Disaggregation Activity Research Articles

Design, synthesis and biological evaluation of 2-styryl-5-hydroxy-4-pyrone derivatives and analogues as multiple functional agents with the potential for the treatment of Alzheimer’s disease

A novel series of 2-styryl-5-hydroxy-4-pyrone derivatives and analogues were designed and synthesized as H3 receptor antagonism based multitarget-directed ligands (MTDLs) for AD therapy using pharmacophore-combine strategy. The 2-styryl-5-hydroxy-4-pyrone pharmacophore with metal ion chelation, antioxidation, and Aβ aggregation inhibition activities was employed as the “eastern part”, and a typical phenoxyalkylamine moiety was used as “central ring + western part” of the H3 receptor antagonist. The biological evaluation revealed that the majority of the target compounds demonstrated desirable multiple functions. The two most promising compounds 8a and 8b exhibited nanomolar IC50 values on H3 receptor antagonism, excellent metal ion chelating capability, more potent ABTS+ scavenging activity than Trolox, efficient Aβ self-aggregation and Cu2+-induced aggregation inhibitory activities, as well as disaggregation activities against Aβ self/Cu2+-induced aggregation.

Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens

This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB’s protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.

Entropic Inhibition: How the Activity of a AAA+ Machine Is Modulated by Its Substrate-Binding Domain.

ClpB is a tightly regulated AAA+ disaggregation machine. Each ClpB molecule is composed of a flexibly attached N-terminal domain (NTD), an essential middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains. The NTD is not well-characterized structurally and is commonly considered to serve as a dispensable substrate-binding domain. Here, we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB’s NTD and reveal its unexpected autoinhibitory function. We find that the NTD fluctuates on the microsecond time scale, and these dynamics result in steric hindrance that limits the conformational space of the MD to restrict its tilting. This leads to significantly inhibited ATPase and disaggregation activities of ClpB, an effect that is alleviated upon binding of a substrate protein or the cochaperone DnaK. This entropic inhibition mechanism, which is mediated by ultrafast motions of the NTD and is not dependent on any strong interactions, might be common in related ATP-dependent proteases and other multidomain proteins to ensure their fast and reversible activation.

Effects of clovamide and its related compounds on the aggregations of amyloid polypeptides.

Alzheimer's disease (AD) and type 2 diabetes (T2D) are common diseases in the elderly, and the increasing number of patients with these diseases has become a serious health problem worldwide. The aggregation and development of plaque of amyloid polypeptides (amyloid β; Aβ and human islet amyloid polypeptide; hIAPP, amylin) are found in the brains of patients with AD and the pancreas of patients with T2D and are considered to be, in part, the causes of both diseases, respectively. Therefore, preventing amyloid aggregation may be a promising therapeutic strategy for preventing AD and T2D. In addition, the disaggregation of the already aggregated amyloid polypeptides is expected to contribute to the prevention and treatment of both diseases as amyloid polypeptide aggregations begin several decades before the onset of disease. Therefore, in this study, we investigated the hIAPP aggregation inhibitory activity and Aβ42/hIAPP disaggregation activity of clovamide which had shown inhibitory activity against Aβ42 aggregation in our previous studies. In addition, active sites were identified (structure-activity relationship analysis) using clovamide-related compounds in which hydroxyl groups of these compounds were either eliminated or methylated. Our results showed that the compounds with one or two catechol moieties showed strong hIAPP aggregation inhibitory activity and Aβ42/hIAPP disaggregation activity; and the non-catechol type compounds showed little or no activity. This suggests that the catechol moiety is important in amyloid polypeptide aggregation inhibition and disaggregation. Thus, clovamide and its related compounds may be promising therapeutic strategies for inhibiting amyloid polypeptide-related pathology in AD and T2D.

Basic mechanism of the autonomous ClpG disaggregase

Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)–ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability.

A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K‐12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K‐12, the 4.88 Mbp Fec10 genome is characterized by distinct single‐nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two‐component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand‐alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI‐Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI‐SG17M. Furthermore, we noted a unique contribution of ClpGGI‐Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food‐borne heat‐resistant strains in the human gut.

Designing Specific HSP70 Substrate Binding Domain Inhibitor for Perturbing Protein Folding Pathways to Inhibit Cancer Mechanism.

HSP70 is a survival factor for tumor cells in transformation and in tumor progression as well as in anti-apoptotic response. Several inhibitors targeting HSP70 ATPase function displayed off-target effects, but PES, which targets the HSP70 substrate binding domain, prevents tumor cell survival prominently. However, PES may not bind HSP70 in the absence of nucleotide. This research aimed to design a unique inhibitor molecule that works both in the presence and absence of nucleotides to amplify inhibition. A set of chimeric coumarine-pyrazole derivatives were determined by in silico techniques and synthesized to elucidate their inhibitory effects. Cell viability experiments displayed KBR1307 as the most efficient inhibitor. A set of characterization experiments were performed, and the results were compared to that of PES agent. Binding constant, ATP hydrolysis rate, and percent aggregation were determined in the presence and absence of inhibitors. In silico docking experiments showed that only KBR1307 binds the HSP70 substrate binding domain and interacts with cochaperone interface. Binding experiments indicated that KBR1307 binds HSP70 both in the presence and absence of nucleotides, but PES does not. Both inhibitors significantly lower HSP70 ATPase activity and substrate protein disaggregation activity. However, KBR1307 displays a lower IC50 value at the MCF-7 cell line compared to PES. Both inhibitors do not alter HSP70 secondary structure composition and overall stability. KBR1307 effectively inhibits HSP70 compared to PES and provides a promising template for novel anticancer drug development.

Inhibition of Amyloid Fibrillation of HEWL by 4-Methylcoumarin and 4-Methylthiocoumarin Derivatives.

Several human diseases like Parkinson's, Alzheimer's disease, and systemic amyloidosis are associated with the misfolding and aggregation of protein molecules. The present study demonstrated the comparison of 4-methyl coumarin and 4-methylthiocoumarin derivative for their anti-amyloidogenic and disaggregation activities. The hen egg-white lysozyme is used as a model system to study protein aggregation and disaggregation under in vitro conditions. Techniques used in the study were Thioflavin T fluorescence assay, intrinsic fluorescence assay, circular dichroism, transmission electron microscopy, and molecular dynamics. Fifteen compounds were screened for their anti-amyloidogenic and disaggregation potential. Six compounds significantly inhibited the fibril formation, whereas ten compounds showed disaggregation property of pre-formed fibrils. Under in vitro conditions, the compound C3 and C7 showed significant inhibition of fibril formation in a concentration-dependent manner as compared to control. C3 and C7 demonstrated 93% and 76% inhibition of fibril formation, respectively. Furthermore, C3 and C7 exhibited 83% and 76% disaggregation activity, respectively, of pre-formed HEWL fibrils at their highest concentration. These anti-amyloidogenic and disaggregation potential of C3 and C7 were validated by intrinsic fluorescence, CD, molecular dynamics, and TEM study. 4-methylthiocoumarins derivatives have shown better anti-amyloidogenic activity as compared to 4-methylcoumarin derivatives for both amyloid formation as well as disaggregation of preformed amyloid fibrils. Structurally, the derivatives of 4-methylthiocoumarins (C3 and C7) contain thio group on 2nd position that might be responsible for anti-amyloidogenic activity as compared to 4- methylcoumarin derivatives (C2 and C4). C3 and C7 are novel 4-methylthiocoumarin derivatives that can be used as a lead for alleviation and symptoms associated with protein aggregation disorders.

Fullerenes as an Effective Amyloid Fibrils Disaggregating Nanomaterial.

Nowadays, determining the disassembly mechanism of amyloids under nanomaterials action is a crucial issue for their successful future use in therapy of neurodegenerative and overall amyloid-related diseases. In this study, the antiamyloid disassembly activity of fullerenes C60 and C70 dispersed in 1-methyl-2-pyrrolidinone (NMP) toward amyloid fibrils preformed from lysozyme and insulin was investigated using a combination of different experimental techniques. Thioflavin T fluorescence assay and atomic force microscopy were applied for monitoring of disaggregation activity of fullerenes. It was demonstrated that both types of fullerene-based complexes are very effective in disassembling preformed fibrils, and characterized by the low apparent half-maximal disaggregation concentration (DC50) in the range of ∼22-30 μg mL-1. Small-angle neutron scattering was employed to monitor the different stages of the disassembly process with respect to the size and morphology of the aggregates. Based on the obtained results, a possible disassembly mechanism for amyloid fibrils interacting with fullerene/NMP complexes was proposed. The study is a principal step in understanding of the fullerenes destruction mechanism of the protein amyloids, as well as providing valuable information on how macromolecules can be engineered to disassemble unwanted amyloid aggregates by different mechanisms.

The HSP110/HSP70 disaggregation system generates spreading‐competent toxic α‐synuclein species

The accumulation and prion‐like propagation of α‐synuclein and other amyloidogenic proteins are associated with devastating neurodegenerative diseases. Metazoan heat shock protein HSP70 and its co‐chaperones DNAJB1 and HSP110 constitute a disaggregation machinery that is able to disassemble α‐synuclein fibrils in vitro, but its physiological effects on α‐synuclein toxicity are unknown. Here, we depleted Caenorhabditis elegans HSP‐110 and monitored the consequences on α‐synuclein‐related pathological phenotypes such as misfolding, intercellular spreading, and toxicity in C. elegans in vivo models. Depletion of HSP‐110 impaired HSP70 disaggregation activity, prevented resolubilization of amorphous aggregates, and compromised the overall cellular folding capacity. At the same time, HSP‐110 depletion reduced α‐synuclein foci formation, cell‐to‐cell transmission, and toxicity. These data demonstrate that the HSP70 disaggregation activity constitutes a double‐edged sword, as it is essential for maintaining cellular proteostasis but also involved in the generation of toxic amyloid‐type protein species.

Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence.

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the ΔclpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.

Tacrine-hydroxamate derivatives as multitarget-directed ligands for the treatment of Alzheimer’s disease: Design, synthesis, and biological evaluation

In order to develop multitarget-directed ligands as potential treatments for Alzheimer's disease, twenty-eight new tacrine-hydroxamate derivatives were designed, synthesized, and biologically evaluated. As expected, most of the compounds exhibited inhibitory activities against cholinesterases (ChEs) and histone deacetylase (HDACs). Among the tested compounds, A10 showed not only potent and selective inhibition on AChE at sub-nanomolar potency (AChEIC50=0.12nM, BChEIC50=361.52nM) but also potent inhibition on HDAC (IC50=0.23nM). Moreover, A10 exhibited inhibitory activity on Aβ1-42 self-aggregation as well as disaggregation activity on pre-formed Aβ fibrils. Furthermore, A10 exhibited antioxidant activity and metal chelating properties. Further mechanistic studies demonstrated that A10 is a pan-inhibitor of HDACs and a mixed-type inhibitor for AChE. It shown that A10 is a BBB penetrant by online prediction. Taken together, the results indicate that A10 can serve as a lead compound to develop promising candidate analogs as AD therapeutics.

Lanosterol modulates proteostasis via dissolving cytosolic sequestosomes/aggresome-like induced structures

Sequestration of misfolded proteins into distinct cellular compartments plays a pivotal role in proteostasis and proteopathies. Cytoplasmic ubiquitinated proteins are sequestered by p62/SQSTM1 to deposit in sequestosomes or aggresome-like induced structures (ALIS). Most aggresome or ALIS regulators identified thus far are recruiters, while little is known about the disaggregases or dissolvers. In this research, we showed that lanosterol synthase and its enzymatic product lanosterol effectively reduced the number and/or size of sequestosomes/ALIS/aggresomes formed by endogenous proteins in the HeLa and HEK-293A cells cultured under both non-stressed and stressed conditions. Supplemented lanosterol did not affect the proteasome and autophagic activities, but released the trapped proteins from the p62-positive inclusions accompanied with the activation of HSF1 and up-regulation of various heat shock proteins. Our results suggested that the coordinated actions of disaggregation by lanosterol and refolding by heat shock proteins might facilitate the cells to recycle proteins from aggregates. The disaggregation activity of lanosterol was not shared by cholesterol, indicating that lanosterol possesses additional cellular functions in proteostasis regulation. Our findings highlight that besides protein modulators, the cells also possess endogenous low-molecular-weight compounds as efficient proteostasis regulators.

The effect of chlorin e<sub>6</sub> drug on platelet aggregation activity

The goal of the study is to evaluate the effect of Radachlorin (OOO “RADA-PHARMA”, Russia) (RC) on platelet aggregation in ex vivo and in vivo experiments. The experiments were conducted on male Wistar rats. Platelet aggregation activity was determined in platelet-rich plasma (PRP) using a turbidimetric method and the aggregation inducer was ADP at a final concentration of 1.25 μM. PRP samples containing RC were irradiated with ALOD-Granat laser device (OOO “Alkom Medika”, Russia) at 662 nm wavelength with 0.05 W/cm2 power density. After a 5-minute incubation of PRP with RC in the dark, dose-dependent inhibition of platelet aggregation was observed. Laser irradiation (12.5 J/cm2 and, especially, 25 J/cm2) increased the inhibitory effect of RC. 3 hours after intravenous administration of RC, the rate and intensity of platelets aggregation did not change, while disaggregation slowed down significantly. Irradiation at a dose of 5 J/cm2 did not affect the platelets aggregation kinetics, and disaggregation slowed down even more at 10 J/cm2, and at 20 J/cm2 the rate and intensity of platelets aggregation decreased, and no disaggregation occurred.In vitro, RC inhibited the ADP-induced platelet aggregation in rats in a dose-dependent manner; after laser irradiation, this effect was enhanced significantly. The effect of RC on circulating platelets leads to a change in their functional state, which manifests in slowing down the disaggregation after exposure to ADP. After laser irradiation (10 J/cm2 and, especially, 20 J/cm2), the severity of the functional changes increases. The role of decreasing the disaggregation activity of platelets in the mechanism of vascular thrombosis in the affected area of photodynamic therapy (PDT) is discussed.

Aggregation induced emission active fluorescent sensor for the sensitive detection of Hg2+ based on organic-inorganic hybrid mesoporous material.

We report the preparation of an organic-inorganic hybrid mesoporous material, PHC-SBA-15, derived from the coupling of a pyrene-based derivative PHC and mesoporous SBA-15 silica. Compared with the stable aggregation-induced emission (AIE) properties of PHC, those of PHC-SBA-15 were more promoted and active due to the fixation of PHC and the space limitation in mesoporous SBA-15. The aggregation and disaggregation activities can be tuned by controlling the concentrations in aqueous media and changing the fluorescence color from yellow to blue. In addition to the controllable AIE properties, PHC-SBA-15 was applied for the highly selective and sensitive detection of Hg2+ through the fluorescence quenching of monomeric pyrene in aqueous media. The fluorescence intensity at 395 nm was linearly proportional to that of Hg2+ in the concentration ranges of 0-1.0 × 10-5 and 1.0 × 10-5-10 × 10-5 M, showing a low detection limit of 1.02 × 10-7 M. This work provides an effective strategy for modulating the AIE properties from non-active to active by introducing AIE stable molecule into mesoporous silica material. This method also favors the development of fluorescent sensors for detecting targets with high sensitivity and selectivity in aqueous media with less synthetic difficulties.

Design, synthesis and preliminary in-vitro studies of novel boronated monocarbonyl analogues of Curcumin (BMAC) for antitumor and β-amiloyd disaggregation activity

Curcumin is currently being investigated for its capacity to treat many types of cancer and to prevent the neuron damage that is observed in Alzheimer’s disease (AD). However, its clinical use is limited by its low stability and solubility in aqueous solutions. In this study, we propose a completely new class of boronated monocarbonyl analogues of Curcumin (BMAC, 6a-c), in which a carbonyl group replaces the Curcumin β-diketone functionality, and an ortho-carborane, an icosahedral boron cluster, substitutes one of the two phenolic rings. BMAC antitumor activity against MCF7 and OVCAR-3 cell lines was assessed in vitro and compared to that of Curcumin and the corresponding MAC derivative. BMAC 6a-c showed efficiencies that are comparable to that of MAC and superior to that of Curcumin in both the cell lines. Moreover, the inhibition of the formation of β-amyloid aggregates by BMAC 6a-c was evaluated and it was shown that compound 6c, which contains two OH moieties, has a better efficiency than Curcumin. The presence of a second –OH group can enhance the compound’s binding efficacy with β-amyloid aggregates. For the future, the presence of at least one carborane group means that the BMAC antitumor effect can be coupled with Boron Neutron Capture Therapy.

Interdomain communication suppressing high intrinsic ATPase activity of Sse1 is essential for its co-disaggregase activity with Ssa1.

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70-like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single-molecule FRET, small angle X-ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70-like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open-lid substrate-binding domain (SBD) in close contact with its nucleotide-binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP-hydrolysis-independent exhaustive interaction between two domains of Hsp110s, NBD-SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1-NBD compared to wild-type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1-mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co-chaperoning activity in disaggregase machinery, and stability of the protein.

Multifunctional indanone–chalcone hybrid compounds with anti-β-amyloid (Aβ) aggregation, monoamine oxidase B (MAO-B) inhibition and neuroprotective properties against Alzheimer’s disease

To discover multifunctional agents for the treatment of Alzheimer’s disease (AD), a series of indanone–chalcone hybrid compounds were designed and synthesized based on the multitarget-directed ligand strategy. Their monoamino oxidases (MAO-A and MAO-B) and Aβ1–42 aggregation inhibitory activities were evaluated. The results were shown that all synthetic compounds exhibited mostly good multifunctional activities. Among all, compound TM-11 represented the best Aβ1–42 aggregation inhibitory potency (IC50 ~1.8 μM) and good disaggregation activity (IC50 ~7.9 μM). Both TEM images and docking studies provided good reasonable explanation to the hypothesis. Meanwhile, compound TM-11 was a selective MAO-B inhibitor, as well as a neuroprotective agent against Aβ1–42-induced toxicity. Based on the structural considerations, the lipophilicity of the compound TM-11 could render to pass through blood−brain barrier (BBB) in vitro in accordance with the Lipinski’s rule of five. In conclusion, these results were suggested that compound TM-11 might be a potential multifunctional agent for the treatment of AD.

Oleuropein derivatives from olive fruit extracts reduce α-synuclein fibrillation and oligomer toxicity

Aggregation of α-synuclein (αSN) is implicated in neuronal degeneration in Parkinson's disease and has prompted searches for natural compounds inhibiting αSN aggregation and reducing its tendency to form toxic oligomers. Oil from the olive tree (Olea europaea L.) represents the main source of fat in the Mediterranean diet and contains variable levels of phenolic compounds, many structurally related to the compound oleuropein. Here, using αSN aggregation, fibrillation, size-exclusion chromatography-multiangle light scattering (SEC-MALS)-based assays, and toxicity assays, we systematically screened the fruit extracts of 15 different olive varieties to identify compounds that can inhibit αSN aggregation and oligomer toxicity and also have antioxidant activity. Polyphenol composition differed markedly among varieties. The variety with the most effective antioxidant and aggregation activities, Koroneiki, combined strong inhibition of αSN fibril nucleation and elongation with strong disaggregation activity on preformed fibrils and prevented the formation of toxic αSN oligomers. Fractionation of the Koroneiki extract identified oleuropein aglycone, hydroxyl oleuropein aglycone, and oleuropein as key compounds responsible for the differences in inhibition across the extracts. These phenolic compounds inhibited αSN amyloidogenesis by directing αSN monomers into small αSN oligomers with lower toxicity, thereby suppressing the subsequent fibril growth phase. Our results highlight the molecular consequences of differences in the level of effective phenolic compounds in different olive varieties, insights that have implications for long-term human health.

Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wild-type α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD Caenorhabditis elegans models, expressing α-synuclein either in muscle or in dopaminergic neurons, significantly reduces the toxicity exerted by α-synuclein. SynuClean-D-treated worms show decreased α-synuclein aggregation in muscle and a concomitant motility recovery. More importantly, this compound is able to rescue dopaminergic neurons from α-synuclein-induced degeneration. Overall, SynuClean-D appears to be a promising molecule for therapeutic intervention in Parkinson's disease.