Structural and mechanistic insights into amyloid-ß and α-synuclein fibril formation and polyphenol inhibitor efficacy in phospholipid bilayers.

Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-ß 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development.

Flavonoids with Potential Anti-Amyloidogenic Effects as Therapeutic Drugs for Treating Alzheimer's Disease.

Alzheimer's disease (AD) is a central neurodegenerative disease generally among the elderly; it accounts for approximately 50-75%of total cases of dementia patients and poses a serious threat to physical and mental health. Currently available treatments for AD mainly relieves its symptoms, and effective therapy is urgently needed. Deposition of amyloid-ß protein in the brain is an early and invariant neuropathological feature of AD. Currently the main efforts in developing anti-AD drugs focus on anti-amyloidogenic therapeutics that prevent amyloid-ß production or aggregation and decrease the occurrence of neurotoxic events. The results of an increasing number of studies suggest that natural extracts and phytochemicals have a positive impact on brain aging. Flavonoids belong to the broad group of polyphenols and recent data indicate a favorable effect of flavonoids on brain aging. In this review, we collect relevant discoveries from 1999 to 2021, discuss 75 flavonoids that effectively influence AD pathogenesis, and summarize their functional mechanisms in detail. The data we have reviewed show that, these flavonoids belong to various subclasses, including flavone, flavanone, biflavone, etc. Our results provide a reference for further study of the effects of flavonoids on AD and the progress of anti-AD therapy.

Mechanisms of amyloid proteins aggregation and their inhibition by antibodies, small molecule inhibitors, nano-particles and nano-bodies.

Protein misfolding and aggregation can be induced by a wide variety of factors, such as dominant disease-associated mutations, changes in the environmental conditions (pH, temperature, ionic strength, protein concentration, exposure to transition metal ions, exposure to toxins, posttranslational modifications including glycation, phosphorylation, and sulfation). Misfolded intermediates interact with similar intermediates and progressively form dimers, oligomers, protofibrils, and fibrils. In amyloidoses, fibrillar aggregates are deposited in the tissues either as intracellular inclusion or extracellular plaques (amyloid). When such proteinaceous deposit occurs in the neuronal cells, it initiates degeneration of neurons and consequently resulting in the manifestation of various neurodegenerative diseases. Several different types of molecules have been designed and tested both in vitro and in vivo to evaluate their anti-amyloidogenic efficacies. For instance, the native structure of a protein associated with amyloidosis could be stabilized by ligands, antibodies could be used to remove plaques, oligomer-specific antibody A11 could be used to remove oligomers, or prefibrillar aggregates could be removed by affibodies. Keeping the above views in mind, in this review we have discussed protein misfolding and aggregation, mechanisms of protein aggregation, factors responsible for aggregations, and strategies for aggregation inhibition.

Novel Multifunctional Ascorbic Triazole Derivatives for Amyloidogenic Pathway Inhibition, Anti-Inflammation, and Neuroprotection.

Alzheimer's disease (AD) is a common neurodegenerative disorder. The number of patients with AD is projected to reach 152 million by 2050. Donepezil, rivastigmine, galantamine, and memantine are the only four drugs currently approved by the United States Food and Drug Administration for AD treatment. However, these drugs can only alleviate AD symptoms. Thus, this research focuses on the discovery of novel lead compounds that possess multitarget regulation of AD etiopathology relating to amyloid cascade. The ascorbic acid structure has been designated as a core functional domain due to several characteristics, including antioxidant activities, amyloid aggregation inhibition, and the ability to be transported to the brain and neurons. Multifunctional ascorbic derivatives were synthesized by copper (I)-catalyzed azide-alkyne cycloaddition reaction (click chemistry). The in vitro and cell-based assays showed that compounds 2c and 5c exhibited prominent multifunctional activities as beta-secretase 1 inhibitors, amyloid aggregation inhibitors, and antioxidant, neuroprotectant, and anti-inflammatory agents. Significant changes in activities promoting neuroprotection and anti-inflammation were observed at a considerably low concentration at a nanomolar level. Moreover, an in silico study showed that compounds 2c and 5c were capable of being permeated across the blood-brain barrier by sodium-dependent vitamin C transporter-2.

Bioactive Phytocompounds: Anti-amyloidogenic Effects Against Hen Egg-White Lysozyme Aggregation.

Amyloidosis is the process of fibril formation responsible for causing several diseases in the human being that involve protein aggregation such as Alzheimer's, Parkinson's, Huntington's disease, and type II diabetes. Natural phytocompounds such as curcumin shown promising anti-amyloidogenic activity. In the present study, selective phytocompounds such as piperine, cinnamaldehyde, eugenol, and cuminaldehyde present in Piper nigrum L, Cinnamomum zeylanicum Blume, Eugenia caryophyllus Thumb, and Cuminum cyminum L, respectively were analyzed for anti-amyloidogenic activity using hen egg white-lysozyme (HEWL) as a model system. Out of the selected phytocompounds, piperine showed the most significant anti-amyloidogenic activity, as evident from in vitro assays that were validated by in silico molecular docking study. Piperine showed 64.7 ± 3.74% inhibition of amyloid formation at 50 µM concentration, as observed by Thioflavin T assay. Subsequently, the anti-amyloidogenic activity of piperine was further validated by congo red, intrinsic fluorescence assay, and transmission electron microscopy analysis. The in silico molecular binding interaction showed piperine with the highest docking score and glide energy. Piperine was found to be interacting with amyloidogenic region residues and Trp62, the most important residue involved in the amyloidogenesis process. In conclusion, piperine can be used as a positive lead for a potential therapeutic role in targeting diseases involved amyloidogenesis.

Review of Evidence and Perspectives of Flavonoids on Metabolic Syndrome and Neurodegenerative Disease.

Flavonoids are commonly found in fruits, vegetables, and plant-derived foods and may promote various health benefits when included in the diet. The biological activity of flavonoids is normally associated to their potent antioxidant and anti-inflammatory effects, since oxidative stress is associated to conditions such as diabetes, obesity, cardiovascular and neurodegenerative diseases. Additionally, flavonoids may be related to metabolic diseases through their effects on inflammatory mediators and pathways, barrier integrity and gut microbiota composition. The extensive metabolism undergone by flavonoids in humans and the individual differences in their bioavailability to target organs hinder the interpretation of results from cell and animal models. Prospective human studies therefore provide an important perspective. In the field of neurodegenerative disease, carefully designed cohort studies have uncovered important associations between flavonoid intake and reduction in dementia risk, especially regarding specific flavonols, but also anthocyanins. Alternative mechanisms of action, such as changes in the gut microbiota or modulation of the production of toxic proteins, such as amyloid and tau, likely account for an important component of their positive effects, and their elucidation may lead to public health benefits of large magnitude.

Pharmacological inhibition and knockdown of O-GlcNAcase reduces cellular internalization of α-synuclein preformed fibrils.

The pathological hallmark of Parkinson's disease (PD) is Lewy bodies that form within the brain from aggregated forms of α-synuclein (α-syn). These toxic α-syn aggregates are transferred from cell to cell by release of fibrils from dying neurons into the extracellular environment, followed by their subsequent uptake by neighboring cells. This process leads to spreading of the pathology throughout the brain in a prion-like manner. Identifying new pathways that hinder the internalization of such α-syn fibrils is of high interest for their downstream potential exploitation as a way to create disease-modifying therapeutics for PD. Here, we show that Thiamet-G, a highly selective pharmacological agent that inhibits the glycoside hydrolase O-GlcNAcase (OGA), blunts the cellular uptake of α-syn fibrils. This effect correlates with increased nucleocytoplasmic levels of O-linked N-acetylglucosamine (O-GlcNAc)-modified proteins, and genetic knockdown of OGA expression closely phenocopies both these effects. These reductions in the uptake of α-syn fibrils caused by inhibition of OGA are both concentration- and time-dependent and are observed in multiple cell lines including mouse primary cortical neurons. Moreover, treatment of cells with the OGT inhibitor, 5SGlcNHex, increases the level of uptake of α-syn PFFs, further supporting O-GlcNAcylation of proteins driving these effects. Notably, this effect is mediated through an unknown mechanism that does not involve well-characterized endocytotic pathways. These data suggest one mechanism by which OGA inhibitors might exert their protective effects in prion-like neuropathologies and support exploration of OGA inhibitors as a potential disease-modifying approach to treat PD.

Sequence and structure-based peptides as potent amyloid inhibitors: A review.

Misfolded and natively disordered globular proteins tend to aggregate together in an interwoven fashion to form fibrous, proteinaceous deposits referred to as amyloid fibrils. Formation and deposition of such insoluble fibrils are the characteristic features of a broad group of diseases, known as amyloidosis. Some of these proteins are known to cause several degenerative disorders in humans, such as Amyloid-Beta (Aß) in Alzheimer's disease (AD), human Islet Amyloid Polypeptide (hIAPP, amylin) in type 2 diabetes, α-synuclein (α-syn) in Parkinson's disease (PD) and so on. The fact that these proteins do not share any significant sequence or structural homology in their native states make therapy quite challenging. However, it is observed that aggregation-prone proteins and peptides tend to adopt a similar type of secondary structure during the formation of fibrils. Rationally designed peptides can be a potent inhibitor that has been shown to disrupt the fibril structure by binding specifically to the amyloidogenic region(s) within a protein. The following review will analyze the inhibitory potency of both sequence-based and structure-based small peptides that have been shown to inhibit amyloidogenesis of proteins such as Aß, human amylin, and α-synuclein.

The Antiaggregative and Antiamyloidogenic Properties of Nanoparticles: A Promising Tool for the Treatment and Diagnostics of Neurodegenerative Diseases.

Due to the progressive aging of the society, the prevalence and socioeconomic burden of neurodegenerative diseases are predicted to rise. The most common neurodegenerative disorders nowadays, such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, can be classified as proteinopathies. They can be either synucleinopathies, amyloidopathies, tauopathies, or TDP-43-related proteinopathies; thus, nanoparticles with a potential ability to inhibit pathological protein aggregation and/or degrade already existing aggregates can be a promising approach in the treatment of neurodegenerative diseases. As it turns out, nanoparticles can be a double-edged sword; they can either promote or inhibit protein aggregation, depending on coating, shape, size, surface charge, and concentration. In this review, we aim to emphasize the need of a breakthrough in the treatment of neurodegenerative disorders and draw attention to nanomaterials, as they can also serve as a diagnostic tool for protein aggregates or can be used in a high-throughput screening for novel antiaggregative compounds.

Diphenylalanin nanofibers-inspired synthesis of fluorescent gold nanoclusters for screening of anti-amyloid drugs.

Protein misfolding and aggregation into amyloid structures is linked with a number of pathophysiological disorders. In the past decade, significant progresses have been made in the drug discovery strategies against toxic aggregates. Although lack of specificity and high sensitivity for in vitro screening system still seen. Here we demonstrate a new targeting probe based on FF diphenylalanine peptide -protected gold nanoclusters (FF AuNCs). Diphenylalanine peptide has previously been shown to self-assemble into well-ordered fiber like the fibers that are observed in amyloid aggregates. We used of the self-assembly properties along with the ability of FF dipeptide in reduction of gold ions for synthesis of novel Au nanoclusters. We used FF AuNCs for monitoring of effectiveness of anti-amyloid drugs. Fluorescence was considerably diminished when drugs at different concentrations added, due to destruction of the amyloid fibers. Furthermore, the analysis of several components demonstrates significant selectivity against the amyloid disrupting molecules. Prepared FF AuNCs will gain possible strategy for in vitro screening of amyloid disrupting molecules.

Non-polyphenolic natural inhibitors of amyloid aggregation.

Protein misfolding diseases (PMDs) are chronic and progressive, with no effective therapy so far. Aggregation and misfolding of amyloidogenic proteins are closely associated with the onset and progression of PMDs, such as amyloid-ß (Aß) in Alzheimer's disease, α-Synuclein (α-Syn) in Parkinson's disease and human islet amyloid polypeptide (hIAPP) in type 2 diabetes. Inhibiting toxic aggregation of amyloidogenic proteins is regarded as a promising therapeutic approach in PMDs. The past decade has witnessed the rapid progresses of this field, dozens of inhibitors have been screened and verified in vitro and in vivo, demonstrating inhibitory effects against the aggregation and misfolding of amyloidogenic proteins, together with beneficial effects. Natural products are major sources of small molecule amyloid inhibitors, a number of natural derived compounds have been identified with great bioactivities and translational prospects. Here, we review the non-polyphenolic natural inhibitors that potentially applicable for PMDs treatment, along with their working mechanisms. Future directions are proposed for the development and clinical applications of these inhibitors.

The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG.

Millions of people around the world suffer from amyloid-related disorders, including Alzheimer's and Parkinson's diseases. Despite significant and sustained efforts, there are still no disease-modifying drugs available for the majority of amyloid-related disorders, and the overall failure rate in clinical trials is very high, even for compounds that show promising anti-amyloid activity in vitro. In this study, we demonstrate that even small changes in the chemical environment can strongly modulate the inhibitory effects of anti-amyloid compounds. Using one of the best-established amyloid inhibitory compounds, epigallocatechin-3-gallate (EGCG), as an example, and two amyloid-forming proteins, insulin and Parkinson's disease-related α -synuclein, we shed light on the previously unexplored sensitivity to solution conditions of the action of this compound on amyloid fibril formation. In the case of insulin, we show that the classification of EGCG as an amyloid inhibitor depends on the experimental conditions select, on the method used for the evaluation of the efficacy, and on whether or not EGCG is allowed to oxidise before the experiment. For α -synuclein, we show that a small change in pH value, from 7 to 6, transforms EGCG from an efficient inhibitor to completely ineffective, and we were able to explain this behaviour by the increased stability of EGCG against oxidation at pH 6.

Chitooligosaccharides as Antibacterial, Antibiofilm, Antihemolytic and Anti-Virulence Agent against Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus nosocomial infections with a high mortality rate in human and animals have been reported to associate with bacterial biofilm formation, along with the secretion of numerous virulence factors. Therefore, the inhibition of biofilm formation and attenuation of virulence determinants are considered as a promising solution to combat the spread of S. aureus infections. Modern trends in antibiofilm therapies have opted for the active agents that are biocompatible, biodegradable, non-toxic and cost-effective. Owning the aforementioned properties, chitosan, a natural N-acetylated carbohydrate biopolymer derived from chitin, has been favorably employed. Recently, the chitosan structure has been chemically modified into Chitooligosaccharides (COS) to overcome its limited solubility in water, thus widening chitosan applications in modern antibiofilm research. In the present study, we have investigated the antibacterial, antibiofilm and anti-virulence activities against S. aureus of COS of different molecular weights dissolved in neutral water. METHODS: The study of bactericidal activity was performed using the micro-dilution method while the biofilm inhibition assay was performed using crystal-violet staining method and confirmed by scanning electron microscopic analysis. The inhibition of amyloid protein production was confirmed by Congo Red staining. RESULTS: Results showed that low molecular weight COS exhibited bactericidal activity and reduced the bacterial amylogenesis, hemolytic activity as well as H2O2 resistance properties, while slightly inhibiting biofilm formation. The present study provides a new insight for further applications of the water-soluble COS as a safe and cost-effective drug for the treatment of S. aureus biofilm-associated infections. CONCLUSION: Reducing the molecular weight of chitosan in the form of COS has become an effective strategy to maintain chitosan biological activity while improving its water solubility. The low molecular weight COS investigated in this study have effectively performed antibacterial, antibiofilm and antivirulence properties against S. aureus.

Autophagy Modulation as a Treatment of Amyloid Diseases.

Amyloids are fibrous proteins aggregated into toxic forms that are implicated in several chronic disorders. More than 30 diseases show deposition of fibrous amyloid proteins associated with cell loss and degeneration in the affected tissues. Evidence demonstrates that amyloid diseases result from protein aggregation or impaired amyloid clearance, but the connection between amyloid accumulation and tissue degeneration is not clear. Common examples of amyloid diseases are Alzheimer's disease (AD), Parkinson's disease (PD) and tauopathies, which are the most common forms of neurodegenerative diseases, as well as polyglutamine disorders and certain peripheral metabolic diseases. In these diseases, increased accumulation of toxic amyloid proteins is suspected to be one of the main causative factors in the disease pathogenesis. It is therefore important to more clearly understand how these toxic amyloid proteins accumulate as this will aide in the development of more effective preventive and therapeutic strategies. Protein homeostasis, or proteostasis, is maintained by multiple cellular pathways-including protein synthesis, quality control, and clearance-which are collectively responsible for preventing protein misfolding or aggregation. Modulating protein degradation is a very complex but attractive treatment strategy used to remove amyloid and improve cell survival. This review will focus on autophagy, an important clearance pathway of amyloid proteins, and strategies for using it as a potential therapeutic target for amyloid diseases. The physiological role of autophagy in cells, pathways for its modulation, its connection with apoptosis, cell models and caveats in developing autophagy as a treatment and as a biomarker is discussed.

Computational Study of Natural Compounds for the Clearance of Amyloid-Βeta: A Potential Therapeutic Management Strategy for Alzheimer's Disease.

Alzheimer's disease (AD) is a widespread dynamic neurodegenerative malady. Its etiology is still not clear. One of the foremost pathological features is the extracellular deposits of Amyloid-beta (Aß) peptides in senile plaques. The interaction of Aß and the receptor for advanced glycation end products at the blood-brain barrier is also observed in AD, which not only causes the neurovascular anxiety and articulation of proinflammatory cytokines, but also directs reduction of cerebral bloodstream by upgrading the emission of endothelin-1 to induce vasoconstriction. In this process, RAGE is deemed responsible for the influx of Aß into the brain through BBB. In the current study, we predicted the interaction potential of the natural compounds vincamine, ajmalicine and emetine with the Aß peptide concerned in the treatment of AD against the standard control, curcumin, to validate the Aß peptide-compounds results. Protein-protein interaction studies have also been carried out to see their potential to inhibit the binding process of Aß and RAGE. Moreover, the current study verifies that ligands are more capable inhibitors of a selected target compared to positive control with reference to ΔG values. The inhibition of Aß and its interaction with RAGE may be valuable in proposing the next round of lead compounds for effective Alzheimer's disease treatment.

Structural Insights into the Inhibition of Amyloid Fibril Formation by Fibrinogen via Interaction with Prefibrillar Intermediates.

Abnormal protein aggregation tends to result in the formation of ß-sheet rich amyloid fibrils, which are related to various kinds of amyloidoses and neurodegenerative diseases. The susceptibility to aggregation of protein molecules is dealt with by proteostasis in living systems, in which molecular chaperones play an important role. Recently, several secreted proteins have been examined as extracellular chaperones with a potency to suppress the formation of amyloid fibrils, although the whole picture that includes their inhibition mechanisms is not yet understood. In this study, we investigated the inhibitory effect of fibrinogen (Fg), one of the extracellular proteins identified as a potential member of the group of chaperones, on fibril formation. Insulin B chain was used as an amyloid formation model system because its prefibrillar intermediate species in the nucleation phase were well characterized. We revealed that Fg efficiently inhibited amyloid fibril formation via a direct interaction with the surface of the prefibrillar intermediates. Small-angle X-ray scattering experiments and a stoichiometry analysis suggested a structural model in which the surface of the rod-shaped prefibrillar intermediates is surrounded by Fg molecules. From such a specific manner of interactions, we propose that the role of Fg is to disturb fibril growth by confining the nuclei even when the nucleation occurs inside the prefibrillar intermediate. The structural property of the B-chain intermediates complexed with Fg would provide insights into the general principles of the functions of chaperones and other potential chaperone-like proteins involved in amyloid-related diseases.

An automated microliter-scale high-throughput screening system (MSHTS) for real-time monitoring of protein aggregation using quantum-dot nanoprobes.

Protein aggregation is the principal component of numerous protein misfolding pathologies termed proteinopathies, such as Alzheimer's disease, Parkinson's disease, prion disease, and AA amyloidosis with unmet treatment needs. Protein aggregation inhibitors have great potential for the prevention and treatment of proteinopathies. Here we report the development of an automated real-time microliter-scale high throughput screening (MSHTS) system for amyloid aggregation inhibitors using quantum-dot nanoprobes. Screening 504 crude extracts and 134 low molecular weight aromatic compounds revealed the relationship of amyloid-ß (Aß) aggregation inhibitory activities of plant extracts using a plant-based classification. Within the eudicots, rosids, Geraniales and Myrtales showed higher activity. Screening low molecular weight aromatic compounds demonstrated that the structure of tropolone endows it with potential Aß aggregation inhibitory activity. The activity of the most active tropolone derivative was higher than that of rosmarinic acid. MSHTS also identified three chaperone molecules as tau aggregation inhibitors. These results demonstrate that our automated MSHTS system is a novel and robust tool that can be adapted to a wide range of compounds and aggregation-prone polypeptides.

Alzheimer's disease (AD) therapeutics - 2: Beyond amyloid - Re-defining AD and its causality to discover effective therapeutics.

Compounds targeted for the treatment of Alzheimer's Disease (AD) have consistently failed in clinical trials despite evidence for target engagement and pharmacodynamic activity. This questions the relevance of compounds acting at current AD drug targets - the majority of which reflect the seminal amyloid and, to a far lesser extent, tau hypotheses - and limitations in understanding AD causality as distinct from general dementia. The preeminence of amyloid and tau led to many alternative approaches to AD therapeutics being ignored or underfunded to the extent that their causal versus contributory role in AD remains unknown. These include: neuronal network dysfunction; cerebrovascular disease; chronic, local or systemic inflammation involving the innate immune system; infectious agents including herpes virus and prion proteins; neurotoxic protein accumulation associated with sleep deprivation, circadian rhythm and glymphatic/meningeal lymphatic system and blood-brain-barrier dysfunction; metabolic related diseases including diabetes, obesity hypertension and hypocholesterolemia; mitochondrial dysfunction and environmental factors. As AD has become increasingly recognized as a multifactorial syndrome, a single treatment paradigm is unlikely to work in all patients. However, the biomarkers required to diagnose patients and parse them into mechanism/disease-based sub-groups remain rudimentary and unvalidated as do non-amyloid, non-tau translational animal models. The social and economic impact of AD is also discussed in the context of new FDA regulatory draft guidance and a proposed biomarker-based Framework (re)-defining AD and its stages as part of the larger landscape of treating dementia via the 2013 G8 initiative to identify a disease-modifying therapy for dementia/AD by 2025.

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds.

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations-an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.