The Nutrients journal retracts the article “Chickpea Peptide: A Nutraceutical Molecule Corroborating Neurodegenerative and ACE-I Inhibition” [...]

Deposits of misfolded tau proteins are leading indicators of cognitive decline in Alzheimer’s disease (AD), and our recent data implicate distinctly misfolded conformers of the tau protein with high seeding potency in rapid progression. We considered prion-like templated propagation of misfolding in neurons as an underlying mechanism and derived sensitive conformational assays to test this concept and identify critical structural drivers. Using novel photochemical hydroxylation monitored with a panel of Europium-labeled monoclonal antibodies, we investigated the structural organization of different microtubule binding domains (MTBDs) in brain-derived tau conformers in AD with different progression rates. We analyzed the impact of structural organization of different MTBDs on seeding potency in vitro and in primary neurons, and on the propagation rate of tau misfolding, compartmentalization, cytotoxicity, and calcium homeostasis in neuronally differentiated SH-SY5Y cells. Within the extensive inter-individual structural variability in all MTBDs and C-terminal tails, the most significant driver of seeding potency and propagation of tau protein misfolding in both in vitro seeding assays and in neuronal cultures was the structural exposure of the fourth MTBD (R4). In contrast, the major driver of calcium influx induced in neurons by the accumulation of misfolded tau was the structural exposure of the R1 domain. The data provide compelling evidence for a major diversity in the structural organization of MTBDs of misfolded AD brain-derived tau protein and implicate the structural exposure of distinct domains in different pathogenetic steps of AD — R4 tau domain in progression rate, and R1 domain in variable synaptic toxicity of misfolded tau, and thus in cognitive decline.

Four copper (II) complexes [Cu(L1)2] (1), [Cu(L2)2] (2), [Cu(L3)2] (3) and [Cu(L4)2] (4) with azo linked O,O donar ligands viz 2-hydroxy-5 (phenyldiazenyl)benzaldehyde (HL1), 1-(2-hydroxy-5-(phenyldiazenyl)phenyl)ethanone (HL2), 2-Hydroxy-5-p-tolylazo-benzaldehyde (HL3), 1-(2-Hydroxy-5-p-tolylazo-phenyl)-ethanone (HL4) have been prepared and characterized by different spectroscopy methods and published earlier by our research group. We investigated the interaction of Human Serum Albumin (HSA) with complexes 1–4 under physiological condition in phosphate buffer solution at pH 7.4 using various spectroscopic techniques. The fluorescence titration spectrum disclosed that the complex 1–4 quench the intrinsic fluorescence of HSA robustly through a static quenching mechanism. Binding constants (Kb) and the number of binding sites (n ≈ 1) were evaluated using modified Stern–Volmer equations. Binding constants were found to be 1.9, 6.6, 0.99 and 1.2x 105 (M−1) for complex 1, 2, 3 and 4 respectively. The CD spectra of free HSA and HSA with complexes 1–4 showed that the complexes have negligibleimpact on the secondary structure of HSA as theyremain helical even after the addition of complexes. Molecular docking study was performed to analyse the binding mode of complexes 1-4with HSA. Docking study revealed that hydrophobic and Vander waals interactions were considered to be the main interaction forces involved in the binding of HSA with complexes 1–4.

Ribonucleic acid (RNA) is a versatile molecule, thus the opportunity to target RNA is abundant. Hence, the current exploration deals with the interaction of a metal complex of hydroxamic acid with t-RNA, along with HDAC8 inhibition activity and antioxidant activity. Binding characteristics of complex 1, Bis(N-phenylbenzohydroxamato)Tungsten(VI), which is represented as [N-PBHA-W(VI)] with Torula yeast RNA (t-RNA) were investigated by using several types of spectroscopic techniques, measurements of viscosity, and computational study through molecular docking. The intrinsic binding constant, Kb was evaluated for complex 1 which shows changes in the shift of absorption spectra, and the intensity of spectra also varies. From fluorescence measurement, the binding constant of complex 1 towards t-RNA is 6.64 +/- 0.05 x 105 M-1. Stern-Volmer quenching constant was calculated. Two displacement method sare performed by using the fluorescence spectroscopic technique which discloses a groove mode of binding. The groove mode of binding is also uncovered by viscosity measurement, which was observed by increasing the complex 1 concentration. An electrochemical investigation was carried out by using cyclic voltammetry. A circular dichroism study was also employed, which further suggests a strong binding occurred between the metal complex used and t-RNA. The docked postures of t-RNA with complex 1 expose the strong interactions which suggest a minor groove mode of binding. All the experimental evidence indicates that the interaction of complex 1 with t-RNA is a minor groove mode of binding which complements the molecular docking results. In-silico studies of HDAC8 inhibition activity of complex 1 reveal that the inhibition takes place mainly by hydrophobic interaction. DPPH-radical scavenging method employed for the antioxidant activity.

Amyloidopathies are associated with a biochemical change in concerned organ or bio-environment known as inflammation (promoted in case of fibrils accumulation due to their stability) that triggered us to explore anti-aggregating and disaggregating property of an anti-inflammatory drug amodiaquine (AD) against human lyso-zyme (HL) fibrillation in this study simultaneously evaluating the mechanism of interaction between AD and HL using biophysical and microscopic techniques. The linear modified Stern-Volmer plot obtained from fluorescence quenching of HL by AD at three temperatures and Ksv dependence on temperature with kq > 2.0 x 1010 supports static quenching mechanism with strong binding (Kb = x 106) and negative & UDelta;G value. The change in Trp and Tyr environment due to AD binding were confirmed via synchronous fluorescence. The studies were further extended to analyse aggregation inhibition and disaggregation of HL by AD. RLS, ThT, CD and DLS results suggested aggregation inhibition and disaggregation which was further confirmed via TEM. Overall, our study suggests that AD is an effective drug for fibrillation inhibition and disaggregation. The possible mechanism behind this behaviour is the stabilization of HL in native state by AD via non-covalent interactions and thus AD will not only reduce fibril burden but will also calm severity of the disease (such as regaining cognitive function due to reduced inflammation). This can be an alternative promising approach that can be exploited for treating amy-loidopathies such as Alzheimer's, Parkinson's disease and systemic amyloidosis.

The conversion of native conformation of protein into proteotoxic insoluble entities is not only confined to the protein misfolding diseases(PMDs) but also associated with pharmaceutically important proteins like insulin during the process of manufacturing/administration. Also all the neurodegenerative diseases are marked with increased level of oxidative stress. Thus finding anti-aggregation agents having anti-oxidant potential is utmost important to combat these complications. Herein, we performed biophysical, computational and imaging tech-niques to decipher the anti-aggregation propensity of a naturally occurring hydroxycinnamate-Ferulic acid ethyl ester(FAEE) that can cross blood brain barrier. Rayleigh light scattering(RLS) measurements, dye binding assays, Circular dichroism(CD) and Dynamic light scattering(DLS) measurements confirmed the efficient inhibition of insulin fibrillation by FAEE. The Stern-Volmer plot analysis reflected both the static and dynamic quenching mechanism of insulin by FAEE. Molecular docking and simulations results showed that FAEE bind and stabilizes the monomeric state of insulin with binding energy-6.3 kcal/mol. Moreover, the inhibiting effect of FAEE on the elongation phase of onward fibrillation in addition with triggering its fibril disassembling capability was also investigated. Both the stabilization of the protein structure and the restriction of monomers recruitment necessary for the fibrillar growth acts synergistically in providing FAEE its anti-aggregation effect. Appearance of significantly lesser and shortened fibrils in FAEE presence was depicted by transmission electron microscopy. Overall, our finding establishes for the first time the anti-amyloidogenic role of lipophilic FAEE which can cross blood brain barrier. These findings may inspire the development of FAEE based formulations that could possibly treat amyloid-related diseases.

Protein aggregation is an underlying cause of many neurodegenerative diseases. Also, the overlapping pathological disturbances between neurodegenerative diseases and type-2 diabetes mellitus have urged the scientific community to explore potential of already available anti-diabetic medications in impeding amyloid formation too. Recent study brief out promising potential of an anti-diabetic drug Glyburide(GLY) as an inhibitor of amyloid fibrillation utilizing several biophysical techniques, computational methods and imaging tools. The mechanism of interaction was elucidated and the structural alterations in human serum albumin(HSA) as well as the microenvironment changes of its fluorophores(tryptophan, tyrosine) upon interacting with GLY were studied by spectroscopic techniques like Circular dichroism and synchronous fluorescence. Binding studies detailing about the GLY-HSA complex distance and the energy transfer efficiency was obtained by Fluorescence resonance energy transfer. For aggregation inhibition studies, the existence and size of aggregates formed in HSA and their inhibition by GLY was determined by Turbidity assay, Dynamic light scattering and Rayleigh light scattering along with dye binding assays. The ThT kinetics measurements analysis suggested that GLY deaccelerates fibrillation by decrement of apparent rate(Kapp) constant. The inhibitory effect of GLY might be attributed to native structure stabilization of HSA by obstruction into β-sheet conversion as confirmed by CD spectroscopy results. Amyloid inhibition and suppression of amyloid-induced hemolysis by GLY was further delineated by TEM and SEM analysis respectively. All these findings for the first time report the new facet of the anti-amyloidogenic potential of GLY, making it a promising candidate to treat neurodegenerative diseases too in the near future.

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.

Protein aggregation leads to several human pathologies such as Alzheimer's disease (AD), type 2 diabetes (T2D), Parkinson's disease (PD), etc. Due to the overlap in the mechanisms of type 2 diabetes and brain disorders, common effective pharmacological interventions to treat both T2D and AD is under extensive research. Therefore, major aim of research is to repurpose already established treatment of diabetes to cure AD as well. This study evaluates mechanistic insight into anti-amyloidogenic potential of anti-diabetic drug Vildagliptin (VLD) on human serum albumin fibrillation (HSA) by using biophysical, calorimetric, imaging techniques along with hemolytic assay. Dynamic light scattering (DLS) and Rayleigh light scattering (RLS) results showed presence of few small-sized aggregates in the presence of VLD which are formed by deaccelerating the amyloidogenesis as shown by thioflavin T (ThT) fluorescence and Congo red (CR) binding assay. Further, Isothermal titration calorimetry (ITC), steady state fluorescence quenching, molecular docking results revealed that VLD form complex with amyloid facilitating state of HSA and consequently mask the hydrophobic residues involved in amyloidogenesis as evident from decrease in ANS fluorescence. Differential scanning calorimetry (DSC) results confirm that VLD stabilizes the amyloid facilitating state of HSA. In addition, SEM images demonstrated that VLD alleviates the hemolytic effect induced by fibrils of HSA. This study reports VLD as a potential inhibitor of amyloid fibrillation and provides promising results to repurpose VLD as a drug candidate for the cure of Alzheimer's diseases along with diabetes.

Loratadine is an important anti-allergic drug. It is a second generation antihistamine drug used to treat allergic rhinitis, hay fever and urticaria. Human serum alpha 1-acid glycoprotein (AG) is an important acute phase protein and its serum concentration is found to increase in inflammation and acute response.The binding interaction between loratadine and AG is studied using spectroscopy and molecular docking techniques. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of AG is quenched by loratadine. Loratadine was found to bind AG with the binding constant of approximate to 10(4) at 298 K. The Gibb's free energy change was found to be negative for the interaction of loratadine with AG indicating the binding process is spontaneous. Binding of loratadine with AG induced ordered structures in the protein. Hydrogen bonding and hydrophobic interactions were the main bonding forces between AG-loratadine as revealed by molecular docking results. This study suggests the importance of binding of anti-allergic drug to AG spatially in the diseases where the plasma concentration of AG increases many folds and interaction with this protein becomes significant. This study will help in design of drug dosage and adjustment accordingly to achieve optimal treatment outcome.

Recent findings of diverse populations of prion-like conformers of misfolded tau protein expand the prion concept to Alzheimer's disease (AD) and monogenic frontotemporal lobar degeneration (FTLD)-MAPT P301L, and suggest that distinct strains of misfolded proteins drive the phenotypes and progression rates in many neurodegenerative diseases. Notable progress in the previous decades has generated many lines of proof arguing that yeast, fungal, and mammalian prions determine heritable as well as infectious traits. The extraordinary phenotypic diversity of human prion diseases arises from structurally distinct prion strains that target, at different progression speeds, variable brain structures and cells. Although human prion research presents beneficial lessons and methods to study the mechanism of strain diversity of protein-only pathogens, the fundamental molecular mechanism by which tau conformers are formed and replicate in diverse tauopathies is still poorly understood. In this review, we summarize up to date advances in identification of diverse tau conformers through biophysical and cellular experimental paradigms, and the impact of heterogeneity of pathological tau strains on personalized structure- and strain-specific therapeutic approaches in major tauopathies.

Chickpea seeds are the source of proteins in human nutrition and attribute some nutraceutical properties. Herein, we report the effects of chickpea seed bioactive peptide on albumin, insulin, lactoglobulin and lysozyme amyloid fibril formation. Employing thioflavin T (ThT) assays and circular dichroism (CD), amyloid structural binding transition was experimented to analyze the inhibition of amyloid fibril formation. The purified active peptide with a molecular mass of 934.53 Da was evaluated in vitro for its ACE-I inhibitory, antibacterial, antifungal and antidiabetic activities. Further, in vivo animal studies were carried out in wistar rats for blood pressure lowering action. In hypertensive rats, chickpea peptide decreased 131 ± 3.57 mm of Hg for systolic blood pressure and 86 ± 1.5 mm of Hg for diastolic blood pressure after 8 h intraperitoneal administration. Additionally, the peptide suppressed the fibrillation of amyloid and destabilized the preformed mature fibrils. Data emphasize efficacy of chickpea peptide vis-a-vis ACE-Inhibitory, antibacterial, antifungal, antidiabetic and anti-amyloidogenic activities, allowing us to propose this novel peptide as a suitable candidate for nutraceutical-based drugs and seems the first kind of its nature.

Amyloidopathies are the consequence of misfolding with subsequent aggregation affecting people worldwide. Irrespective of speedy advancement in the field of therapeutics no agent for treating amyloidopathies has been discovered and thus targeting amyloid fibrillation process via repositioning of small molecules can be fruitful. According to previous reports potential amyloid inhibitors possess unique features like, hydrophobicity, aromaticity, charge etc. Herein, we have explored the effect of Cholic acid (CA) on amyloid fibrillation irrespective of the charge (determined by Zetasizer) using four proteins Human Serum Albumin, Bovine Serum Albumin, Human Insulin and Beta-lactoglobulin (HSA, BSA, HI and BLG) employing biophysical, imaging and computational techniques. ThT results revealed that CA in both protonated and deprotonated form is potent to curb HSA, BSA, BLG aggregation ~50% and HI aggregation ~96% in a dose dependent manner (in accord with CD, ANS and Congo red assay). Interestingly, CA treated samples displayed reduced cytotoxicity (Hemolytic assay) with altered morphology (TEM) and mechanism behind inhibition may be the interaction of CA with proteins via hydrophobic interactions and hydrogen bonding (supported by molecular docking results). This study proved CA (irrespective of the pH) a potential inhibitor of amyloidosis thus can be helpful in generalizing and repurposing the related drugs/compounds for their anti-aggregation behavior as an implication towards treating amyloidopathies.

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques—mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures.

Oxidative stress, amyloid formation, impaired proteasomal degradation are hallmarks of neurodegenerative diseases like Alzheimer's (AD) and are targets for developing therapeutics against such diseases. Thionamide antibiotics are second-line anti-TB drugs. We studied the inhibitory action of two thionamide drugs, prothionamide and ethionamide, against amyloid formation using various biophysical techniques. We also studied the ability of these drugs to act as antioxidants. Here, we show that both drugs are potent inhibitors of in-vitro amyloid formation of human insulin and Aβ42 and protect cultured neuroblastoma cells against the toxic effects of amyloids. Various biophysical techniques like thioflavin-T binding assays, dynamic light-scattering (DLS), circular dichroism (CD), and transmission electron microscopy studies confirm that these drugs prevent amyloid fibril formation. CD and DLS measurements reveal that these drugs exert their anti-amyloid potency by stabilising the proteins in their native state. Their cytoprotective behaviour could be attributed to their antioxidant properties and their ability to inhibit in vitro lipid peroxidation, as confirmed by various antioxidant assays. This study reports for the first time a new facet of thionamide antibiotics as potential amyloid and oxidation inhibitors, with implications in reducing oxidative stress-related manifestations of AD, thereby opening avenues to be used as a therapeutic in AD.

Fluorescence and circular dichroism spectroscopic techniques and molecular docking were used to study binding of azelastine with human serum albumin (HSA). Time resolve fluorescence spectroscopy results indicated that the quenching mechanism is dynamic. Fluorescence quenching results demonstrated that the binding of azelastine to HSA is weak, binding reaction is spontaneous. There is fluorescence energy transfer from tryptophan of HSA to bound azelastine. 2.34 nm is the binding distance calculated from FRET data. Molecular docking results suggested that the binding site for azelastine in HSA is located in subdomain II A. Interaction of azelastine to HSA induced ordered secondary structure in HSA. Binding of azelastine to HSA can affect pharmacokinetics of drug. Hence, rationalizing drug dosage is important for clinical application of azelastine. (C) 2019 Elsevier B.V. All rights reserved.

Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer's and Parkinson's diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha helical structure of protein during fibrillation, reduces the formation of beta sheet rich structure. Furthermore, Dynamic Light Scattering (DLS) and Transmission Electronic Microscopy (TEM) analysis confirmed that CN treated samples exhibited different size distribution and morphology, respectively. In addition, molecular docking results revealed that CN interacts with insulin through hydrophobic interactions as well as hydrogen bonding, and the Hemolytic assay confirmed the non-hemolytic activity of CN on human RBCs. For future research, this study may assist in the rational designing of molecules against amyloid formation.