Inhibition Of Protein Aggregation Research Articles

Single-molecule observations of human small heat shock proteins in complex with aggregation-prone client proteins.

Small heat shock proteins (sHsps) are molecular chaperones that act to prevent the aberrant aggregation of misfolded proteins. Whilst it is suggested that sHsps prevent aggregation by binding to misfolded client proteins, the dynamic and heterogeneous nature of sHsps has hindered attempts to establish the mechanistic details of how sHsp-client protein complexes form. Single-molecule approaches have emerged as a powerful tool to investigate dynamic and heterogeneous interactions such as those that can occur between sHsps and their client proteins. Here, we use total internal reflection fluorescence microscopy to observe and characterise the complexes formed between model aggregation-prone client proteins [firefly luciferase (FLUC), rhodanese, and chloride intracellular channel 1 protein (CLIC)], and the human sHsps αB-crystallin (αB-c; HSPB5) and Hsp27 (HSPB1). We show that small (monomeric or dimeric) forms of both αB-c and Hsp27 bind to misfolded or oligomeric forms of the client proteins at early stages of aggregation, resulting in the formation of soluble sHsp-client complexes. Stoichiometric analysis of these complexes revealed that additional αB-c subunits accumulate onto pre-existing sHsp-client complexes to form larger species - this does not occur to the same extent for Hsp27. Instead, Hsp27-client interactions tend to be more transient than those of αB-c. Elucidating these mechanisms of sHsp function is crucial to our understanding of how these molecular chaperones act to inhibit protein aggregation and maintain cellular proteostasis.

Development of carbazole-based molecules for inhibition of mutant hSOD1 protein aggregation in Amyotrophic Lateral Sclerosis.

Development of carbazole-based molecules for inhibition of mutant hSOD1 protein aggregation in Amyotrophic Lateral Sclerosis.

Overview of Metformin and Neurodegeneration: A Comprehensive Review.

This comprehensive review examines the therapeutic potential of metformin, a well-established diabetes medication, in treating neurodegenerative disorders. Originally used as a first-line treatment for type 2 diabetes, recent studies have begun investigating metformin's effects beyond metabolic disorders, particularly its neuroprotective capabilities against conditions like Parkinson's disease, Alzheimer's disease, Huntington's disease, and multiple sclerosis. Key findings demonstrate that metformin's neuroprotective effects operate through multiple pathways: AMPK activation enhancing cellular energy metabolism and autophagy; upregulation of antioxidant defenses; suppression of inflammation; inhibition of protein aggregation; and improvement of mitochondrial function. These mechanisms collectively address common pathological features in neurodegeneration and neuroinflammation, including oxidative stress, protein accumulation, and mitochondrial dysfunction. Clinical and preclinical evidence supporting metformin's association with improved cognitive performance, reduced risk of dementia, and modulation of pathological hallmarks of neurodegenerative diseases is critically evaluated. While metformin shows promise as a therapeutic agent, this review emphasizes the need for further investigation to fully understand its mechanisms and optimal therapeutic applications in neurodegenerative diseases.

Plant-Based Inhibitors of Protein Aggregation.

The assembly of amyloidogenic proteins and peptides into toxic oligomeric and fibrillar aggregates is closely connected to the onset and progression of more than 50 protein diseases, such as Alzheimer's disease, Parkinson's disease, prion disease, and type 2 diabetes, to name only a few. Considerable research efforts at identifying the therapeutic strategies against these maladies are currently focused on preventing and inhibiting pathogenic protein aggregation by various agents. Plant-based extracts and compounds have emerged as promising sources of potential inhibitors due to their dual role as nutraceuticals as part of healthy diets and as specific pharmaceuticals when administered at higher concentrations. In recent decades, several plant extracts and plant-extracted compounds have shown potential to modulate protein aggregation. An ever-growing body of research on plant-based amyloid inhibitors requires a detail analysis of existing data to identify potential knowledge gaps. This review summarizes the recent progress in amyloid inhibition using 17 flavonoids, 11 polyphenolic non-flavonoid compounds, 23 non-phenolic inhibitors, and 59 plant extracts, with the main emphasis on directly modulating the fibrillation of four amyloid proteins, namely amyloid-β peptide, microtubule-associated protein tau, α-synuclein, and human islet amyloid polypeptide.

Nafamostat Mesylate Regulates Glycosylation to Alleviate Aristolochic Acid Induced Kidney Injury.

Acute kidney injury (AKI) is a condition with a poor prognosis, exacerbated by the lack of effective therapeutic options and inadequately understood underlying mechanisms. Glycosylation, a post-translational modification of proteins, is essential for maintaining protein stability and function, and its dysregulation leads to protein misfolding and amyloid aggregation. Glycosylation dynamics are implicated in several pathologies, including inflammation, cancer, and AKI, highlighting the therapeutic potential of regulating glycosylation and preventing aggregation in AKI treatment. This study investigates the effect of nafamostat mesylate (NM) on protein glycosylation and amyloid aggregation in vivo. Using optical spectroscopy and other analytical techniques, we demonstrate that NM restores glycosylation levels and inhibits protein aggregation in aristolochic-acid-induced acute kidney injury. The mechanism likely involves enzymatic modulation that corrects hypoglycosylation and prevents amyloid aggregation, promoting proper protein folding and enhancing its stability. These findings suggest that NM may provide a novel therapeutic strategy for AKI and other glycosylation-related diseases, underscoring the potential for early intervention and treatment of these conditions.

Clay-Polymer Nanocomposites Mediated Inhibition of Protein Aggregation: Possible Role in the Prevention of Proteinopathies.

The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies). To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed. These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy. Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions. This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

Inhibition of amyloid formation of prion fragment (106-128) by polyphenolic compounds.

Inhibition of amyloid formation of prion fragment (106-128) by polyphenolic compounds.

YAT2150 is irresistible in Plasmodium falciparum and active against Plasmodium vivax and Leishmania clinical isolates

We recently characterized the potent antiplasmodial activity of the aggregated protein dye YAT2150, whose presumed mode of action is the inhibition of protein aggregation in the malaria parasite. Using single-dose and ramping methods, assays were done to select Plasmodium falciparum parasites resistant to YAT2150 concentrations ranging from 3× to 0.25× the in vitro IC50 of the compound (in the two-digit nM range) and performed a cross-resistance assessment in P. falciparum lines harboring mutations that make them resistant to a variety of antimalarial drugs. Resistant parasites did not emerge in vitro after 60 days of incubation, which postulates YAT2150 as an ‘irresistible’ antimalarial. The lyophilized compound is stable for at least one year stored at 25 °C. Tests performed in clinical isolates indicated that YAT2150 had also strong activity against Plasmodium vivax (IC50 between 4 and 36 nM) and Leishmania infantum (1.27 and 1.11 µM), placing it as a unique compound with perspectives of becoming the first drug to be used against both malaria and leishmaniasis.

Continuous selection in E. coli to identify cyclic peptide inhibitors of protein aggregation.

Continuous selection in E. coli to identify cyclic peptide inhibitors of protein aggregation.

Rapid discovery of cyclic peptide protein aggregation inhibitors by continuous selection.

Protein aggregates are associated with numerous diseases. Here we report a platform for the rapid phenotypic selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in Escherichia coli based on phage-assisted continuous evolution (PACE). We developed a new PACE-compatible selection for protein aggregation inhibition and used it to identify cyclic peptides that suppress amyloid-β42 and human islet amyloid polypeptide aggregation. Additionally, we integrated a negative selection that removes false positives and off-target hits, greatly improving cyclic peptide selectivity. We show that selected inhibitors are active when chemically resynthesized in in vitro assays. Our platform provides a powerful approach for the rapid discovery of cyclic peptide inhibitors of protein aggregation and may serve as the basis for the future evolution of cyclic peptides with a broad spectrum of inhibitory activities.

The role of amphipathic and cationic helical peptides in Parkinson's disease

AbstractPeptides are attracting a growing interest for therapeutic applications in biomedicine. In Parkinson's disease (PD), different human endogenous peptides have been associated with beneficial effects, including protein aggregation inhibition, reduced inflammation, or the protection of dopaminergic neurons. Such effects seem to be connected to the spatial arrangement of peptide side chains, and many of these human molecules share common conformational traits, displaying a distinctive amphipathic and cationic helical structure, which is believed to be crucial for their activities. This review delves into the relationship between these structural properties and the current evidence connecting biogenic peptides to the amelioration of PD symptoms. We discuss their implications in the disease, the different mechanisms of action, their state of validation, and their therapeutic potential.

Polymeric Nanodiscs Comprising 5-Fluorouracil for Inhibition of Protein Aggregation and Their Anti-Alzheimer's Activity in the Drosophila Model.

Nanoconjugates are promising for therapeutic drug delivery and targeted applications due to the numerous opportunities to functionalize their surface. The present study reports the synthesis of 5-fluorouracil (5-FU)-entrapped polyvinylpyrrolidone (PVP) nanoconjugates, precisely 5-FU-PVP and 5-FU-PVP-Au, and the evaluation of protein aggregation inhibition efficiency. The 5-FU-loaded polymer nanoconjugates were functionalized with gold nanoparticles and analyzed using characterization techniques like dynamic light scattering, UV-visible spectroscopy, Fourier-transform infrared spectroscopy, and zeta potential analysis. These conjugates exhibit consistent morphology with a spherical, flat, disc-like structure. The 5-FU-PVP and 5-FU-PVP-Au nanoconjugates exhibited a high drug loading, up to 81% and 90%, respectively. The nanoconjugates exhibited prolonged drug delivery of 5-FU from 5-FU-PVP and 5-FU-PVP-Au, wherein 5-FU-PVP-Au depicted a higher drug release. They were investigated for inhibiting the protein hen egg white lysozyme (HEWL) aggregation by ThT fibril size measurement, binding assay, and electron microscopy, and the results showed that conjugates repressed the fibrillogenesis in HEWL. The prominent activity of amyloid aggregation inhibition for HEWL using 5-FU-PVP and 5-FU-PVP-Au was found to be 29 μg.mL-1 and 27 μg.mL-1, respectively. The dissociation of amyloid aggregates was achieved against 5-FU-PVP and 5-FU-PVP-Au at 27 μg.mL-1 and 25 μg.mL-1, respectively. Furthermore, the nanoconjugates were investigated for anti-Alzheimer's activity in the Drosophila model. A Drosophila model of Alzheimer's disease (AD) was developed that expressed Aβ42 peptides in the neuronal secretory system to comprehend the pathogenic effects of Aβ42 in vivo. All the results demonstrate that polymer nanoconjugates exhibit more effective inhibition of protein aggregation than bare drugs.

Development of tau targeted protein degraders for Alzheimer disease

AbstractBackgroundAlzheimer disease (AD) is a progressive neurodegenerative disease that is accountable for the leading case of dementia in elder people. Before, only symptomatic treatments are available for AD. Since 2021, two anti‐amyloid antibodies aducanumab and lecanemab have been approved by the US Food and Drug Administration. However, controversies are still around these two antibodies regarding their efficacy, safety, and substantial economic burden. They also have restricted brain delivery and could not pass through intact neurons to target intracellular proteins. There is still an urgent need for effective, easily administered (oral) and low‐cost drugs for AD.The intraneuronal accumulation of tau paired helical filaments (PHFs) is one of the hallmarks of AD brain pathology. Lowered efficiency of degradation pathways, such as ubiquitin proteasome system, further exacerbates tau pathologies. Instead of inhibiting protein aggregation, a class of molecules that promote the degradation of targeted protein is known as proteolysis‐targeting chimeras (PROTACs). Since 2016, PROTACs have been applied to resolve tau pathologies, progressing from being based on peptides to fully synthetic small molecules. This exploration is still at the early stage. Here, we aim to develop novel small‐molecule PROTACs as tau targeted protein degraders for AD treatment.MethodsWe performed global molecular docking of tau binders from a public database (bindingdb.org) into the available PHFs 3D structure and selected top ranked compounds. Together with them, ligands binding to E3 ligase, for instance cereblon (CRBN) and carboxyl terminus of Hsp70 interacting protein (CHIP), were used with different linkers to design synthetically accessible PROTACs. The modelling of ternary complex (tau‐PROTACs‐E3 ligase) are performed to rank designed PROTACs and the highly scored will be synthesised. Protein binding affinities and tau‐reducing effects will be evaluated in different cell models: human neuroblastoma SH‐SY5Y cells and mouse embryonic primary neurons.ResultsWe perform the development of tau targeted protein degraders according to the methods listed above. We selected the top ranked tau binders and designed new PROTACs.ConclusionsThe most potent PROTACs with least off‐target effects will move on to in vivo study and provide pre‐clinical evidence for novel treatment of AD tauopathies.

Development of Diverse Carbon Dots to Inhibit Protein Aggregation in Alzheimer’s Disease

Development of Diverse Carbon Dots to Inhibit Protein Aggregation in Alzheimer’s Disease

Characterization of a novel Carbon Dot Inhibitor of Tau and Amyloid Beta Aggregation in Alzheimer’s Disease

AbstractBackgroundAlzheimer’s Disease (AD) is a neurodegenerative disorder whose pathological hallmarks include tau and amyloid beta aggregation, a phenomenon that has been linked to inflammation and degradation of brain tissue. Prior data published in the Wang lab suggests that carbon dots (CDs) synthesized from citric acid and urea can inhibit aggregation. We sought to characterize the inhibitory effects of a new class of CDs synthesized from varied ratios of Congo red and citric acid. Since Congo red carbon dots (CRCDs) can cross the blood‐brain barrier (BBB), they have the potential to be used as a therapeutic.MethodA new type of CD was synthesized via the muffle furnace method using citric acid, urea, thiourea, selenourea, formamide, and congo red. The CDs have a diameter less than 20 nanometers, enabling them to cross both the BBB and the cell membrane, which was confirmed experimentally with zebrafish models. The newly synthesized CDs were used in Thioflavin T (ThT) aggregation assays to assess protein aggregation inhibition. This was verified visually by Atomic Force Microscopy (AFM).ResultAll three CRCDs inhibited tau aggregation, with CRCD1 being the most effective. CRCD1 had the lowest IC50 of 0.21ug/mL, while CRCD2 and CRCD3 had an IC50 of 0.50ug/mL and 2.55ug/mL, respectively. All three CRCDs also inhibited amyloid beta aggregation, with CRCD being the most effective. CRCD1 had the lowest IC50 of 1.76 ug/mL, while CRCD2 and CRCD3 had an IC50 of 3.34 ug/mL and 5.61 ug/mL, respectively.ConclusionThe ability to reduce tau aggregation in the brain has the potential to slow the growth of the disease and potentially alleviate symptoms. There are currently limited treatments available for AD, but CRCDs show great promise.

Imidazo[2,1-b][1,3]thiazine Derivatives as Potential Modulators of Alpha-Synuclein Amyloid Aggregation.

Insoluble amyloid fibrils accumulate in the intercellular spaces of organs and tissues, leading to various amyloidosis-related disorders in the human body. Specifically, Parkinson's disease is associated with the aggregation of alpha-synuclein. However, current treatments for Parkinson's primarily focus on managing motor symptoms and slowing disease progression. Efforts to prevent and halt the progression of these diseases involve the search for small molecular compounds. In this work, we synthesized imidazo[2,1-b][1,3]thiazines in an atom-economic way by cyclization of 2-alkynylthioimidazoles using 10% AuCl as the catalyst. We identified several compounds with specific functional groups capable of both inhibiting the aggregation of alpha-synuclein and redirecting the fibril formation pathway. The investigation into how these substances function revealed that imidazo[2,1-b][1,3]thiazine derivatives can influence alpha-synuclein aggregation in several ways. They not only inhibit the primary nucleation process and maintain a balance toward nonaggregated protein states but also stabilize smaller oligomeric species of alpha-synuclein and cause the formation of fibrils with unique structures and forms. These imidazo[2,1-b][1,3]thiazines could potentially be used in developing highly efficient, small molecular weight protein aggregation inhibitors.

Research progress on antioxidants and protein aggregation inhibitors in cataract prevention and therapy (Review).

Cataracts are primarily caused by aging or gene mutations and are the leading cause of blindness globally. As the older population increases, the number of patients with a cataract is expected to grow rapidly. At present, cataract surgery to replace the lens with an artificial intraocular lens is the principal treatment method. However, surgery has several drawbacks, including economic burdens and complications such as inflammation, xerophthalmia, macular edema and posterior capsular opacification. Thus, developing an effective non‑surgical treatment strategy is beneficial to both patients and public health. Mechanistically, cataract formation may be due to various reasons but is primarily initiated and promoted by oxidative stress and is closely associated with crystallin aggregation. In the present review, the current research progress on anti‑cataract drugs, including antioxidants and protein aggregation inhibitors is examined. It summarizes strategies for preventing and treating cataract through cell apoptosis and protein aggregation inhibition while discussing their limitations and further prospects.

Replenishing Cation-π Interactions for the Fabrication of Mesoporous Levodopa Nanoformulations for Parkinson Remission.

Directly assembling drugs into mesoporous nanoformulations will be greatly favored due to the combination of enhanced drug delivery efficiency and mesostructure-enabled nanobio interactions. However, such an approach is hindered due to the lack of understanding of polymer nanoparticles' formation mechanism, especially the relationship between polymerization, self-assembly, and the nucleation process. Here, by investigating the levodopa and dopamine polymerization process, we identify π-cation interaction as pivotal in the self-assembly and nucleation control of dopa molecules. Thus, through manipulation of the π-cation interaction, we present the direct assembly of a commercial drug, levodopa, into mesoporous nanoformulations. The synthesized nanospheres, approximately 200 nm in diameter, exhibit uniform mesopores of around 8 nm. These nanoformulations, abundant in mesopores, enhance chiral phenylalanine interaction with α-synuclein (Syn), curbing aggregation, safeguarding neurons, and alleviating Parkinson's pathology. When combating α-synuclein, the nanoformulation achieved ∼100% inhibition of protein aggregation and sustained neuron viability up to 300%. We believe that this study may advance mesoscale self-assembly knowledge, guiding future nanopharmaceutical developments.

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

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

Amyotrophic Lateral Sclerosis: Insights and New Prospects in Disease Pathophysiology, Biomarkers and Therapies.

Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disorder marked by the gradual loss of motor neurons, leading to significant disability and eventual death. Despite ongoing research, there are still limited treatment options, underscoring the need for a deeper understanding of the disease's complex mechanisms and the identification of new therapeutic targets. This review provides a thorough examination of ALS, covering its epidemiology, pathology, and clinical features. It investigates the key molecular mechanisms, such as protein aggregation, neuroinflammation, oxidative stress, and excitotoxicity that contribute to motor neuron degeneration. The role of biomarkers is highlighted for their importance in early diagnosis and disease monitoring. Additionally, the review explores emerging therapeutic approaches, including inhibitors of protein aggregation, neuroinflammation modulators, antioxidant therapies, gene therapy, and stem cell-based treatments. The advantages and challenges of these strategies are discussed, with an emphasis on the potential for precision medicine to tailor treatments to individual patient needs. Overall, this review aims to provide a comprehensive overview of the current state of ALS research and suggest future directions for developing effective therapies.