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Studies of the structure and dynamics of oligomeric aggregates of amyloidogenic peptides pose challenges due to their transient nature. This concept article provides a brief overview of various nucleation mechanisms with reference to the classical nucleation theory and illustrates the advantages of incubating amyloidogenic peptides in reverse micelles (RMs). The use of RMs not only facilitates size regulation of oligomeric aggregates but also provides an avenue to explore protein-protein interactions among the oligomeric aggregates of various amyloidogenic peptides. Additionally, we envision the feasibility of preparing brain tissue-derived oligomeric aggregates using RMs, potentially advancing the development of monoclonal antibodies with enhanced potency against these pathological species in vivo., (© 2024 Wiley-VCH GmbH.)

Proteinopathies or amyloidoses are a group of life-threatening disorders that result from misfolding of proteins and aggregation into toxic insoluble amyloid aggregates. Amyloid aggregates have low clearance from the body due to the insoluble nature, leading to their deposition in various organs and consequent organ dysfunction. While amyloid deposition in the central nervous system leads to neurodegenerative diseases that mostly cause dementia and difficulty in movement, several other organs, including heart, liver and kidney are also affected by systemic amyloidoses. Regardless of the site of amyloid deposition, misfolding and structural alteration of the precursor proteins play the central role in amyloid formation. Kinetic stabilizers are an emerging class of drugs, which act like pharmacological chaperones to stabilize the native state structure of amyloidogenic proteins and to increase the activation energy barrier that is required for adopting a misfolded structure or conformation, ultimately leading to the inhibition of protein aggregation. In this review, we discuss the kinetic stabilizers that stabilize the native quaternary structure of transthyretin, immunoglobulin light chain and superoxide dismutase 1 that cause transthyretin amyloidoses, light chain amyloidosis and familial amyotrophic lateral sclerosis, respectively., (© 2024 Wiley-VCH GmbH.)

Amyloid peptides (AMYs) and antimicrobial peptides (AMPs) are considered as the two distinct families of peptides, characterized by their unique sequences, structures, biological functions, and specific pathological targets. However, accumulating evidence has revealed intriguing pathological connections between these peptide families in the context of microbial infection and neurodegenerative diseases. Some AMYs and AMPs share certain structural and functional characteristics, including the ability to self-assemble, the presence of β-sheet-rich structures, and membrane-disrupting mechanisms. These shared features enable AMYs to possess antimicrobial activity and AMPs to acquire amyloidogenic properties. Despite limited studies on AMYs-AMPs systems, the cross-seeding phenomenon between AMYs and AMPs has emerged as a crucial factor in the bidirectional communication between the pathogenesis of neurodegenerative diseases and host defense against microbial infections. In this review, we examine recent developments in the potential interplay between AMYs and AMPs, as well as their pathological implications for both infectious and neurodegenerative diseases. By discussing the current progress and challenges in this emerging field, this account aims to inspire further research and investments to enhance our understanding of the intricate molecular crosstalk between AMYs and AMPs. This knowledge holds great promise for the development of innovative therapies to combat both microbial infections and neurodegenerative disorders.

Neurodegenerative diseases (NDDs) refer to a complex heterogeneous group of diseases which are associated with the accumulation of amyloid fibrils or plaques in the brain leading to progressive loss of neuronal functions. Alzheimer's disease is one of the major NDD responsible for 60-80 % of all dementia cases. Currently, there are no curative or disease-reversing/modifying molecules for many of the NDDs except a few such as donepezil, rivastigmine, galantamine, carbidopa and levodopa which treat the disease-associated symptoms. Similarly, there are very few FDA-approved tracers such as flortaucipir (Tauvid) for tau fibril imaging and florbetaben (Neuraceq), flutemetamol (Vizamyl), and florbetapir (Amyvid) for amyloid imaging available for diagnosis. Recent advances in the cryogenic electron microscopy reported distinctly different microstructures for tau fibrils associated with different tauopathies highlighting the possibility to develop tauopathy-specific imaging agents and therapeutics. In addition, it is important to identify the proteins that are associated with disease development and progression to know about their 3D structure to develop various diagnostics, therapeutics and theranostic agents. The current article discusses in detail the disease-associated amyloid and non-amyloid proteins along with their structural insights. We comprehensively discussed various novel proteins associated with NDDs and their implications in disease pathology. In addition, we document various emerging chemical compounds developed for diagnosis and therapy of different NDDs with special emphasis on theranostic agents for better management of NDDs., (© 2024 Wiley-VCH GmbH.)

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-β 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., (© 2021 Federation of European Biochemical Societies.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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.

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-β 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-β 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.

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (© 2020 Federation of European Biochemical Societies.)

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 (Aβ) 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 Aβ, human amylin, and α-synuclein., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Monika Pichla et al.)

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.

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-β (Aβ) 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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.

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.

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 (Aβ) peptides in senile plaques. The interaction of Aβ 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 Aβ into the brain through BBB. In the current study, we predicted the interaction potential of the natural compounds vincamine, ajmalicine and emetine with the Aβ peptide concerned in the treatment of AD against the standard control, curcumin, to validate the Aβ peptide-compounds results. Protein-protein interaction studies have also been carried out to see their potential to inhibit the binding process of Aβ 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 Aβ and its interaction with RAGE may be valuable in proposing the next round of lead compounds for effective Alzheimer's disease treatment.

Abnormal protein aggregation tends to result in the formation of β-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.

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-β (Aβ) 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 Aβ 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.

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 H 2 O 2 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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Amyloid fibrils are linear polypeptide aggregates with a cross-β structure. These fibrils are best known for their association with neurodegenerative diseases, such as Alzheimer's or Parkinson's, but they may also be used by living organisms as functional units, e.g. in the synthesis of melanin or in the formation of bacterial biofilms. About a decade ago, in a search for semen factors that modulate infection by HIV-1 (a sexually transmitted virus and the causative agent of the acquired immune deficiency syndrome (AIDS)), it was demonstrated that semen harbors amyloid fibrils capable of markedly increasing HIV infection rates. This discovery not only created novel opportunities to prevent sexual HIV-1 transmission but also stimulated research to unravel the natural role of these factors. We discuss here the identification of these intriguing structures, their molecular properties, and their effects on both sexually transmitted diseases and reproductive health. Moreover, we review strategies to antagonize semen amyloid to prevent sexual transmission of viruses.

Aggregation and accumulation of amyloid β and tau proteins to plaques and neurofibrillary tangles are the key pathogenic events in Alzheimer's disease. Here, we studied the capability of the cyclic d,l-α-peptide CP-2 as a conformational inhibitor of different amyloids to cross-interact with tau-derived AcPHF6 peptide and inhibit its aggregation, membrane perturbation and toxicity.

The increased incidence of neurodegenerative diseases represents a huge challenge for societies. These diseases are characterized by neuronal death and include several different pathologies, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease and transmissible spongiform encephalopathies. Most of these pathologies are often associated with the aggregation of misfolded proteins, such as amyloid-ß, tau, α-synuclein, huntingtin and prion proteins. However, the precise mechanisms that lead to neuronal dysfunction and death in these diseases remain poorly understood. Nucleic acid aptamers represent a new class of ligands that could be useful to better understand these diseases and develop better diagnosis and therapy. In this review, several of these aptamers are presented as well as their applications for neurodegenerative diseases., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC 50 of 47.5 µM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure-activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel anti-amyloidogenic reagents based on specific-binding ligands.

Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC 50 ) of PRM for HI and HL was estimated to be 114.81±1.21μM and 186.20±1.03μM, respectively. Microscopic techniques revealed the absence of fibrillar structures when HI and HL was co-incubated with PRM. Cytoprotective behavior of PRM was investigated by cell based cytotoxicity assay performed on SH-SY5Y neuronal cell lines. The half maximal disaggregation concentration (DC 50 ) was calculated as 21.37±0.89μM and 45.70±0.76μM, signifying that PRM is much potent to disaggregate pre formed fibrils rather than to inhibit fibrillation. Thus, PRM could be beneficial as therapeutic agent that can aid in the cure of amyloid related diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-β (Aβ) aggregation in vitro and suppress the Alzheimer's-like phenotypes in a transgenic mouse model of Aβ deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation., Competing Interests: The authors declare no conflict of interest.

Protein misfolding and inclusion body formations are common events in neurodegenerative diseases characterized by deposition of misfolded proteins inside or outside of neurons, and are commonly referred to as "protein misfolding neurodegenerative diseases" (PMNDs). These phenotypically diverse but biochemically similar aggregates suggest a highly conserved molecular mechanism of pathogenesis. These challenges are magnified by presence of mutations that render individual proteins subject to misfolding and/or aggregation. Cell proteostasis network and molecular chaperoning are maintaining cell proteome to preserve the protein folding, refolding, oligomerization, or disaggregation, and play formidable tasks to maintain the health of organism in the face of developmental changes, environmental insults, and rigors of aging. Maintenance of cell proteome requires the orchestration of major pathways of the cellular proteostasis network (heat shock response (HSR) in the cytosol and the unfolded protein response (UPR) in the endoplasmic reticulum). Proteostasis responses culminate in transcriptional and post-transcriptional programs that up-regulate the homeostatic mechanisms. Proteostasis is strongly influenced by the general properties of individual proteins for folding, misfolding, and aggregation. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We first try to introduce some new chemical approaches to prevent the misfolding or aggregation of specific proteins via direct binding interactions. We then start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organ proteostasis. © 2016 BioFactors, 43(6):737-759, 2017., (© 2016 International Union of Biochemistry and Molecular Biology.)

Many chronic human diseases, including multiple neurodegenerative diseases, are associated with deleterious protein aggregates, also called protein amyloids. One common therapeutic strategy is to develop protein aggregation inhibitors that can slow down, prevent, or remodel toxic amyloids. Natural products are a major class of amyloid inhibitors, and several dozens of natural product-based amyloid inhibitors have been identified and characterized in recent years. These plant- or microorganism-extracted compounds have shown significant therapeutic potential from in vitro studies as well as in vivo animal tests. Despite the technical challenges of intrinsic disordered or partially unfolded amyloid proteins that are less amenable to characterizations by structural biology, a significant amount of research has been performed, yielding biochemical and pharmacological insights into how inhibitors function. This review aims to summarize recent progress in natural product-based amyloid inhibitors and to analyze their mechanisms of inhibition in vitro. Major classes of natural product inhibitors and how they were identified are described. Our analyses comprehensively address the molecular interactions between the inhibitors and relevant amyloidogenic proteins. These interactions are delineated at molecular and atomic levels, which include covalent, non-covalent, and metal-mediated mechanisms. In vivo animal studies and clinical trials have been summarized as an extension. To enhance natural product bioavailability in vivo, emerging work using nanocarriers for delivery has also been described. Finally, issues and challenges as well as future development of such inhibitors are envisioned., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Alzheimer's disease characterized by misfolding, aggregation, and accumulation of amyloid fibrils in an insoluble form in the brain, is often known as amyloidosis. The process of aggregation follows a mechanism of seeded polymerization. For decades, a great number of failures in Alzheimer's disease (AD) drug development, with both small molecules and immunotherapies failing to establish a drug/placebo difference or having an unacceptable toxicity have led to the therapeutic research interest towards a group of anti-amyloidogenic compounds originated from plants called biophenols. A number of in vitro and in vivo studies have demonstrated that the plant biophenols bind with amyloid beta (Aβ) toxic oligomers and reducing the fibril formation and toxicity. The exact mechanism of biophenols action against Aβ toxicity is unknown, while studies have suggested the amyloid-binding affinity of biophenols affecting Aβ on various levels, e.g. by direct inhibiting fibril formation or steering oligomer formation into unstructured, inhibiting Aβ aggregation, and promoting nontoxic pathways. Furthermore, biophenols involved in the inhibition of Aβ progression (e.g., oxidative stress and neuroinflammation) and effecting the amyloid precursor protein processing through the direct or indirect inhibition of β-secretase (BACE-1), γ-secretase and/or activation of α-secretase. This critical review account for the biophenols as magic bullet targeting against Aβ, and simulation the results on how biophenols interact with the Aβ monomers and oligomers, highly desirable knowledge for predicting new efficient nutraceutical drugs., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Epigallocatechin gallate (EGCG), the most abundant flavanoid in green tea, is currently being evaluated in the clinic due to its benefits in the treatment of amyloid disorders. Its anti-amyloidogenic effect has been attributed to direct interaction of the intact molecule with misfolded polypeptides. In addition, antioxidant activity is also involved in the anti-amyloidogenic role. The detailed molecular mechanism is still unclear and requires further investigation. In the present study, the kinetics of EGCG oxidation and the anti-amyloidogenic effect of the resultant oxidation substances have been examined. The results indicate that EGCG degrades in a medium at pH 8.0 with a half-life less than 2h. By utilizing lysozyme as an in vitro model, the oxidized EGCG demonstrates a more potent anti-amyloidogenic capacity than the intact molecule, as shown by ThT and ANS fluorescence, TEM determination, and hemolytic assay. The oxidized EGCG also has a stronger disruptive effect on preformed fibrils than the native form. Ascorbic acid eliminates the disruptive role of native EGCG on the fibrils, suggesting that oxidation is a prerequisite in fibril disruption. The results of this work demonstrate that oxidized EGCG plays a more important role than the intact molecule in anti-amyloidogenic activity. These insights into the action of EGCG may provide a novel route to understand the anti-amyloidogenic activity of natural polyphenols., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic intranasal administration of fibrillar structures of proinflammatory S100A9 protein impaired passive avoidance learning in old C57Bl/6 mice. Combined treatment with S100A9 fibrils and antibodies to glutamate was followed by an increase in horizontal locomotor activity of animals in the open-field test and did not disturb spatial memory.

The first description of Alzheimer's disease (AD) was made in 1907 by Alois Alzheimer (Allgemeine Zeitschrift fur Psyciatrie und Psychisch-Gerichtliche Medizin 64, 3, 1907), although other contemporary physicians had made similar, and rather more complete, assessments of the neuropathological changes present in the AD brain (Fischer, Monatsschr Psychiat Neurol 22, 17, 1907). Our knowledge of AD has increased dramatically and continues to accelerate. This year is 25 years after the publication of a series of papers that, in various ways, articulated the amyloid cascade hypothesis (ACH) for AD (Beyreuther and Masters, Brain Pathol 1, 241-251, 1991; Hardy and Allsop, Trends Pharmacol Sci 12, 383-388, 1991; Selkoe, Neuron 6, 487-498, 1991; Hardy and Higgins, Science 256, 184-185, 1992). This review will cover some familiar territory, but we shall also place the ACH into a wider context, compare it with other hypotheses for AD, explore the evolution of the hypothesis to encompass new findings, and determine, irrespective of the merits of the hypothesis itself, whether it has been useful for the research field, both in academia and in industry. Finally, we shall review how the ACH has led to a number of therapeutic approaches, all of which have, to date, failed to reach their primary efficacy end-points in clinical trials and reflect upon what the future may hold. We review the amyloid cascade hypothesis (ACH) and compare it with other hypotheses that have been posited to explain the initiation and progression Alzheimer's disease. We document the data that support the ACH, and also reflect upon its deficiencies. We list the recent clinical failures of amyloidocentric drugs and anticipate the results that new therapeutic approaches may deliver. This article is part of the 60th Anniversary special issue., (© 2016 International Society for Neurochemistry.)

Alzheimer's disease (AD) is a neurodegenerative disorder that affects memory, behavior, thinking and emotion. Current therapies to treat AD patients are only capable for temporarily slowing-down the cognitive decline, as they are focused on ameliorating symptoms instead of targeting its underlying causes. The aim of this review is to describe what is known about the protein structures implicated in AD pathogenesis, amyloid cascade members, as well as those structures involved in Aβ clearance. Thus, structural information available for APP, α- β- and γ-secretases, CTFβ and derived Aβ peptides, AICDs, apoE and apoJ, LRP-1 and RAGE, and neprilysin and insulin-degrading enzyme is provided. The recently solved structure for the γ-secretase complex opens the rational design of a new generation of inhibitors, whereas that for Aβ oligomers offers a putative mechanism explaining why monoclonal antibodies targeted to the N-terminus are effective. Then, an overview on therapies targeting some of these molecules presents their benefits and drawbacks. As a general conclusion our knowledge on the protein structures involved in AD has recently substantially advanced, allowing for the rational design of different therapeutic approaches. Hopefully, we are getting closer to finding a strong disease-modifying drug to cure this devastating disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The "molecular tweezer" CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer's disease and other amyloidoses, here we studied its safety and pharmacokinetics., Methods: Toxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer's disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration., Results: Examination of CLR01's effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound's putative "process-specific" mechanism of action.A single-injection of 100 mg/kg CLR01 in mice - 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin.The brain penetration of CLR01 was found to be 1 - 3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h., Conclusion: Estimation of brain levels compared to amyloid β-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01.The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses.

Alzheimer's disease (AD) is the most common form of dementia and currently affects 5.4 million Americans. A number of anti-Aβ (beta amyloid) therapeutic agents have been developed for AD, but so far all of them failed in clinic. Here we used peptoid chemistry to develop ligands selective for Aβ42. Peptoids are N-substituted glycine oligomers, a class of peptidomimics. We synthesized an on-bead peptoid library consisting of 38,416 unique peptoids. The generated peptoid library was screened and arrays of Aβ42-selective peptoid ligands were identified. One of those peptoid ligands, IAM1 (inhibitor of amyloid), and the dimeric form (IAM1)2 were synthesized and evaluated in a variety of biochemical assays. We discovered that IAM1 selectively binds to Aβ42, while the dimeric derivative (IAM1)2 has a higher affinity for Aβ42. Furthermore, we demonstrated that IAM1 and (IAM1)2 were able to inhibit the aggregation of Aβ42 in a concentration-dependent manner, and that (IAM1)2 protected primary hippocampal neurons from the Aβ-induced toxicity in vitro. These results suggest that IAM1 and (IAM1)2 are specific Aβ42 ligands with antiaggregation and neuroprotective properties. IAM1, (IAM1)2, and their derivatives hold promise as Aβ42 detection agents and as lead compounds for the development of AD therapeutic agents.

Bacteria establish stable communities, known as biofilms, that are resistant to antimicrobials. Biofilm robustness is due to the presence of an extracellular matrix, which for several species-among them Bacillus subtilis-includes amyloid-like protein fibers. In this work, we show that B. subtilis biofilms can be a simple and reliable tool for screening of molecules with antiamyloid activity. We identified two molecules, AA-861 and parthenolide, which efficiently inhibited biofilms by preventing the formation of amyloid-like fibers. Parthenolide also disrupted pre-established biofilms. These molecules also impeded the formation of biofilms of other bacterial species that secrete amyloid proteins, such as Bacillus cereus and Escherichia coli. Furthermore, the identified molecules decreased the conversion of the yeast protein New1 to the prion state in a heterologous host, indicating the broad range of activity of the molecules., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Amyloid formation is associated with several human diseases including Alzheimer's disease (AD), Parkinson's disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease-modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high-resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti- cancer, anti-viral and anti-bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone-tryptophan hybrids which are capable of inhibiting amyloid-beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α-synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti-amyloid drugs., (Copyright © 2012 Wiley Periodicals, Inc.)

Unveiling interactions between labeling molecules and amyloid fibrils is essential to develop new detection methods for studying amyloid structures under various conditions. This review endeavours to reflect the progress in studying interactions between molecular inhibitors and amyloid peptides using a series of experimental approaches, such as X-ray diffraction, nuclear magnetic resonance, scanning probe microscopy, and electron microscopy. The revealed binding mechanisms of anti-amyloid drugs and target proteins could benefit the rational design of drugs for prevention or treatment of amyloidal diseases., (This journal is © The Royal Society of Chemistry 2012)

Abnormal protein assembly causes multiple devastating disorders in the central nervous system (CNS), such as Alzheimer's, Parkinson's, Huntington's, and prion diseases. Due to the now extended human lifespan, these diseases have been increasing in prevalence, resulting in major public health problems and the associated financial difficulties worldwide. The wayward proteins that lead to disease self-associate into neurotoxic oligomers and go on to form fibrillar polymers through multiple pathways. Thus, a range of possible targets for pharmacotherapeutic intervention exists along these pathways. Many compounds have shown different levels of effectiveness in inhibiting aberrant self-assembly, dissociating existing aggregates, protecting cells against neurotoxic insults, and in some cases ameliorating disease symptoms in vivo, yet achieving efficient, disease-modifying therapy in humans remains a major unattained goal. To a large degree, this is because the mechanisms of action for these drugs are essentially unknown. For successful design of new effective drugs, it is crucial to elucidate the mechanistic details of their action, including the actual target(s) along the protein aggregation pathways, how the compounds modulate these pathways, and their effect at the cellular, tissue, organ, and organism level. Here, the current knowledge of major mechanisms by which some of the more extensively explored drug candidates work are discussed. In particular, we focus on three prominent strategies: 1) stabilizing the native fold of amyloidogenic proteins, 2) accelerating the aggregation pathways towards the fibrillar endpoint thereby reducing accumulation of toxic oligomers, and 3) modulating the assembly process towards nontoxic oligomers/aggregates. The merit of each strategy is assessed, and the key points to consider when analyzing the efficacy of possible drug candidates and their mechanism of action are discussed., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein co-secreted with insulin in response to glucose levels. The formation of hIAPP amyloid plaques near islet cells has been linked to the death of insulin-secreting β-cells in humans and the progression of type II diabetes. Since both healthy individuals and those with type II diabetes produce and secrete hIAPP, it is reasonable to look for factors involved in storing hIAPP and preventing amyloidosis. We have previously shown that zinc inhibits the formation of insoluble amyloid plaques of hIAPP; however, there remains significant ambiguity in the underlying mechanisms. In this study, we show that zinc binds unaggregated hIAPP at micromolar concentrations similar to those found in the extracellular environment. By contrast, the fibrillar amyloid form of hIAPP has low affinity for zinc. The binding stoichiometry obtained from isothermal titration calorimetry experiments indicates that zinc favors the formation of hIAPP hexamers. High-resolution NMR structures of hIAPP bound to zinc reveal changes in the electron environment along residues that would be located along one face of the amphipathic hIAPP α-helix proposed as an intermediate for amyloid formation. Results from electrospray ionization mass spectroscopy investigations showed that a single zinc atom is predominantly bound to hIAPP and revealed that zinc inhibits the formation of the dimer. At higher concentrations of zinc, a second zinc atom binds to hIAPP, suggesting the presence of a low-affinity secondary binding site. Combined, these results suggest that zinc promotes the formation of oligomers while creating an energetic barrier for the formation of amyloid fibers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

TTR (transthyretin), a β-sheet-rich protein, is the precursor protein of familial amyloidotic polyneuropathy and senile systemic amyloidosis. Although it has been widely accepted that protein misfolding of the monomeric form of TTR is a rate-limiting step for amyloid formation, no effective therapy targeting this misfolding step is available. In the present study, we focused on CyDs (cyclodextrins), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various branched β-CyDs, GUG-β-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD] showed potent inhibition of TTR amyloid formation. Far-UV CD spectra analysis showed that GUG-β-CyD reduced the conformational change of TTR in the process of amyloid formation. In addition, tryptophan fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized the TTR conformation via interaction with the hydrophobic amino acids of TTR, especially tryptophan. Moreover, GUG-β-CyD exerted its inhibitory effect by reducing TTR deposition in transgenic rats possessing a human variant TTR gene in vivo. Collectively, these results indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.