-immunoprecipitation, and the influence of SMOC1 on Aβ aggregation kinetics was assessed using Thioflavin-T assays and electron microscopy. SMOC1 strongly colocalized with a subpopulation of amyloid plaques in AD (43.8 ± 2.4%), MCI (32.8 ± 5.4%), and preclinical AD (28.3 ± 6.4%). SMOC1 levels in the brain strongly correlated with plaque load, irrespective of disease stage. SMOC1 also colocalized with a subpopulation of phosphorylated tau aggregates in AD (9.6 ± 2.6%). Co-immunoprecipitation studies showed that SMOC1 strongly interacted with Aβ in human MCI and AD brain tissue and with phosphorylated tau in human AD brain tissue. Thioflavin-T aggregation assays showed that SMOC1 significantly delayed Aβ aggregation in a dose-dependent manner, and electron microscopy confirmed that the Aβ fibrils generated in the presence of SMOC1 had an altered morphology. Overall, our results emphasize the importance of SMOC1 in the onset and progression of AD and suggest that SMOC1 may influence pathology development in AD.

Cross-Interactions of Aβ Peptides Implicated in Alzheimer's Disease Shape Amyloid Oligomer Structures and Aggregation.

A defining hallmark of Alzheimer's disease (AD) is the synaptic aggregation of the amyloid β (Aβ) peptide. In vivo, Aβ production results in a diverse mixture of variants, of which Aβ40, Aβ42, and Aβ43 are profusely present in the AD brain, and their relative abundance is recognized to play a role in disease onset and progression. Nonetheless, the occurrence of Aβ40, Aβ42, and Aβ43 hetero-oligomerization and the subsequent effects on Aβ aggregation remain elusive and were investigated here. Using thioflavin-T (ThT)-monitored aggregation assays and native mass spectrometry coupled to ion mobility analysis (IM-MS), we first show that all Aβ peptides are aggregation-competent and self-assemble into homo-oligomers with distinct conformational populations, which are more pronounced between Aβ40 than the longer variants. ThT assays were then conducted on binary mixtures of Aβ variants, revealing that Aβ42 and Aβ43 aggregate independently from Aβ40 but significantly speed up Aβ40 fibrillation. Aβ42 and Aβ43 were observed to aggregate concurrently and mutually accelerate fibril formation, which likely involves hetero-oligomerization. Accordingly, native MS analysis revealed pairwise oligomerization between all variants, with the formation of heterodimers and heterotrimers. Interestingly, IM-MS indicates that hetero-oligomers containing longer Aβ variants are enriched in conformers with lower collision cross-sections when compared to their homo-oligomer counterparts. This suggests that Aβ42 and Aβ43 are capable of remodeling the oligomer structure toward a higher compaction level. Altogether, our findings provide a mechanistic description for the hetero-oligomerization of Aβ variants implicated in AD, contributing to rationalizing their in vivo proteotoxic interplay.

Evaluation of Alpha-Synuclein and Tau Antiaggregation Activity of Urea and Thiourea-Based Small Molecules for Neurodegenerative Disease Therapeutics.

Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial, chronic diseases involving neurodegeneration. According to recent studies, it is hypothesized that the intraneuronal and postsynaptic accumulation of misfolded proteins such as α-synuclein (α-syn) and tau, responsible for Lewy bodies (LB) and tangles, respectively, disrupts neuron functions. Considering the co-occurrence of α-syn and tau inclusions in the brains of patients afflicted with subtypes of dementia and LB disorders, the discovery and development of small molecules for the inhibition of α-syn and tau aggregation can be a potentially effective strategy to delay neurodegeneration. Urea is a chaotropic agent that alters protein solubilization and hydrophobic interactions and inhibits protein aggregation and precipitation. The presence of three hetero atoms (O/S and N) in proximity can coordinate with neutral, mono, or dianionic groups to form stable complexes in the biological system. Therefore, in this study, we evaluated urea and thiourea linkers with various substitutions on either side of the carbamide or thiocarbamide functionality to compare the aggregation inhibition of α-syn and tau. A thioflavin-T (ThT) fluorescence assay was used to evaluate the level of fibril formation and monitor the anti-aggregation effect of the different compounds. We opted for transmission electron microscopy (TEM) as a direct means to confirm the anti-fibrillar effect. The oligomer formation was monitored via the photoinduced cross-linking of unmodified proteins (PICUP). The anti-inclusion and anti-seeding activities of the best compounds were evaluated using M17D intracellular inclusion and biosensor cell-based assays, respectively. Disaggregation experiments were performed with amyloid plaques extracted from AD brains. The analogues with indole, benzothiazole, or N,N-dimethylphenyl on one side with halo-substituted aromatic moieties had shown less than 15% cutoff fluorescence obtained with the ThT assay. Our lead molecules 6T and 14T reduced α-syn oligomerization dose-dependently based on the PICUP assays but failed at inhibiting tau oligomer formation. The anti-inclusion effect of our lead compounds was confirmed using the M17D neuroblastoma cell model. Compounds 6T and 14T exhibited an anti-seeding effect on tau using biosensor cells. In contrast to the control, disaggregation experiments showed fewer Aβ plaques with our lead molecules (compounds 6T and 14T). Pharmacokinetics (PK) mice studies demonstrated that these two thiourea-based small molecules have the potential to cross the blood-brain barrier in rodents. Urea and thiourea linkers could be further improved for their PK parameters and studied for the anti-inclusion, anti-seeding, and disaggregation effects using transgenic mice models of neurodegenerative diseases.

Cavity Lasing of Thioflavin T in the Condensed Phase for Discrimination between Surface Interaction and β-Sheet Groove Binding in Alzheimer-Linked Peptides.

This study investigates the lasing effects in a Fabry-Perot cavity to discern the binding interactions of thioflavin T (ThT) with various peptides associated with Alzheimer's disease, including Aβ(1-42), KLVFFA, and diphenylalanine (FF) in the condensed phase. Utilizing kinetic lasing measurements, the research explores ThT emission enhancements due to specific groove binding in β-sheet structures and highlights additional contributions from weak surface interactions and solvent-solute interactions. Lasing spectroscopy reveals a lack of transition of the FF system from its native state to an amyloid-like structure, challenging traditional ThT assay interpretations. These findings show the potential of lasing spectroscopy in elucidating the molecular basis of amyloid fibril formation and the development of diagnostic tools for amyloidogenic diseases.

The islet tissue plasminogen activator/plasmin system is upregulated with human islet amyloid polypeptide aggregation and protects beta cells from aggregation-induced toxicity

Aims/hypothesisApart from its fibrinolytic activity, the tissue plasminogen activator (tPA)/plasmin system has been reported to cleave the peptide amyloid beta, attenuating brain amyloid deposition in Alzheimer’s disease. As aggregation of human islet amyloid polypeptide (hIAPP) is toxic to beta cells, we sought to determine whether activation of the fibrinolytic system can also reduce islet amyloid deposition and its cytotoxic effects, which are both observed in type 2 diabetes.MethodsThe expression of Plat (encoding tPA) and plasmin activity were measured in isolated islets from amyloid-prone hIAPP transgenic mice or non-transgenic control islets expressing non-amyloidogenic mouse islet amyloid polypeptide cultured in the absence or presence of the amyloid inhibitor Congo Red. Plat expression was also determined in hIAPP-treated primary islet endothelial cells, bone marrow-derived macrophages (BMDM) and INS-1 cells, in order to determine the islet cell type(s) producing tPA in response to hIAPP aggregation. Cell-free thioflavin-T assays and MS were used to respectively monitor hIAPP aggregation kinetics and investigate plasmin cleavage of hIAPP. Cell viability was assessed in INS-1 beta cells treated with hIAPP with or without plasmin. Finally, to confirm the findings in human samples, PLAT expression was measured in freshly isolated islets from donors with and without type 2 diabetes.ResultsIn isolated islets from transgenic mice, islet Plat expression and plasmin activity increased significantly with the process of amyloid deposition (p≤0.01, n=5); these effects were not observed in islets from non-transgenic mice and were blocked by Congo Red (p≤0.01, n=4). In response to hIAPP exposure, Plat expression increased in BMDM and INS-1 cells vs vehicle-treated cells (p≤0.05, n=4), but not in islet endothelial cells. Plasmin reduced hIAPP fibril formation in a dose-dependent manner in a cell-free system, and restored hIAPP-induced loss of cell viability in INS-1 beta cells (p≤0.01, n=5). Plasmin cleaved monomeric hIAPP, inducing a rapid decrease in the abundance of full-length hIAPP and the appearance of hIAPP 1–11 and 12–37 fragments. hIAPP 12–37, which contains the critical amyloidogenic region, was not toxic to INS-1 cells. Finally, PLAT expression was significantly increased by 2.4-fold in islets from donors with type 2 diabetes (n=4) vs islets from donors without type 2 diabetes (n=7) (p≤0.05).Conclusions/interpretationThe fibrinolytic system is upregulated in islets with hIAPP aggregation. Plasmin rapidly degrades hIAPP, limiting its aggregation into amyloid and thus protecting beta cells from hIAPP-induced toxicity. Thus, increasing islet plasmin activity might be a strategy to limit beta cell loss in type 2 diabetes.Graphical

Upcycling of Defatted Sesame Seed Meal via Protein Amyloid-Based Nanostructures: Preparation, Characterization, and Functional and Antioxidant Attributes.

Herein, the possibility of valorizing defatted sesame seed meal (DSSM) as a viable source for valuable plant proteins and amyloid-based nanostructure was investigated. Sesame seed protein isolate (SSPI) and the major storage protein globulin (SSG) were prepared by alkaline extraction-isoelectric point precipitation as well as fractionation in the case of SSG. The protein samples were characterized for their physicochemical attributes. SSPI and SSG were also evaluated for their ability to form amyloid structures under heating (90 °C) at low pH (2.0). Additionally, the functional attributes, antioxidant activity, and biocompatibility of the proteins and amyloid nanostructures were also examined. SSPI and SSG were both successfully prepared from DSSM. The data showed that the physicochemical attributes of both protein samples were quite similar, except for the fact that SSG was mostly composed of 11S globulin, as evinced by Tricine-SDS-PAGE analysis. TEM micrographs revealed that SSG was able to form curly-shaped fibrillar amyloid structures, whereas those derived from SSPI were mostly amorphous. Thioflavin-T assay and Tricine-SDS-PAGE analysis indicated that acidic heating promoted protein hydrolysis and self-aggregation of the hydrolyzed peptides into a β-sheet rich amyloid structure. Importantly, the amyloid preparations displayed commendable solubility, superior water and oil holding capacities, and antioxidant activity against DPPH and ABTS. The protein amyloid nanostructures were found to be non-toxic against RAW264.7 cells, HaCaT cells, and red blood cells. These findings indicate that DSSM could be upcycled into valuable protein amyloid structures with good potentialities as novel food ingredients.

Monitoring insulin fibrillation kinetics using chromatographic analysis

Insulin is a small protein widely used to treat patients with diabetes and is a commonly used model for protein fibrillation studies. Under specific conditions, such as low pH and high temperature, insulin monomers aggregate to form fibrils. This aggregation is problematic for manufacturing and storage of insulin. The thioflavin T (ThT) assay is commonly used to study amyloid fibrillation but suffers from several limitations, such as the effect of protein concentration, the size of the amyloid fibrillar bundles, competitive binding, and fibril aggregation, all of which hinder precise quantitative analysis. Here, we present a method for studying the kinetics of insulin fibrillation utilizing ultra-performance liquid chromatography (UPLC). This method enables the quantitative detection of soluble insulin components, including chemically modified components. The formation of a deamidated species could be monitored at the early stage of fibrillation, and this species was likely included in the fibrils. In addition, in the presence of inhibitors known to compete with ThT for binding to fibrils, UPLC analysis showed the disappearance of soluble components even though the ThT assay did not indicate the presence of fibrils. These results suggest that the UPLC-based analysis presented here can complement the ThT assay for investigating the kinetics of protein fibrillation.

Lead optimization based design, synthesis, and pharmacological evaluation of quinazoline derivatives as multi-targeting agents for Alzheimer's disease treatment

The complexity and multifaceted nature of Alzheimer's disease (AD) have driven us to further explore quinazoline scaffolds as multi-targeting agents for AD treatment. The lead optimization strategy was utilized in designing of new series of derivatives (AK-1 to AK-14) followed by synthesis, characterization, and pharmacological evaluation against human cholinesterase's (hChE) and β-secretase (hBACE-1) enzymes. Amongst them, compounds AK-1, AK-2, and AK-3 showed good and significant inhibitory activity against both hAChE and hBACE-1 enzymes with favorable permeation across the blood-brain barrier. The most active compound AK-2 revealed significant propidium iodide (PI) displacement from the AChE-PAS region and was non-neurotoxic against SH-SY5Y cell lines. The lead molecule (AK-2) also showed Aβ aggregation inhibition in a self- and AChE-induced Aβ aggregation, Thioflavin-T assay. Further, compound AK-2 significantly ameliorated Aβ-induced cognitive deficits in the Aβ-induced Morris water maze rat model and demonstrated a significant rescue in eye phenotype in the Aꞵ-phenotypic drosophila model of AD. Ex-vivo immunohistochemistry (IHC) analysis on hippocampal rat brains showed reduced Aβ and BACE-1 protein levels. Compound AK-2 suggested good oral absorption via pharmacokinetic studies and displayed a good and stable ligand-protein interaction in in-silico molecular modeling analysis. Thus, the compound AK-2 can be regarded as a lead molecule and should be investigated further for the treatment of AD.

C9-Functionalized Doxycycline Analogs as Drug Candidates to Prevent Pathological α-Synuclein Aggregation and Neuroinflammation in Parkinson's Disease Degeneration.

Doxycycline, a semi-synthetic tetracycline, is a widely used antibiotic for treating mild-to-moderate infections, including skin problems. However, its anti-inflammatory and antioxidant properties, combined with its ability to interfere with α-synuclein aggregation, make it an attractive candidate for repositioning in Parkinson's disease. Nevertheless, the antibiotic activity of doxycycline restricts its potential use for long-term treatment of Parkinsonian patients. In the search for non-antibiotic tetracyclines that could operate against Parkinson's disease pathomechanisms, eighteen novel doxycycline derivatives were designed. Specifically, the dimethyl-amino group at C4 was reduced, resulting in limited antimicrobial activity, and several coupling reactions were performed at position C9 of the aromatic D ring, this position being one of the most reactive for introducing substituents. Using the Thioflavin-T assay, we found seven compounds were more effective than doxycycline in inhibiting α-synuclein aggregation. Furthermore, two of these derivatives exhibited better anti-inflammatory effects than doxycycline in a culture system of microglial cells used to model Parkinson's disease neuroinflammatory processes. Overall, through structure-activity relationship studies, we identified two newly designed tetracyclines as promising drug candidates for Parkinson's disease treatment.

Design, synthesis, and biological evaluation of some 2-(3-oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-yl)-N-phenylacetamide hybrids as MTDLs for Alzheimer's disease therapy

Inspite of established symptomatic relief drug targets, a multi targeting approach is highly in demand to cure Alzheimer's disease (AD). Simultaneous inhibition of cholinesterase (ChE), β secretase-1 (BACE-1) and Dyrk1A could be promising in complete cure of AD. A series of 18 diaryl triazine based molecular hybrids were successfully designed, synthesized, and tested for their hChE, hBACE-1, Dyrk1A and Aβ aggregation inhibitory potentials. Compounds S-11 and S-12 were the representative molecules amongst the series with multi-targeted inhibitory effects. Compound S-12 showed hAChE inhibition (IC50 value = 0.486 ± 0.047 μM), BACE-1 inhibition (IC50 value = 0.542 ± 0.099 μM) along with good anti-Aβ aggregation effects in thioflavin-T assay. Only compound S-02 of the series has shown Dyrk1A inhibition (IC50 value = 2.000 ± 0.360 μM). Compound S-12 has also demonstrated no neurotoxic liabilities against SH-SY5Y as compared to donepezil. The in vivo behavioral studies of the compound S-12 in the scopolamine- and Aβ-induced animal models also demonstrated attanuation of learning and memory functions in rats models having AD-like characteristics. The ex vivo studies, on the rat hippocampal brain demonstrated reduction in certain biochemical markers of the AD brain with a significant increase in ACh level. The Western blot and Immunohistochemistry further revealed lower tau, APP and BACE-1 molecular levels. The drosophilla AD model also revealed improved eyephenotype after treatment with compound S-12. The molecular docking studies of the compounds suggested that compound S-12 was interacting with the ChE-PAS & CAS residues and catalytic dyad residues of the BACE-1 enzymes. The 100 ns molecular dynamics simulation studies of the ligand-protein complexed with hAChE and hBACE-1 also suggested stable ligand–protein confirmation throughout the simulation run.

Structural insights into the interactions of repositioning and known drugs for Alzheimer’s disease with hen egg white lysozyme by MM-GBSA

Six drugs (dapsone, diltiazem, timolol, rosiglitazone, mesalazine, and milnacipran) that were predicted by network-based polypharmacology approaches as potential anti-Alzheimer’s drugs, have been subjected in this study for in silico and in vitro evaluation to check their potential against protein fibrillation, which is a causative factor for multiple diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington disease, cardiac myopathy, type-II diabetes mellitus and many others. Molecular docking and thereafter molecular dynamics (MD) simulations revealed that diltiazem, rosiglitazone, and milnacipran interact with the binding residues such as Asp52, Glu35, Trp62, and Asp101, which lie within the fibrillating region of HEWL. The MM-GBSA analysis revealed −7.86, −5.05, and −10.29 kcal/mol as the binding energy of diltiazem, rosiglitazone, and milnacipran. The RMSD and RMSF calculations revealed significant stabilities of these ligands within the binding pocket of HEWL. While compared with two reported ligands inhibiting HEWL fibrillation, milnacipran depicted almost similar binding potential with one of the known ligands (Ligand binding affinity −10.66 kcal/mol) of HEWL. Furthermore, secondary structure analyses revealed notable inhibition of the secondary structural changes with our candidate ligand; especially regarding retention of the 3/10 α-helix both by DSSP simulation, Circular dichroism, and FESEM-based microscopic image analyses. Taking further into experimental validation, all three ligands inhibited fibrillation in HEWL in simulated conditions as revealed by blue shift in Congo red assay and later expressing percentage inhibition in ThioflavinT assay as well. However, dose-dependent kinetics revealed that the antifibrillatory effects of drugs are more pronounced at low protein concentrations. Communicated by Ramaswamy H. Sarma

Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aβ) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aβ oligomers (AβOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AβOs have proven challenging because AβOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aβ peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aβ peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aβ core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a β-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AβOs. Our approach represents a significant advancement in the isolation and characterization of β-sheet rich AβOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

1,4-Diurea- and 1,4-Dithiourea-Substituted Aromatic Derivatives Selectively Inhibit α-Synuclein Oligomer Formation In Vitro.

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting the elderly population worldwide. In PD, the misfolding of α-synuclein (α-syn) results in the formation of inclusions referred to as Lewy bodies (LB) in midbrain neurons of the substantia nigra and other specific brain localizations, which is associated with neurodegeneration. There are no approved strategies to reduce the formation of LB in the neurons of patients with PD. Our drug discovery program focuses on the synthesis of urea and thiourea compounds coupled with aminoindole moieties to abrogate α-syn aggregation and to slow down the progression of PD. We synthesized several urea and thiourea analogues with a central 1,4-phenyl diurea/thiourea linkage and evaluated their effectiveness in reducing α-syn aggregation with a special focus on the selective inhibition of oligomer formation among other proteins. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), as well as M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. Our results identified compound 1 as the best compound in reducing α-syn fibril formation via ThT assays. The antioligomer formation of compound 1 was subsequently superseded by compound 2. Both compounds selectively curtailed the oligomer formation of α-syn but not tau 4R isoforms (0N4R, 2N4R) or p-tau (isoform 1N4R). Compounds 1 and 2 failed to abrogate tau 0N3R fibril formation by ThT and atomic force microscopy. Compound 2 was best at reducing the formation of recombinant α-syn fibrils by TEM. In contrast to compound 2, compound 1 reduced the formation of α-syn inclusions in M17D neuroblastoma cells in a dose-dependent manner. Compound 1 may provide molecular scaffolds for the optimization of symmetric molecules for its α-syn antiaggregation activity with potential therapeutic applications and development of small molecules in PD.

In silico and in vitro assessment of the anti-β-amyloid aggregation and anti-cholinesterase activities of Ptaeroxylon obliquum and Bauhinia bowkeri extracts

BackgroundAlzheimer’s disease (AD) is the most common form of dementia and dementia constitutes the fifth leading cause of mortality across the globe. Available treatment modalities and drugs have abysmally failed to curtail AD. This study evaluated the mitigation of Aβ aggregation and anti-cholinesterase activities with the crude extracts of Ptaeroxylon obliquum and Bauhinia bowkeri. Computational studies of the most abundant phytochemicals from the crude extracts of both plants with proteins were investigated. The phytochemical composition of the different crude extracts (hexane, DCM, and ethanol) of the plants were analyzed with FTIR and GC-MS. The inhibitory potential of the extracts on BACE-1 and cholinesterase activities was determined with both computational molecular docking studies and in vitro enzyme assays. Their anti-aggregation properties were confirmed with Thioflavin-T assay and TEM. ResultsThe in silico studies revealed that though thunbergol and cyclotetradecatriene (the major constituents of the extracts) inhibited all the proteins, the latter exhibited the best inhibitory potential. The in vitro results showed that while the dichloromethane (DCM) extract of P. obliquum had the highest butyrylcholinesterase (BuChE) inhibitory activity (1.77 µg/ml), the hexane and ethanol extract of B. bowkeri exhibited the highest β-site amyloid precursor cleaving enzymes-1 (BACE-1) (30.4 µg/ml) and acetylcholinesterase (AChE) (58.11 µg/ml) inhibitory efficacy, respectively. The ethanol extract (160 μg/ml) of B. bowkeri had the most efficacious anti-aggregation activity. ConclusionsThis study suggests that the plants could possess neuroprotective effects and could also be sources of anti-AD novel drugs.How to cite: Ojo MC, Mosa RA, Osunsanmi FO, et al. In silico and in vitro assessment of the anti-β-amyloid aggregation and anti-cholinesterase activities of Ptaeroxylon obliquum and Bauhinia bowkeri extracts. Electron J Biotechnol 2024;68. https://doi.org/10.1016/j.ejbt.2023.11.004.

Multi-Target Actions of Flavonoid Derivatives from Mesua ferrea Linn Flower against Alzheimer’s disease Pathogenesis

OBJECTIVE Kaempferol-3-O-rhamnoside (compound 1) and quercetin- 3-O-rhamnoside (compound 2), two flavonoids isolated from Mesua ferrea L. flowers, were examined for their activities related Alzheimer’s disease (AD) pathogenesis including antioxidant, acetylcholinesterase (AChE) inhibition, anti-beta amyloid (Ab) aggregation and neuroprotection. METHODS The two flavonoids were isolated from M. ferrea L. flowers using the column chromatography technique. Both compounds were evaluated for their effects on AD pathogenesis, including antioxidant action by ABTS assay, AChE inhibition by Ellman’s method, and anti-Ab aggregation by thioflavin T (ThT) assay and neuroprotection by cell base assay. To explain the mechanism of AChE inhibition and anti-Ab aggregation, binding interactions between the test compounds and AChE and Ab were studied in-silico. RESULTS Compounds 1 and 2 showed an ability to scavenge ABTS radicals, with IC50 values of 424.57±2.97 and 308.67±9.90 µM, respectively, and to inhibit AChE function with IC50 values of 769.23±6.23 and 520.64±5.94, respectively. ThT assay indicated that both compounds inhibited Ab aggregation with IC50 values of 406.43±9.95 and 300.69 ±1.18 µM, respectively. The neuroprotection study revealed that the two flavonoids could reduce human neuroblastoma (SH-SY5Y) cell death induced by H2O2. The in-silico study showed that both compounds bound AChE at catalytic anionic and peripheral anionic sites. In addition, the test compounds prevented Ab aggregation by interacting at the central hydrophobic core, the C-terminal hydrophobic region, and the important residues of Ile41. CONCLUSIONS Together, the results showed that kaempferol-3-O- rhamnoside and quercetin-3-O- rhamnoside exhibit multiple mechanisms of action that are involved in the pathogenesis of AD including antioxidant, AChE inhibition, anti-Ab aggregation, and neuroprotection. KEYWORDS flavonoid rhamnosides, Alzheimer’s disease, oxidation, beta amyloid, acetylcholinesterase, molecular docking

The role of the biofilm of Porphyromonas gingivalis in driving amyloid beta secretion and aggregation

AbstractBackgroundOver the past five years, the hypothesis linking periodontitis to Alzheimer’s disease (AD) has gained significant traction even though little is known about the underlying mechanisms driving this association. Periodontitis is caused by the dysbiosis of the dental biofilm, an extra‐cellular matrix produced by bacteria, which is mostly caused by the Gram‐negative bacterium Porphyromonas gingivalis. P. gingivalis has been found to infect the brain of AD patients and drive amyloid beta (Aβ) production while biofilm components can be identified interwoven with Aβ plaques1,2. We therefore hypothesize that P. gingivalis drives Aβ production in neural cells which co‐aggregates with bacterial biofilm to expedite plaque formation and downstream neuroinflammation.MethodTo assess if the biofilm of co‐aggregation of Aβ and the matrix was then analyzed by fluorescence microscopy, atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assays. To examine whether P. gingivalis stimulates Aβ production, we infected BE(2)‐M17 human neuroblastomas at a mean of infection of 1:100 before collecting cells and supernatant proteins to observe the cell response through protein and transcription assays.ResultsWhen co‐cultured, P. gingivalis and Aβ1‐42 co‐aggregated in plaque‐like structures, Interestingly, colocalization analysis revealed that Aβ1‐42 particularly associate with the extracellular component of biofilm (Figure 1). ThT assays and AFM imaging demonstrated faster aggregation of Aβ1‐42 when co‐incubated with fragments of P. gingivalis biofilm for 8 h, which suggests a significant cross‐seeding phenomenon between the two components (Figure 2). Furthermore, infection of human neuroblastomas resulted in a significant increase in Aβ production that could be detected by western blot, thus offering a workable model to study the link between AD and periodontitis (Figure 3).ConclusionOur project offers significant insights into the relationship between periodontitis and AD, allowing us to understand how P. gingivalis triggers Aβ production and how its biofilm drives plaque aggregation. This research greatly contributes to a better understanding of external AD risk factors and how to mediate their impacts on neurodegeneration. 1Dominy et al. Science Advances (2019) 2Mirzaei et al. Microbial Pathogenesis (2020)

Anti-Glycation, Anti-β-Amyloid Aggregation, and Antioxidant Effect of Cassia Seed-Derived Secondary Metabolites

Aggregation and non-enzymatic glycation of amyloidogenic peptides, amyloid-beta (Aβ), and insulin are key features of neuropathology. In this study, we evaluate the anti-glycation effect of Cassia seed-derived secondary metabolites on human insulin and bovine serum albumin, as well as their anti-Aβ aggregation and antioxidant effects using in vitro spectrofluorometric method, thioflavin T fluorescence, and peroxynitrite (ONOO‒) scavenging assay. Furthermore, molecular docking simulation was performed to investigate the binding characteristics of test compounds and Aβ42 peptide. Among 38 compounds, anthraquinones 9–12 and 14–18; naphthopyrones 24, 25, 27, and 33–36; and 38 from the naphthalenes and naphthalenic lactone groups showed moderate-to-good inhibition of AGE formation with IC50 values ranging from 7.52 ± 1.19 to 155.86 ± 0.79 µM. Likewise, compounds 5, 24, 15, 16, 27, and 20 showed good inhibition of D-ribose-mediated glycation of human insulin, with an IC50 value range of 46.37 ± 4.06 to 97.69 ± 7.88 µM. In the thioflavin-T assay, compounds 8 and 12 showed promising inhibition of Aβ aggregation, comparable to that of the reference compound morin. Molecular docking simulations confirmed that these active compounds have strong potential to interact with Aβ42 peptides and interrupt their self-assembly and conformational transformation, thereby inhibiting Aβ42 aggregation. In addition, compounds 5, 8, 10, 14, and 35 scavenged ONOO− at low concentrations. Overall, Cassia compounds’ anti-glycation, anti-Aβ aggregation, and antioxidant effects warrant further in vivo studies to evaluate their potential neuroprotective effects against comorbid AD and diabetes.

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-β (Aβ) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated α-synuclein (α-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant Aβ plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing α-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated Aβ-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of α-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant α-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of α-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller Aβ-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies.

Effect of cycloastragenol and punicalagin on Prp(106–126) and Aβ(25–35) oligomerization and fibrillizaton

Numerous neurological disorders, including prion, Parkinson's, and Alzheimer's disease (AD), are identified as being caused by alterations in protein conformation, aggregation, and metal ion dyshomeostasis. Recent years have seen a significant increase in the exploration and study of natural products (NPs) from plant and microbial sources for their therapeutic potential against several diseases, including cancer, diabetes, cardiovascular disease, and neurodegenerative diseases. In this study, we have examined the effect of two NPs, cycloastragenol (CAG) and punicalagin (PCG), on the metal-induced oligomerization and aggregation of Aβ25–35 and PrP106–126 peptides. The peptide aggregation and inhibitory properties of both NPs were examined by the thioflavin-T (ThT) assay, MALDI-TOF, circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM). Among the two NPs, PCG significantly binds to the peptides, chelates metal ions (Cu2+ and Zn2+), inhibits peptide aggregation, substantially reduces oxidative stress, and controls the production of reactive oxygen species (ROS). Both NPs exhibited low cytotoxicity and prominently mitigated peptide-mediated cell cytotoxicity in hippocampal neuronal HT-22 cells by covalent bonding and hydrophobic interactions.