Amyloid Inhibitors Research Articles

Discovery of novel deoxyvasicinone derivatives with benzenesulfonamide substituents as multifunctional agents against Alzheimer's disease

A series of deoxyvasicinone derivatives with benzenesulfonamide substituents were designed and synthesized to find a multifunctional anti-Alzheimer's disease (AD) drug. The results of the biological activity evaluation indicated that most compounds demonstrated selective inhibition of acetylcholinesterase (AChE). Among them, g17 exhibited the most potent inhibitory effect on AChE (IC50 = 0.24 ± 0.04 μM). Additionally, g17 exhibited promising properties as a metal chelator and inhibitor of amyloid β peptides self-aggregation (68.34 % ± 1.16 %). Research on oxidative stress has shown that g17 displays neuroprotective effects and effectively suppresses the intracellular accumulation of reactive oxygen species. Besides, g17 demonstrated remarkable anti-neuroinflammatory effects by significantly reducing the production of pro-inflammatory cytokines (such as NO, IL-1β, and TNF-α) and inhibiting the expression of inflammatory mediators iNOS and COX-2. In vivo studies showed that g17 significantly improved AD model mice's cognitive and memory abilities. Histological examination of mouse hippocampal tissue sections using hematoxylin and eosin staining revealed that g17 effectively mitigates neuronal damage. Considering the multifunctional properties of g17, it is regarded as a promising lead compound for treating AD.

Catechol-Amyloid Interactions.

This review introduces multifaceted mutual interactions between molecules containing a catechol moiety and aggregation-prone proteins. The complex relationships between these two molecular species have previously been elucidated primarily in a unidirectional manner, as demonstrated in cases involving the development of catechol-based inhibitors for amyloid aggregation and the elucidation of the role of functional amyloid fibers in melanin biosynthesis. This review aims to consolidate scattered clues pertaining to catechol-based amyloid inhibitors, functional amyloid scaffold of melanin biosynthesis, and chemically designed peptide fibers for providing chemical insights into the role of the local three-dimensional orientation of functional groups in manifesting such interactions. These orientations may play crucial, yet undiscovered, roles in various supramolecular structures.

Anti-Alzheimer potential of coumarin derivatives: A review

Alzheimer’s is a type of neurodegenerative diseases (NDs) found in old age people which main causes the dementia and various brain disorders. FDA approved drugs used commonly in treatment of moderate to severe Alzheimer's disease are Donepezil, Rivastigmine, Galantamine and Memantine. However, these approved drugs provide only palliative support not complete cure. So, urgency of new molecules has been becoming so important to cure this complex Disease. Coumarin or 2-H-Chromen-2-one is an oxygenated heterocyclic compound, which is isolated from plants named Dipteryx odorata Willd, (Fabaceae). Synthetic coumarin derivatives has been well presence in literature due to existence of their potential pharmacological activities in literature. This review covers the anti-Alzheimer activities of chemically synthesized coumarin derivatives from 2016-2023. Coumarin derivatives profound found exhibits potential anti-Alzheimer activities in literature. Coumarin derivatives showed their anti-Alzheimer potential through various pharmacological pathways such as monoamine oxidase (MAO) inhibitor, acetylcholinesterase (AChE) & Butyl cholinesterase (BuChE) inhibitory activity, Aβ amyloid inhibition, Beta secretase 1 (BACE1 inhibitors). Multi-target directed ligands (MTDLs) approach also extensively reported to design and synthesis of novel coumarin scaffold as potential multifunctional Anti-Alzheimer activities in last one decade. We also covered literature of some coumarin hybrids derivatives as potential Anti-Alzheimer activities. Some common synthetic methods of coumarin structure also covered in this review article. This review supported that coumarin derivatives found excellent anti-Alzheimer activities and it can be play major role to solve the problem of complex AD disease.

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion.

Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis.

Understanding the Molecular Mechanisms of Polyphenol Inhibition of Amyloid β Aggregation.

Alzheimer's disease (AD) is highly associated with self-aggregation of amyloid β (Aβ) proteins into fibrils. Inhibition of Aβ aggregation by polyphenols is one of the major therapeutic strategies for AD. Among them, four polyphenols (brazilin, resveratrol, hematoxylin, and rosmarinic acid) have been reported to be effective at inhibiting Aβ aggregation, but the inhibition mechanisms are still unclear. In this work, these four polyphenols were selected to explore their interactions with the Aβ17-42 pentamer by molecular dynamics simulation. All four polyphenols can bind to the pentamer tightly but prefer different binding sites. Conversion of the β-sheet to the random coil, fewer interchain hydrogen bonds, and weaker salt bridges were observed after binding. Interestingly, different Aβ17-42 pentamer destabilizing mechanisms for resveratrol and hematoxylin were found. Resveratrol inserts into the hydrophobic core of the pentamer by forming hydrogen bonds with Asp23 and Lys28, while hematoxylin prefers to bind beside chain A of the pentamer, which leads to β-sheet offset and dissociation of the β1 sheet of chain E. This work reveals the interactions between the Aβ17-42 pentamer and four polyphenols and discusses the relationship between inhibitor structures and their inhibition mechanisms, which also provides useful guidance for screening effective Aβ aggregation inhibitors and drug design against AD.

Structure-based design of nanobodies that inhibit seeding of Alzheimer’s patient–extracted tau fibrils

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

The structurally diverse phytocannabinoids cannabichromene, cannabigerol and cannabinol significantly inhibit amyloid β-evoked neurotoxicity and changes in cell morphology in PC12 cells.

Phytocannabinoids (pCBs) have been shown to inhibit the aggregation and neurotoxicity of the neurotoxic Alzheimer's disease protein beta amyloid (Aβ). We characterized the capacity of six pCBs-cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidiol (CBD) and Δ9 -tetrahydrocannabinol (Δ9 -THC)-to disrupt Aβ aggregation and protect against Aβ-evoked neurotoxicity in PC12 cells. Neuroprotection against lipid peroxidation and Aβ-induced cytotoxicity was assessed using the MTT assay. Transmission electron microscopy was used to visualize pCB effects on Aβ aggregation and fluorescence microscopy, with morphometrics and principal component analysis to assess PC12 cell morphology. CBD inhibited lipid peroxidation with no significant effect on Aβ toxicity, whilst CBN, CBDV and CBG provided neuroprotection. CBC, CBG and CBN inhibited Aβ1-42 -induced neurotoxicity in PC12 cells, as did Δ9 -THC, CBD and CBDV. CBC, CBN and CBDV inhibited Aβ aggregation, whilst Δ9 -THC reduced aggregate density. Aβ1-42 induced morphological changes in PC12 cells, including a reduction in neuritic projections and rounded cell morphology. CBC and CBG inhibited this effect, whilst Δ9 -THC, CBD and CBDV did not alter Aβ1-42 effects on cell morphology. These findings highlight the neuroprotective activity of CBC, CBG and CBN as novel pCBs associated with variable effects on Aβ-evoked neurite damage and inhibition of amyloid β aggregation.

Towards Optimal Control of Amyloid Fibrillation.

Epigallocatechin-3-gallate, as a representative amyloid inhibitors, has shown a promising ability against A[Formula: see text] fibrillation by directly degradating the mature fibrils. Most previous studies have been focusing on its functional mechanisms, meanwhile its optimal dosage has been seldom considered. To solve this critical issue, we refer to the generalized Logistic model for amyloid fibrillation and inhibition and adopt the optimal control theory to balance the effectiveness and cost (or toxicity) of inhibitors. The optimal control trajectory of inhibitors is analytically solved, based on which the influence of model parameters, the difference between the optimal control strategy and several other traditional drug dosing strategies are systematically compared and validated through experiments. It is found that the strategy of multiple-times adding is more suitable for a long-term disease treatment, while single high-dose therapy is preferred for a short-term treatment. We hope our findings can shed light on the rational usage of amyloid inhibitors in clinic.

Polymeric curcumin nanospheres for lysozyme aggregation inhibition, antibacterial, and wound healing applications.

The present study reports highly stable polymeric nanoparticles comprising curcumin and polyvinylpyrrolidone, and then conjugated with gold nanoparticles, resulting in C-PVP and C-PVP-Au, respectively. The synthesized conjugates C-PVP and C-PVP-Au were investigated for amyloid aggregation inhibition activity, antimicrobial activity, and wound healing applications.The anti-amyloidogenic capacity of nanoconjugates were studied for model protein, hen egg-white lysozyme (HEWL). The ThT binding assay, fibril size measurement, and electron microscopy results revealed that conjugates suppress fibrillogenesis in HEWL. The highest amyloid inhibition activity obtained against C-PVP and C-PVP-Au was 31μg.mL-1 and 30μg.mL-1, respectively. The dissociation activity for amyloid aggregation was observed against Q-PVP and Q-PVP-Au at 29μg.mL-1 and 27μg.mL-1, respectively. The antibacterial studies show significant efficacy against Escherichia coli (E. coli) in the presence of C-PVP and C-PVP-Au. The substantial antibacterial potential of C-PVP@PVA and C-PVP-Au@PVA membranes shows promising wound healing applications. The PVA membranes with nanoparticles promote the antibacterial activity and wound healing activity in the Drosophila model. C-PVP-Au@PVA membrane healed the wound faster than the C-PVP@PVA, and it can be used for better results in wound healing. Thus, C-PVP-Au and C-PVP have higher bioavailability and stability and can act as multifunctional therapeutic agents for amyloid-related diseases and as wound healing agents. Graphical abstract C-PVP, and C-PVP-Au conjugates for inhibition of HEWL aggregation, antibacterial and wound healing activity.

Inhibition of Amyloid β Aggregation and Cytotoxicity by Berbamine Hydrochloride.

Alzheimer's disease (AD) continues to be a major global health challenge, and the recent approval of Aduhelm and Leqembi has opened new avenues for its treatment. Small-molecule inhibitors targeting Aβ aggregation hold promise as an alternative to monoclonal antibodies. In this study, we evaluated the ability of berbamine hydrochloride (BBMH), a member of the bisbenzylisoquinoline alkaloids, to reduce Aβ aggregation and cytotoxicity. Thioflavin T kinetics, circular dichroism spectroscopy, and atomic force microscopy results indicated that BBMH effectively inhibited Aβ aggregation. Surface plasmon resonance and molecular docking results further revealed that BBMH could bind to Aβ fibrils, thereby hindering the aggregation process. This physical picture has been confirmed in a quantitative way by chemical kinetics analysis, which showed BBMH tends to bind with the fibril ends and thus prevents the transition from protofibrils to mature fibrils as well as the elongation process. Additionally, our MTT results showed that BBMH was able to reduce the cytotoxicity of Aβ40 on N2a cells. Our results demonstrate, for the first time, the potential of BBMH to inhibit Aβ aggregation and cytotoxicity, offering a promising direction for further research and drug development efforts in the fight against Alzheimer's disease.

Repurposing Antimicrobial Protegrin-1 as a Dual-Function Amyloid Inhibitor via Cross-seeding.

Amyloids and antimicrobial peptides have traditionally been recognized as distinct families with separate biological functions and targets. However, certain amyloids and antimicrobial peptides share structural and functional characteristics that contribute to the development of neurodegenerative diseases. Specifically, the aggregation of amyloid-β (Aβ) and microbial infections are interconnected pathological factors in Alzheimer's disease (AD). In this study, we propose and demonstrate a novel repurposing strategy for an antimicrobial peptide of protegrin-1 (PG-1), which exhibits the ability to simultaneously prevent Aβ aggregation and microbial infection both in vitro and in vivo. Through a comprehensive analysis using protein, cell, and worm assays, we uncover multiple functions of PG-1 against Aβ, including the following: (i) complete inhibition of Aβ aggregation at a low molar ratio of PG-1/Aβ = 0.25:1, (ii) disassembly of the preformed Aβ fibrils into amorphous aggregates, (iii) reduction of Aβ-induced cytotoxicity in SH-SY5Y cells and transgenic GMC101 nematodes, and (iv) preservation of original antimicrobial activity against P.A., E.coli., S.A., and S.E. strains in the presence of Aβ. Mechanistically, the dual anti-amyloid and anti-bacterial functions of PG-1 primarily arise from its strong binding to distinct Aβ seeds (KD = 1.24-1.90 μM) through conformationally similar β-sheet associations. This work introduces a promising strategy to repurpose antimicrobial peptides as amyloid inhibitors, effectively targeting multiple pathological pathways in AD.

Ambient Temperature Affects Protein Self-Assembly by Interfering with the Interfacial Aggregation Behavior.

Amyloid fibrillation is known to be associated with degenerative diseases, and mature fibrils are also considered as valuable biomedical materials. Thus, the mechanism and influencing factors of fibrillation have always been the focus of research. However, in vitro studies are always plagued by low reproducibility of kinetics and the molecular mechanism of amyloid fibrillation is under debate until now. Here, we identified the ambient temperature (AT) as a non-negligible interfering factor in in vitro self-assembly of globular protein hen egg-white lysozyme for the first time. By multimodal molecular spectroscopy methods, not only the effect of ATs on the kinetics of protein aggregation was described but also the conformational changes of the molecular structure with different ATs were captured. Through investigating the dependence of interfacial area and catalysis, the reason for this influence was construed by the various aggregation behaviors of protein molecules in the two-phase interface. The results suggest that in vitro mechanism research on protein fibrillation needs to first clarify the AT for a more accurate comparative analysis. The proposal of this concept will provide a new clue for a deeper understanding of the mechanism of protein self-assembly and may have an impact on evaluating the efficiency of amyloid accelerators or inhibitors based on the comparative analysis of protein self-assembly.

Realization of Amyloid-like Aggregation as a Common Cause for Pathogenesis in Diseases.

Amyloids were conventionally referred to as extracellular and intracellular accumulation of Aβ42 peptide, which causes the formation of plaques and neurofibrillary tangles inside the brain leading to the pathogenesis in Alzheimer's disease. Subsequently, amyloid-like deposition was found in the etiology of prion diseases, Parkinson's disease, type II diabetes, and cancer, which was attributed to the aggregation of prion protein, α-Synuclein, islet amyloid polypeptide protein, and p53 protein, respectively. Hence, traditionally amyloids were considered aggregates formed exclusively by proteins or peptides. However, since the last decade, it has been discovered that other metabolites, like single amino acids, nucleobases, lipids, glucose derivatives, etc., have a propensity to form amyloid-like toxic assemblies. Several studies suggest direct implications of these metabolite assemblies in the patho-physiology of various inborn errors of metabolisms like phenylketonuria, tyrosinemia, cystinuria, and Gaucher's disease, to name a few. In this review, we present a comprehensive literature overview that suggests amyloid-like structure formation as a common phenomenon for disease progression and pathogenesis in multiple syndromes. The review is devoted to providing readers with a broad knowledge of the structure, mode of formation, propagation, and transmission of different extracellular amyloids and their implications in the pathogenesis of diseases. We strongly believe a review on this topic is urgently required to create awareness about the understanding of the fundamental molecular mechanism behind the origin of diseases from an amyloid perspective and possibly look for a common therapeutic strategy for the treatment of these maladies by designing generic amyloid inhibitors.

Identification of Novel Peptides as Potential Modulators of Aβ42 Amyloidogenesis: An in silico Approach.

Alzheimer's disease is a neurodegenerative disease for which no cure is available. The presence of amyloid plaques in the extracellular space of neural cells is the key feature of this fatal disease. The proteolysis of Amyloid Precursor Protein by presenilin leads to the formation of Amyloid-beta peptides (Aβ 42/40). Deposition of 42 residual Aβ peptides forms fibril's structure, disrupting neuron synaptic transmission, inducing neural cell toxicity, and ultimately leading to neuron death. Various novel peptides have been investigated via molecular docking and molecular dynamic simulation studies to investigate their effects on Aβ amyloidogenesis. The sequence-based peptides were rationally designed and investigated for their interaction with Aβ42 monomer and fibril, and their influence on the structural stability of target proteins was studied. Analyzed docking results suggest that the peptide YRIGY (P6) has the highest binding affinity with Aβ42 fibril amongst all the synthetic peptides, and the peptide DKAPFF (P12) similarly shows a better binding with the Aβ42 monomer. Moreover, simulation results also suggest that the higher the binding affinity, the better the inhibitory action. These findings indicate that both the rationally designed peptides can modulate amyloidogenesis, but peptide (P6) has better potential for the disaggregation of the fibrils. In contrast, peptide P12 stabilizes the native structure of the Aβ42 monomer more effectively and hence can serve as a potential amyloid inhibitor. Thus, these peptides can be explored as therapeutic agents against Alzheimer's disease. Experimental testing of these peptides for immunogenicity, stability in cellular conditions, toxic effects and membrane permeability can be the future research scope of this study.

Self-Assembly of Cysteine into Nanofibrils Precedes Cystine Crystal Formation: Implications for Aggregation Inhibition.

The self-association of metabolites into well-ordered assemblies at the nanoscale has significant biological and medical implications. The thiol-containing amino acid cysteine (CYS) can assemble into amyloid-like nanofibrils, and its oxidized form, the disulfide-bonded cystine (CTE), forms hexagonal crystals as those found in cystinuria due to metabolic disorder. Yet, there have been no attempts to connect these two phenomena, especially the fibril-to-crystal transition. Here, we reveal that these are not separated events, and the CYS-forming amyloid fibrils are mechanistically linked to hexagonal CTE crystals. For the first time, we demonstrated that cysteine fibrils are a prerequisite for forming cystine crystals, as observed experimentally. To further understand this mechanism, we studied the effects of thiol-containing cystinuria drugs (tiopronin, TIO; and d-penicillamine, PEN) and the canonical epigallocatechin gallate (EGCG) amyloid inhibitor on fibril formation by CYS. The thiol-containing drugs do not solely interact with monomeric CYS via disulfide bond formation but can disrupt amyloid formation by targeting CYS oligomers. On the other hand, EGCG forms inhibitor-dominant complexes (more than one EGCG molecule per cysteine unit) to prevent CYS fibril formation. Interestingly, while CYS can be oxidized into CTE, the thiol drugs can reduce CTE back to CYS. We thus suggest that the formation of crystals in cystinuria could be halted at the initial stage by targeting CYS fibril formation as an alternative to solubilizing the water-insoluble hexagonal CTE crystals at a later stage. Taken together, we depicted a complex hierarchical organization in a simple amino acid assembly with implications for therapeutic intervention.

Identification of new pentapeptides as potential inhibitors of amyloid–β42 aggregation using virtual screening and molecular dynamics simulations

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease mainly characterized by extracellular accumulation of amyloid-β (Aβ) peptide. Previous studies reported pentapeptide RIIGL as an effective inhibitor of Aβ aggregation and neurotoxicity induced by Aβ aggregates. In this work, a library of 912 pentapeptides based on RIIGL has been designed and assessed for their efficacy to inhibit Aβ42 aggregation using computational techniques. The top hit pentapeptides revealed by molecular docking were further assessed for their binding affinity with Aβ42 monomer using MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method. The MM-PBSA analysis identified RLAPV, RVVPI, and RIAPA, which bind to Aβ42 monomer with a higher binding affinity −55.80, −46.32, and −44.26 kcal/mol, respectively, as compared to RIIGL (ΔGbinding = −41.29 kcal/mol). The residue-wise binding free energy predicted hydrophobic contacts between Aβ42 monomer and pentapeptides. The secondary structure analysis of the conformational ensembles generated by molecular dynamics (MD) depicted remarkably enhanced sampling of helical and no β–sheet conformations in Aβ42 monomer on the incorporation of RVVPI and RIAPA. Notably, RVVPI and RIAPA destabilized the D23–K28 salt bridge in Aβ42 monomer, which plays a crucial role in Aβ42 oligomer stability and fibril formation. The MD simulations highlighted that the incorporation of proline and arginine in pentapeptides contributed to their strong binding with Aβ42 monomer. Furthermore, RVVPI and RIAPA prevented conformational conversion of Aβ42 monomer to aggregation-prone structures, which, in turn, resulted in a lower aggregation tendency of Aβ42 monomer.

Rivastigmine-DHA ion-pair complex improved loading in hybrid nanoparticles for better amyloid inhibition and nose-to-brain targeting in Alzheimer’s

Rivastigmine hydrogen tartrate (RIV-HT) is given orally for Alzheimer's disease. However, oral therapy shows low brain bioavailability, short half-life and gastrointestinal-mediated adverse effects. RIV-HT intranasal delivery can avoid these side effects, but its low brain bioavailability remains challenging. These issues could be solved with hybrid lipid nanoparticles with enough drug loading to enhance RIV-HT brain bioavailability while avoiding oral route side effects. The RIV-HT and docosahexaenoic acid (DHA) ion-pair complex (RIV:DHA) was prepared to improve drug loading into lipid-polymer hybrid (LPH) nanoparticles. Two types of LPH, i.e., cationic (RIV:DHA LPH(+ve)) and anionic LPH (RIV:DHA LPH(-ve)) were developed. The effect of LPH surface charge on in-vitro amyloid inhibition, in-vivo brain concentrations and nose-to-brain drug targeting efficiency were investigated. LPH nanoparticles showed concentration dependant amyloid inhibition. RIV:DHA LPH(+ve) demonstrated relatively enhanced Aβ1-42 peptide inhibition. The thermoresponsive gel embedded with LPH nanoparticles improved nasal drug retention. LPH nanoparticles gel significantly improved pharmacokinetic parameters compared to RIV-HT gel. RIV:DHA LPH(+ve) gel showed better brain concentrations than RIV:DHA LPH(-ve) gel. The histological examination of nasal mucosa treated with LPH nanoparticles gel showed that the delivery system was safe. In conclusion, the LPH nanoparticle gel was safe and efficient in improving the nose-to-brain targeting of RIV, which can potentially be utilized in managing Alzheimer's.

Dissecting the Self-assembly Dynamics of Imperfect Repeats in α-Synuclein.

The pathological aggregation of α-synuclein (αS) into amyloid fibrils is the hallmark of Parkinson's disease (PD). The self-assembly and membrane interactions of αS are mainly governed by the seven imperfect 11-residue repeats of the XKTKEGVXXXX motif around residues 1-95. However, the particular role of each repeat in αS fibrillization remains unclear. To answer this question, we studied the aggregation dynamics of each repeat with up to 10 peptides in silico by conducting multiple independent micro-second atomistic discrete molecular dynamics simulations. Our simulations revealed that only repeats R3 and R6 readily self-assembled into β-sheet-rich oligomers, while the other repeats remained as unstructured monomers with weak self-assembly and β-sheet propensities. The self-assembly process of R3 featured frequent conformational changes with β-sheet formation mainly in the non-conserved hydrophobic tail, whereas R6 spontaneously self-assembled into extended and stable cross-β structures. These results of seven repeats are consistent with their structures and organization in recently solved αS fibrils. As the primary amyloidogenic core, R6 was buried inside the central cross-β core of all αS fibrils, attracting the hydrophobic tails of adjacent R4, R5, and R7 repeats forming β-sheets around R6 in the core. Further away from R6 in the sequence but with a moderate amyloid aggregation propensity, the R3 tail could serve as a secondary amyloidogenic core and form independent β-sheets in the fibril. Overall, our results demonstrate the critical role of R3 and R6 repeats in αS amyloid aggregation and suggest their potential as targets for the peptide-based and small-molecule amyloid inhibitors.

Inhibition of primary and secondary nucleation alongwith disruption of amyloid fibrils and alleviation of associated cytotoxicity: A biophysical insight of a novel property of Chlorpropamide (an anti-diabetic drug)

Aggregation of physiologically synthesized soluble proteins to insoluble, cytotoxic fibrils is a pre-requisite for pathogenesis of amyloid associated disorders including Alzheimer's disease, non-systemic amyloidosis, Parkinson's disease, etc. Considerable advancement has been made to understand the mechanism behind aggregation process but till date we have no efficient cure and preventive therapy for associated diseases. Strategies to prevent protein aggregation are nevertheless many which have been proved promisingly successful in vitro. One of those is repurposing already approved drugs that saves time and money too and has been employed in this study. Here, for the first time we are reporting the effectiveness of an anti-diabetic drug chlorpropamide (CHL) under dosage conditions, a novel property to inhibit aggregation in human lysozyme (HL) in vitro. Spectroscopic (Turbidity, RLS, ThT, DLS, ANS) and microscopic (CLSM) results demonstrates that CHL has the potency to suppress aggregation in HL up to 70 %. CHL is shown to affect the elongation of fibrils with IC50 value of 88.5 μM as clear from the kinetics results, may be by interacting near/with aggregation prone regions of HL. Hemolytic assay also revealed the reduced cytotoxicity in the presence of CHL. Disruption of amyloid fibrils and inhibition of secondary nucleation in the presence of CHL was also evidenced by ThT, CD and CLSM results with reduced cytotoxicity as confirmed by hemolytic assay. We also performed preliminary studies on α-synuclein fibrillation inhibition and surprisingly found that CHL is not just inhibiting the fibrillation but also stabilizing the protein in its native state. These findings insinuate that CHL (anti-diabetic) possess multiple roles and can be a promising drug for developing therapeutic against non-systemic amyloidosis, Parkinson's disease and other amyloid associated disorders.

Design and synthesis of piano-stool ruthenium(II) complexes and their studies on the inhibition of amyloid β (1–42) peptide aggregation

Misfolding and protein aggregation have been linked to numerous human neurodegenerative disorders such as Alzheimer's, prion, and Parkinson's diseases. Ruthenium (Ru) complexes have received considerable attention in studying protein aggregation due to their interesting photophysical and photo properties. In this study, we have synthesized the novel Ru complexes ([Ru(p-cymene)Cl(L-1)][PF6](Ru-1), and [Ru(p-cymene)Cl(L-2)][PF6](Ru-2)) and investigated their inhibitory activity against the bovine serum albumin (BSA) aggregation and the Aβ1–42 peptides amyloid formation. Several spectroscopic methods were used to characterize these complexes, and the molecular structure of the complex was determined by X-ray crystallography. Amyloid aggregation and inhibition activities were examined using the Thioflavin-T (ThT) assay, and the secondary structures of the protein were analyzed by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The cell viability assay was carried out on the neuroblastoma cell line, revealing that the complex Ru-2 showed better protective effects against Aβ1–42 peptide toxicity on neuro-2a cells than the complex Ru-1. Molecular docking studies elucidate the binding sites and interactions between the Ru-complexes and Aβ1–42 peptides. The experimental studies revealed that these complexes significantly inhibited the BSA aggregation and Aβ1–42 amyloid fibril formation at 1:3 and 1:1 molar concentrations, respectively. Antioxidant assays demonstrated that these complexes act as antioxidants, protecting from amyloid-induced oxidative stress. Molecular docking studies with the monomeric Aβ1–42 (PDB: 1IYT) show hydrophobic interaction, and both complexes bind preferably in the central region of the peptide and coordinate with two binding sites of the peptide. Hence, we suggest that the Ru-based complexes could be applied as a potential agent in metallopharmaceutical research against Alzheimer's disease.